Thèses : « Farmacologic resistance »

1

Bassani, Nicklas. « New targets in tumor angiogenesis to block tumor re-growth and therapeutic resistance ». Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/462953.

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Antiangiogenic drugs are used clinically for treatment of different types of cancers and in the case of renal cell carcinoma (RCC) are now standard first-line treatments (Rini, 2009). Nevertheless, these agents mainly serve to stabilize the disease but are not able to eliminate all tumor cells and resistance eventually develops concomitant with progression (Kerber and Folkman, 2002). Using different orthoxenograft mouse models of RCC we confirmed that inhibitors of the VEGF pathway have a therapeutic window of effectiveness but unfortunately adaptation and tumor relapse always occur. Several different mechanisms of resistance to antiangiogenics have been already described, many concerning the activation of compensatory signals that produce re-vascularization and tumor re-growth. (Bergers and Hanahan, 2008). In our study we determined that even if resistance is characterized by the up-regulation of the pro-angiogenic enzyme ECGF1 (previous data from the lab); re-vascularization does not occur, the vascular trimming is maintained by the treatment pointing to an alternative mode of adaptation. Using two different approaches, we demonstrated that PD-ECGF1 is responsible of the acquisition of resistance to anti-anigiogenics. In fact, its enzymatic inhibition as second-line treatment post resistance resulted in the arrest and stabilization of tumor growth. PD-ECGF1 inhibition did not increase the anti-vascular effect of DC101, confirming that resistance was vessel independent, but dramatically affected tumor cell proliferation and apoptosis. Considering the numerous proprieties ascribed to its final metabolite 2-deoxy-D-ribose (Ikeda et al., 2006; Bjinsdorp et al., 2008), we evaluated its role, finding that recombinant 2-deoxy-D-ribose nullified the effect of AEAC on 786O- and Ren28 tumor growth rescuing tumor cell proliferation and apoptosis, without promoting re-vascularization. Overall these findings, confirmed also in an in vitro setting, demonstrate that tumor stroma plays a minor role in this model of anti-angiogenics resistance, and that PD-ECGF1 acts mainly intracellularly supporting tumor cell proliferation and protecting from apoptosis by its enzymatic activity and final metabolite 2-deoxy-D-ribose. Using a genetic approach in which PD-ECGF1 protein expression was silence as a second line of treatment post DC101-resistance, mimicking what done pharmacologically, we confirmed and improved the anti-tumoral response described, without perceiving any doxycycline toxic effect. In fact, PD-ECGF1 genetic knock down resulted in a complete arrest of tumor progression enhancing the merely partial stabilization produce by AEAC treatment in 786O- tumor bearing mice. Unfortunately VEGFR-blockers can’t be used in vitro on cancer cells, and to verify this hypothesis we decided to mimic the final effects of anti-angiogenic treatments, finding that under nutrient deprivation conditions cancer cells significantly up-regulate PD-ECGF1 expression, and metabolizing thymidine acquired considerable growth advantages. Finally, the analysis of plasma and tissue samples of ccRCC patients from the Bellvitge Hospital confirm in a clinical set that PD-ECGF1 is exclusively found in pathologic conditions, where if highly expressed correlates with poor prognosis, suggesting that might represent a good therapeutic predictor factor. Moreover, the analysis of tissues biopsies before and after anti-angiogenic treatment revealed that whilst PD-ECGF1 treatment-induce up-regulation was a common feature, the patients that unfortunately didn’t respond showed the sharp difference, confirming PD-ECGF1 as a possible therapeutic target.
Los fármacos antiangiogénicos se usan clínicamente para el tratamiento de diferentes tipos de cáncer y en el caso del carcinoma de células renales (CCR) representan la primera diana terapéutica (Rini, 2009). Sin embargo, no son capaces de eliminar todas las células tumorales y muchos pacientes desarrollan resistencia al tratamiento y recrecimiento tumoral (Kerber y Folkman, 2002). Mediante el uso de diferentes modelos de ratón hemos confirmado que los inhibidores de la vía VEGF tienen una limitada ventana terapéutica de efectividad, seguida desafortunadamente de adaptación y recaída tumoral que en nuestro estudio esta` caracterizada por la regulación positiva de la enzima PD-ECGF1. Usando dos enfoques diferentes, demostramos que PD-ECGF1 es responsable de la adquisición de resistencia a anti-anigiogénicos. De hecho, su inhibición enzimática como tratamiento de segunda línea después de la resistencia dio como resultado la detención y la estabilización del crecimiento tumoral. La inhibición de PD-ECGF1 no aumentó el efecto antivascular de DC101, pero afectó de manera dramática la proliferación y apoptosis de las células tumorales. Teniendo en cuenta las numerosas propiedades atribuidas a su metabolito final 2-deoxy-D-ribose (Ikeda et al., 2006; Bjinsdorp et al., 2008), evaluamos su papel, encontrando que la 2-deoxy-D-ribose anuló el efecto de la inhibición de PD-ECGF1 rescatando el crecimiento tumoral. Experimentos en vitro demostraron como las células tumorales incrementaban la expresión de PD-ECGF1 en condiciones de falta de nutrientes haciéndonos especular que esta proteína tenga un papel en la adaptación metabolica. Finalmente, el análisis de muestras de plasma y tejido de pacientes con ccRCC del Hospital de Bellvitge confirma en un conjunto clínico que PD-ECGF1 se encuentra exclusivamente en condiciones patológicas, donde se correlaciona con mal pronóstico, sugiriendo que podría representar un buen factor predictor terapéutico.

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Ravegnini, Gloria <1984&gt. « Mechanisms Contributing to Tyrosin Kinase Inhibitor Resistance in GISTs : Toward a Personalized Therapy ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6331/1/Ravegnini_tesi.pdf.

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Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. This work considers the pharmacological response in GIST patients treated with imatinib by two different angles: the genetic and somatic point of view. We analyzed polymorphisms influence on treatment outcome, keeping in consideration SNPs in genes involved in drug transport and folate pathway. Naturally, all these intriguing results cannot be considered as the only main mechanism in imatinib response. GIST mainly depends by oncogenic gain of function mutations in tyrosin kinase receptor genes, KIT or PDGFRA, and the mutational status of these two genes or acquisition of secondary mutation is considered the main player in GIST development and progression. To this purpose we analyzed the secondary mutations to better understand how these are involved in imatinib resistance. In our analysis we considered both imatinib and the second line treatment, sunitinib, in a subset of progressive patients. KIT/PDGFRA mutation analysis is an important tool for physicians, as specific mutations may guide therapeutic choices. Currently, the only adaptations in treatment strategy include imatinib starting dose of 800 mg/daily in KIT exon-9-mutated GISTs. In the attempt to individualize treatment, genetic polymorphisms represent a novelty in the definition of biomarkers of imatinib response in addition to the use of tumor genotype. Accumulating data indicate a contributing role of pharmacokinetics in imatinib efficacy, as well as initial response, time to progression and acquired resistance. At the same time it is becoming evident that genetic host factors may contribute to the observed pharmacokinetic inter-patient variability. Genetic polymorphisms in transporters and metabolism may affect the activity or stability of the encoded enzymes. Thus, integrating pharmacogenetic data of imatinib transporters and metabolizing genes, whose interplay has yet to be fully unraveled, has the potential to provide further insight into imatinib response/resistance mechanisms.

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Ravegnini, Gloria <1984&gt. « Mechanisms Contributing to Tyrosin Kinase Inhibitor Resistance in GISTs : Toward a Personalized Therapy ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6331/.

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Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. This work considers the pharmacological response in GIST patients treated with imatinib by two different angles: the genetic and somatic point of view. We analyzed polymorphisms influence on treatment outcome, keeping in consideration SNPs in genes involved in drug transport and folate pathway. Naturally, all these intriguing results cannot be considered as the only main mechanism in imatinib response. GIST mainly depends by oncogenic gain of function mutations in tyrosin kinase receptor genes, KIT or PDGFRA, and the mutational status of these two genes or acquisition of secondary mutation is considered the main player in GIST development and progression. To this purpose we analyzed the secondary mutations to better understand how these are involved in imatinib resistance. In our analysis we considered both imatinib and the second line treatment, sunitinib, in a subset of progressive patients. KIT/PDGFRA mutation analysis is an important tool for physicians, as specific mutations may guide therapeutic choices. Currently, the only adaptations in treatment strategy include imatinib starting dose of 800 mg/daily in KIT exon-9-mutated GISTs. In the attempt to individualize treatment, genetic polymorphisms represent a novelty in the definition of biomarkers of imatinib response in addition to the use of tumor genotype. Accumulating data indicate a contributing role of pharmacokinetics in imatinib efficacy, as well as initial response, time to progression and acquired resistance. At the same time it is becoming evident that genetic host factors may contribute to the observed pharmacokinetic inter-patient variability. Genetic polymorphisms in transporters and metabolism may affect the activity or stability of the encoded enzymes. Thus, integrating pharmacogenetic data of imatinib transporters and metabolizing genes, whose interplay has yet to be fully unraveled, has the potential to provide further insight into imatinib response/resistance mechanisms.

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Silva, Aleksandra Alves. « Efeito de inibidores farmacologicos da iNOS na sensibilidade e sinalização de insulina em animais obesos ». [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311208.

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Orientador: Mario Jose Abdalla Saad
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-14T18:10:42Z (GMT). No. of bitstreams: 1 Silva_AleksandraAlves_M.pdf: 835795 bytes, checksum: 33a3294673868ddb2965c3532c08b534 (MD5) Previous issue date: 2009
Resumo: As óxido nítrico sintases (NOS) são divididas em dois grandes grupos de enzimas, NOS induzível (iNOS) e NOS constitutivas (cNOS). Embora o óxido nítrico (NO) seja um importante mediador de defesa do organismo, a produção excessiva de NO está envolvida na patogênese de muitas doenças inflamatórias e metabólicas. Alguns estudos demonstram que o óxido nítrico exógeno e o NO produzido pela iNOS pode induzir resistência à insulina em músculo e desempenha um papel importante na hiperglicemia de jejum. Este estudo teve como objetivo sintetizar e investigar o efeito de um potente e seletivo inibidor de atividade da iNOS, o Iodato de S-Metilisotiouréia (I-SMT) 5 mg/kg por dia, na hiperglicemia de jejum e na resistência à insulina em um modelo de obesidade induzida por dieta hiperlipídica. Foram observados os parâmetros metabólicos e de sinalização celular da Proteína quinase B/Akt (Akt) e os resultados fornecem evidências de que o grupo tratado com I-SMT foi protegido contra o desenvolvimento de resistência à insulina, e intolerância à glicose induzida por dieta hiperlipídica. Portanto, propomos que potentes inibidores farmacológicos, com seletividade significativa pela iNOS podem representar uma nova abordagem terapêutica para o tratamento da resistência à insulina e suas complicações como o diabetes tipo 2.
Abstract: Nitric oxide synthase (NOS) has been divided into two major sub-enzymes, inducible NOS (iNOS) and constitutive NOS (cNOS). Although nitric oxide (NO) is an important defense mediator, the excessive production of NO has been involved in the pathology of many inflammatory and metabolic diseases. Some studies demonstrate that exogenous nitric oxide (NO) and the NO produced by iNOS can induce insulin resistance in muscle and plays an important role in fasting hyperglycemia. This study investigates the effect of a potent and selective iNOS activity inhibitor, the S-Methylisothiourea Iodide (SMT-I) 5 mg/kg per day, in fasting hyperglycemia and insulin resistance in diet-induced obesity model. We observed the metabolic parameters and Akt signalization and these findings provide evidence that the SMT-I treated group are protected against the development of insulin resistance, glucose intolerance and diet-induced obesity. Therefore, we propose that highly selective inhibitors of iNOS activity may represent a novel therapeutic approach for the therapy of insulin resistance and its complications as type 2 diabetes.
Mestrado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Mestre em Fisiopatologia Médica

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Giommarelli, C. « APPROACHES OF MODULATION OF THERAPEUTIC TARGETS RELEVANT TO DRUG RESISTANCE ». Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/150271.

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An emerging anticancer strategy is to identify molecular targets that when inhibited pharmacologically, result in pleiotropic downstream effects on pathways relevant to the malignant phenotype, with particular reference to the development of resistance. In recent years, the molecular chaperone Hsp90 has emerged as leading example of such a target-specific therapy. Heat-shock proteins are found at increased levels in many solid tumors and haematological malignancies. Their expression may account for the ability of malignant cells to maintain protein homeostasis even in the hostile hypoxic microenvironment of the tumor. Particularly, Hsp90 has emerged as a target for cancer therapy due to its critical roles in retaining the conformation and the function of its client proteins, many of which are associated with cancer pathology. There are 12 HSP90 inhibitors in clinical trials; all have potencies on the order of 7-35 nM in a cell-based assay and half-lives of 1-3 h in the plasma of rodents. These inhibitors may distribute preferentially to the tumors and may have longer half-lives in the tumors than in the plasma, as in the case of 17-AAG and its active metabolite. However, side effects and poor formulability of 17-AAG restricted its potential of clinical application and motivated many groups to synthesize new compounds. The aim of this project is to identify new Hsp90 inhibitors which could overcome the limitations of available inhibitors and exhibit therapeutic advantages in terms of specificity, therapeutic index and antitumor efficacy. Several compounds have been tested during these years, especially a series of mold metabolites of Ascomycetes, structurally belonging to the class of azaphilones, were found to inhibit the heat shock protein Hsp90. In particular, bulgarialactone B was tested for its binding to Hsp90 using surface plasmon resonance and limited proteolysis assays and for its effects on Hsp90 client proteins expression in a series of human tumor cell lines. This compound showed high affinity for Hsp90, interacting with the 90–280 region of the N-terminal domain and down-regulated the Hsp90 clientproteins Raf-1, survivin, Cdk4, Akt, and EGFR. Bulgarialactone B and other natural azaphilones showed antiproliferative activity in a panel of human tumor cell lines; their conversion into semisyntheticderivatives by reaction with primary amines increased the antiproliferative activity. Preliminary results indicated in vivo activity of bulgarialactone B against an ascitic ovarian carcinoma xenograft, thus supporting the therapeutic potential of this novel series of Hsp90 inhibitors. Moreover, to identify favourable interactions between Hsp90 inhibitors and target-specific agents, combinations between curcumin and two well-known HDAC inhibitors (vorinostat and panobinostat), have been tested. Curcumin, a natural polyphenol, has been described to exhibit effects on signaling pathways, leading to induction of apoptosis. In this study, we observed that curcumin inhibited Hsp90 activity causing depletion of client proteins implicated in survival pathways. Based on this observation, the study was designed to investigate the cellular effects of curcumin combination with the pan- HDAC inhibitors, vorinostat and panobinostat, which induce hyperacetylation of Hsp90, resulting in inhibition of its chaperone function. The results showed that, at subtoxic concentrations, curcumin markedly sensitized tumor cells to vorinostat- and panobinostat-induced growth inhibition and apoptosis. The sensitization was associated with persistent depletion of Hsp90 client proteins (EGFR, Raf-1, Akt, and survivin). In conclusion, our findings document a novel mechanism of action of curcumin and support the therapeutic potential of curcumin/HDAC inhibitors combination, because the synergistic interaction was observed at pharmacologically achievable concentrations, which were ineffective when each drug was used alone. Another new therapeutic target is GGT (gamma-glutamyl transferase), the interest in this enzyme is related to recent evidence supporting that it is implicated in tumor progression and in drug resistance to stress-inducing agents (i.e. platinum compounds). The extracellular -glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. Therefore the objective of this approach was to investigate the role of GGT in response to various stress-inducing agents. The study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H2O2. Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents. Again, in an attempt to develop novel strategies for overcoming the mechanisms of cellular protection against oxidative stress, we have explored the efficacy of the combination of two prooxidant agents in the two human melanoma cell clones differently expressing GGT. The γ-glutamyltransferase-overexpressing clone exhibited a low susceptibility to arsenic trioxide-induced apoptosis, associated with low reactive oxygen species induction and increased catalase activity. The combination of arsenic trioxide with subtoxic concentrations of ascorbic acid resulted in a sensitization to apoptotic cell death. The expression of protective mechanisms, in particular catalase activity, accounted for the behavior of the resistant clone. The sensitization achieved by the combination was associated with a cellular response involving the ASK1/p38 axis, which is implicated in the regulation of catalase expression and the activation of apoptotic signals. In conclusion, the results of our study provide evidence that a rational combination of prooxidant agents may be effective in overcoming cellular tolerance to oxidative stress. Moreover, as a number of recent observations have suggested a potential role for membrane-bound gammaglutamyltransferase (GGT) in tumor progression through redox interactions leading to production of reactive oxygen species, we performed a study to evaluate whether such pro-oxidant activity of GGT can promote oxidative DNA damage, thus contributing to cancer genomic instability. Human GGT-transfected melanoma cells were studied, and DNA damage was measured by using the alkaline comet assay. Our results indicate that higher levels of GGT activity are associated with higher levels of background DNA damage and oxidized bases. This association cannot be explained by differences in cell cycle distribution or apoptotic rates. GGT-over-expressing cells also presented with a markedly higher glucose uptake, a phenomenon potentially leading to higher metabolic rate and oxidative DNA damage. Anyway, when GGT-over-expressing cells were incubated in the presence of GGT substrates and a source of catalytic iron, increased levels of DNA damage and oxidized bases were observed, an effect completely prevented in the presence of GGT inhibitors or various antioxidants. The findings reported indicate that GGT activity is able to promote iron-dependent DNA oxidative damage, thus potentially representing an important mechanism in initiation/progression of neoplastic transformation.

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Cocetta, Veronica. « New insights in cisplatin-resistance : role of metabolic reprogramming in cancer cells ». Doctoral thesis, Università degli studi di Padova, 2019. http://hdl.handle.net/11577/3424833.

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Cisplatin is one of the most potent anticancer agents used in the treatment of various solid tumors. Unfortunately, the onset of resistance still limits its use in therapy and severely compromises the treatment effectiveness. Although several studies have been performed, the molecular mechanisms involved in cisplatin resistance are not completely understood. Recently, a metabolic rewiring has been shown to play a prominent role in the response of cancer cells to first-line chemotherapeutic agents, indicating the metabolic pathways as powerful mediators of resistance to cancer treatments. Previous studies of our laboratory had already demonstrated a metabolic switch of cisplatin-resistant cells toward glycolysis and to an altered mitochondrial functionality and morphology. So, in order to better characterize the metabolic fingerprint of cisplatin-resistant cells, in this work the lipid metabolic pathway and the glutamine metabolism have been investigated in models of gynecological cancer cells and Triple-negative breast cancer cells, sensitive and resistant to cisplatin. Aerobic glycolysis, mitochondrial reprogramming, and deregulation of lipid metabolism are not independent pathways but rather they cooperate to sustain cells proliferation and to allow cells survival in a challenging environment induced also by chemotherapeutic drugs. The characterization of the metabolic fingerprint of cells and the interconnection between the different pathways is essential to identify specific targets in cisplatin-resistant cancer cells useful to design pharmacological strategies to bypass resistance. Results indicate a deregulation of the lipid homeostasis in resistant cells which induces a significant lipid accumulation; moreover, preliminary results indicate that resistant cells rely more on glutamine for their survival. From this study and previous observations of our laboratory, different possible metabolic targets specific of cisplatin-resistant cells have been identified, and different pharmacological approaches able to target the identified alterations in lipid metabolism and mitochondria remodeling have been tested. This work fits in the research panorama aimed at providing new insights into the differential metabolic dependencies of cisplatin-resistant tumors. Results may provide novel therapeutic targets exploitable to overcome cisplatin resistance and to enhance the efficacy of the current chemotherapy.
Il cisplatino è uno dei più potenti agenti antitumorali utilizzati nel trattamento di vari tumori solidi. L’insorgenza di fenomeni di resistenza al farmaco è uno dei fattori che ne limita l’utilizzo in terapia e compromette gravemente l’efficacia del trattamento. Nonostante numerosi studi siano stati condotti negli ultimi anni, i meccanismi molecolari coinvolti nella resistenza al cisplatino non sono ancora stati completamente elucidati. Recentemente è stato dimostrato che un ruolo chiave nella risposta cellulare ai farmaci antitumorali è svolto da una riprogrammazione del metabolismo cellulare, indicando le vie metaboliche come potenti mediatori della resistenza ai trattamenti chemioterapici. Studi precedentemente condotti nel nostro laboratorio su cellule di carcinoma ovarico avevano già dimostrato uno shift metabolico verso la glicolisi e alterazioni a livello di funzionalità/morfologia mitocondriale nei cloni resistenti al cisplatino (C13). L’obiettivo di questo lavoro quindi è stato quello di studiare i pathways del metabolismo lipidico e della glutammina allo scopo di ottenere una caratterizzazione più completa del profilo metabolico delle cellule resistenti. Per questo studio sono stati utilizzati diversi modelli cellulari di tumore ginecologico e diverse linee cellulari di tumore mammario triplo negativo, sensibili e resistenti al cisplatino. È importante sottolineare come glicolisi aerobica, riprogrammazione mitocondriale e del metabolismo lipidico non sono vie indipendenti, ma cooperano per sostenere l’omeostasi cellulare e consentire la sopravvivenza delle cellule in ambienti ostili, indotti anche da farmaci chemioterapici. Caratterizzare il profilo metabolico delle cellule e le interconnessioni tra i diversi pathways è essenziale per l’identificazione di target molecolari tumore-resistente specifici sfruttabili per approcci farmacologici innovativi. I risultati ottenuti indicano come le cellule resistenti al cisplatino presentino una alterazione dell’omeostasi lipidica che induce un significativo accumulo intracellulare di lipidi; inoltre gli studi preliminari riguardanti il ruolo della glutammina indicano come la sopravvivenza delle cellule resistenti sia particolarmente dipendente dal metabolismo di questo aminoacido. Da questo studio, e da precedenti osservazioni del nostro laboratorio, sono stati identificati diversi possibili target metabolici specifici delle cellule cisplatino-resistenti, sia a livello di vie metaboliche lipidiche sia a livello mitocondriale. Questo lavoro si inserisce nel panorama di ricerca volto ad approfondire le specifiche dipendenze metaboliche di tumori cisplatino-resistenti. I risultati ottenuti potranno fornire nuovi target terapeutici sfruttabili al fine di superare la resistenza al cisplatino e migliorare l’efficacia dell’attuale trattamento chemioterapico.

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De, Giorgi Ugo Federico Francesco <1970&gt. « CYP17A1 polymorphisms and clinical outcome of patients with metastatic castration-resistant prostate cancer treated with abiraterone ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6436/1/De_Giorgi_Ugo_Tesi.pdf.

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Background. Abiraterone acetate is a potent inhibitor of cytochrome P450 17 α-hydrolase (CYP17A1) that causes a reduction in the synthesis of testosterone in the adrenal glands, testes and tumor microenvironment. Blocking androgen production, abiraterone has been shown to prolong progression-free survival (PFS) and overall survival (OS) in patients with metastatic castration-resistant prostate cancer (CRPC) previously submitted to chemotherapy. The aim of our study was to verify the role of single nucleotide polymorphisms (SNPs) in predicting clinical outcome in CRPC patients treated with abiraterone after chemotherapy. Methods. We analyzed 48 CRPC consecutive patients treated with abiraterone after at least one chemotherapeutic regimen with docetaxel. DNA was extracted from peripheral blood and genotyped for four polymorphisms in the CYP17A1 gene (rs743572, rs10883783, rs17115100, rs284849). PFS and OS survival curves were used to identify statistical associations between haplotypes and clinical outcome. Results. Forty-eight Caucasian patients with metastatic CRPC treated with abiraterone were genotyped for polymorphisms in the CYP17A1 gene. All samples were evaluable for both sequencing and TaqMan Genotyping assay. The CRPC patients treated with abiraterone had a median PFS and OS of 7.6 months (95% CI: 4.3-10.5) and 17.6 months (95% CI: 10.5-19.0), respectively Statistical analyses highlighted a difference approaching statistical significance (log-rank test p = 0.0534) between rs10883783 and PFS. Other polymorphisms were not associated with a benefit from treatment with abiraterone. Conclusions. In our case series of 48 treated patients, rs10883783 only was identified as a possible predictive marker, results showing a trend toward statistical significance. Further analysis of this polymorphism is needed in larger series of patients to confirm our findings.

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De, Giorgi Ugo Federico Francesco <1970&gt. « CYP17A1 polymorphisms and clinical outcome of patients with metastatic castration-resistant prostate cancer treated with abiraterone ». Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6436/.

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Background. Abiraterone acetate is a potent inhibitor of cytochrome P450 17 α-hydrolase (CYP17A1) that causes a reduction in the synthesis of testosterone in the adrenal glands, testes and tumor microenvironment. Blocking androgen production, abiraterone has been shown to prolong progression-free survival (PFS) and overall survival (OS) in patients with metastatic castration-resistant prostate cancer (CRPC) previously submitted to chemotherapy. The aim of our study was to verify the role of single nucleotide polymorphisms (SNPs) in predicting clinical outcome in CRPC patients treated with abiraterone after chemotherapy. Methods. We analyzed 48 CRPC consecutive patients treated with abiraterone after at least one chemotherapeutic regimen with docetaxel. DNA was extracted from peripheral blood and genotyped for four polymorphisms in the CYP17A1 gene (rs743572, rs10883783, rs17115100, rs284849). PFS and OS survival curves were used to identify statistical associations between haplotypes and clinical outcome. Results. Forty-eight Caucasian patients with metastatic CRPC treated with abiraterone were genotyped for polymorphisms in the CYP17A1 gene. All samples were evaluable for both sequencing and TaqMan Genotyping assay. The CRPC patients treated with abiraterone had a median PFS and OS of 7.6 months (95% CI: 4.3-10.5) and 17.6 months (95% CI: 10.5-19.0), respectively Statistical analyses highlighted a difference approaching statistical significance (log-rank test p = 0.0534) between rs10883783 and PFS. Other polymorphisms were not associated with a benefit from treatment with abiraterone. Conclusions. In our case series of 48 treated patients, rs10883783 only was identified as a possible predictive marker, results showing a trend toward statistical significance. Further analysis of this polymorphism is needed in larger series of patients to confirm our findings.

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GonÃalves, Walterlene de Carvalho. « Perfil da ResistÃncia aos FÃrmacos Antituberculose em Pacientes Atendidos nos ServiÃos de SaÃde no Estado do PiauÃ ». Universidade Federal do CearÃ, 2012. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=9768.

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nÃo hÃ
A tuberculose Ã uma doenÃa infectocontagiosa, que persiste ao longo da histÃria mundial como um sÃrio problema de saÃde pÃblica. Ã causada pelo Complexo Mycobacterium tuberculosis. O MinistÃrio da SaÃde considera a tuberculose como uma doenÃa prioritÃria no Brasil, pois o mesmo ocupa o 17Â lugar entre os 22 paÃses em desenvolvimento, responsÃveis por 80% dos casos mundiais dessa enfermidade. TÃm-se verificado um aumento no nÃmero de casos de resistÃncia bacteriana Ãs drogas antituberculose. Este estudo teve carÃter descritivo e foi retrospectivo dos anos de 2005 a 2007 e prospectivo de 2008 a 2009, tendo por objetivo avaliar o diagnÃstico da tuberculose e o perfil de resistÃncia Ãs drogas antituberculose em 563 exames de pacientes atendidos nos ServiÃos de SaÃde do Estado no LACEN-PI, atravÃs da cultura e teste de sensibilidade de variadas espÃcimes biolÃgicas. De 563 casos encontrados avaliados, 123 apresentaram cultura positiva, dos quais cerca de 91,8 % apresentaram a forma pulmonar da doenÃa, sendo 65 % da populaÃÃo avaliada do sexo masculino. A mÃdia da idade em anos foi de 43,97Â16,81 (1-89). Em todos os anos, os casos tratados superaram em 92,6% os casos novos. Identificou-se qualquer resistÃncia em 68 casos (55,3%). A taxa de resistÃncia primÃria e adquirida foi, 3% e 97%, respectivamente. Foi encontrado 39 casos (31,7%) de multirresistÃncia, 15 casos (12,1%) de monorresistÃncia, 14 casos (11,3%) de polirresistÃncia. Conclui-se que os nÃveis de resistÃncia encontrados sÃo elevados. Dessa forma, faz-se necessÃrios o monitoramento dos nÃveis de resistÃncia, o controle adequado dos casos de tuberculose no PiauÃ, com a ampliaÃÃo dos exames de cultura e teste de sensibilidade para o diagnÃstico precoce. TambÃm deve ser encorajado o desenvolvimento de mais pesquisas para padronizaÃÃo de novos testes de diagnÃsticos que sejam prÃtico, rÃpido, de fÃcil execuÃÃo, baixo custo que reÃna alta especificidade e sensibilidade para a doenÃa
Tuberculosis is an infectious and contagious disease which persists throughout world history as a serious public health problem. It is caused by the complex Mycobacterium tuberculosis. The Ministry of Health considers tuberculosis a priority disease in Brazil, because the country occupies the 17th place among 22 developing countries, all together responsible for 80% of the worldwide cases of this disease. There has been an increase in the number of bacterial resistance cases to antituberculosis drugs. This study was descriptive and retrospective study of the years 2005 to 2007 and prospective from 2008 to 2009, aiming to assess the diagnosis of tuberculosis and the profile of antituberculosis drug resistance tests in 563 patients treated in the Health Services of the State in LACEN -PI, through culture and sensitivity testing of various biological specimens. From 563 cases, 123 were positive to culture, of which about 91.8% had the pulmonary form of the disease, being 65% of the population in focus consisting of male sex individuals. The mean age in years was 43.97 Â 16.81 (1 - 89). In all years, the cases treated exceeded in 92.6% the new cases. No resistance was identified in 68 cases (55.3%). The rate of primary and acquired resistance was 3% and 97% respectively. It was found 39 cases (31.7%) of multi-drug resistance, 15 cases (12.1%) of single drug resistance, and 14 cases (11.3%) of full range drug resistance. The conclusion is that levels of resistance found are high. Thus, it is necessary to monitor the resistance levels, in addition to adequate control of tuberculosis cases in PiauÃ, by expanding the culture testing and sensitivity testing for early diagnosis. It should also be encouraged to develop more research to standardize new diagnostic tests that are practical, fast, easily run, low cost and possessing high specificity and sensitivity for the disease

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Catanzaro, Daniela. « Metabolic and mitochondrial remodelling in cisplatin resistance : studies on ovarian cancer cells and derived cytoplasmic hybrids ». Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423389.

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The onset of resistance to cisplatin still limits its use in the chemotherapy of ovarian cancer and, despite several mechanisms of resistance have been discovered, they are not exhaustive. The aim of this study was to identify which other pathways are exploited by cancer cells to escape cisplatin cytotoxicity, to possibly prevent or overcome the phenomenon with new pharmacological approaches. The increase of anaerobic glycolysis, even in the presence of oxygen (Warburg effect), is the first observation indicating the alteration of energetic metabolism used by tumor cells as a strategy to adapt and grow independently from the availability of the substrate. This evidence suggested us to investigate the hypothesis that a similar metabolic strategy might be of relevance in resistance to cisplatin. Recently it has been shown that only approximately 1% of intracellular platinum is bound to nuclear DNA, while the great majority of the intracellular drug is available to interact with other nucleophilic sites including but not limited to phospholipids, cytosolic, cytoskeletal and membrane proteins, RNA and mitochondrial DNA. mtDNA, unlike nDNA, does not possess efficient repair systems and is therefore more susceptible to the onset of mutations often associated to cancer development, loss of tumor suppressor, activation of oncogenes and mitochondrial dysfunctions often related with an increase of glycolytic activity. Therefore, the aim of this study was to investigate the energetic metabolism and the mitochondrial function of cisplatin-resistant (C13) and sensitive (2008) ovarian cancer cells with different experimental approaches. Results revealed that resistant cells present a significant reduced respiratory chain activity correlated to a lower mitochondrial mass, altered mitochondrial morphology as well as a metabolomic profile typical of a lipogenic phenotype. To investigate the role of mtDNA and nDNA in the mitochondrial and metabolic remodeling of cisplatin-resistant line, cancer cells (2008-C13) were used to generate transmitochondrial hybrids (H2008-HC13). Mitochondrial DNA of parental and hybrid cells was sequenced, showing similar, almost non pathological, polymorphisms. Interestingly, investigating the energetic metabolism and the mitochondrial structure of hybrids, no differences were observed between H2008 and HC13. These data demonstrated that the metabolic reprogramming of C13 cells was not dependent from mtDNA, but was controlled by nuclear factors. Having regard to these data, the activity of some nuclear transcription factors (HIF-1α, and c-Myc) involved in the metabolic reprogramming of tumor cells, has been evaluated and it has been highlighted a different expression of some of their target genes involved in the glycolytic flux. Finally, the metabolic profile of 2008-C13 cells has been outlined by LC-MS which evidenced some interesting differences in aminoacids, phospholipids and antioxidants content.
L’utilizzo del cisplatino nel trattamento del cancro all’ovaio è tutt’oggi limitato dall’insorgenza di chemioresistenza. Nonostante siano stati evidenziati numerosi meccanismi implicati nella resistenza al cisplatino, questo fenomeno non è ancora stato del tutto chiarito. Lo scopo di questo studio è stato quindi quello di identificare quali altre strategie vengano sfruttate dalle cellule tumorali per sfuggire alla citotossicità indotta dal cisplatino in modo da prevenire o superare la resistenza attraverso l’utilizzo di nuovi approcci farmacologici. L'aumento della glicolisi anaerobica, anche in presenza di alte concentrazioni di ossigeno (effetto Warburg), è la più nota e meglio conosciuta alterazione del metabolismo energetico utilizzato dalle cellule tumorali come strategia per adattarsi e crescere in modo indipendente dalla disponibilità del substrato. Queste evidenze scientifiche ci hanno suggerito di indagare l'ipotesi che una simile strategia possa essere rilevante nell’insorgenza della resistenza al cisplatino. Recentemente è stato dimostrato che solo ~1% del platino intracellulare si lega al DNA nucleare, mentre la gran parte del farmaco interagisce con altri siti nucleofili tra cui fosfolipidi, proteine di membrana, RNA e DNA mitocondriale. Il mtDNA, a differenza del nDNA, non possiede sistemi di riparazione efficaci ed è quindi più suscettibile all’insorgenza di mutazioni e trasformazioni oncogeniche spesso associate allo sviluppo del cancro, perdita di oncosoppressori, attivazione di oncogeni e ad alterazioni della funzionalità mitocondriale correlata ad aumento dell'attività glicolitica. Pertanto, l'obiettivo di questo studio è stato quello di studiare il metabolismo energetico e la funzione mitocondriale delle cellule di cancro ovarico sensibili (2008) e resistenti (C13) al cisplatino utilizzando diversi approcci sperimentali. I risultati hanno evidenziato che le cellule resistenti presentano una significativa riduzione dell'attività della catena respiratoria correlata ad una minore massa mitocondriale, alterata morfologia mitocondriale nonché un profilo metabolico tipico di un fenotipo lipogenico. Per studiare il ruolo del mtDNA e del nDNA nel rimodellamento metabolico e mitocondriale osservato nella linea resistente al cisplatino, le cellule tumorali (2008-C13) sono state utilizzate per generare ibridi transmitocondriali (H2008-HC13). Il DNA mitocondriale delle cellule parentali e degli ibridi citoplasmatici è stato quindi sequenziato, mostrando polimorfismi simili, ma non patologici. È interessante notare che, le differenze metaboliche e mitocondriali evidenziate nelle linee tumorali, non vengono mantenute nei rispettivi cibridi dimostrando che la riprogrammazione metabolica delle cellule C13 non è influenzata dal mtDNA, ma è controllata da fattori nucleari. Alla luce di tali dati, è stata quindi valutata l’attività di alcuni noti fattori di trascrizione (HIF-1α e c-Myc) coinvolti nella riprogrammazione metabolica delle cellule tumorali ed è stata evidenziata una diversa espressione di alcuni dei loro geni target implicati nel flusso glicolitico. Infine il profilo metabolico delle linee 2008-C13 è stato delineato mediante LC-MS da cui sono emerse interessanti differenze nel contenuto lipidico, amminoacidico e di alcuni noti agenti antiossidanti.

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Vianello, Caterina. « Innovative mitochondrial and metabolic targets to enhance cisplatin response : studies on cancer cells with acquired and intrinsic resistance ». Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3425345.

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Cisplatin is one of the most potent anticancer agents used in the treatment of various solid tumors. Unfortunately the onset of resistance is the main limit of this therapy and severely compromises the treatment effectiveness. Although several studies regarding cisplatin resistance have been performed, the molecular mechanisms are not completely understood. Classically, cisplatin is studied as a DNA-damaging chemotherapy agent, but more recent investigations showed that only 5-10% of intracellular platinum is bound to nuclear DNA, while the great majority of the intracellular drug can interact with a variety of cellular component including proteins, RNA and mitochondrial DNA. MtDNA, unlike nDNA, does not possess efficient repair systems; therefore it is more susceptible to the onset of mutations often associated to cancer development, loss of tumor suppressor, activation of oncogenes and mitochondrial dysfunctions related with an increase of glycolytic activity. The Warburg effect indicates the alteration of energetic metabolism used by tumor cells as a strategy to adapt and grow independently from the substrate availability. This evidence suggested us to verify the hypothesis that a similar metabolic strategy might be of relevance in cisplatin resistance. Therefore, our aim was to investigate the energetic metabolism and the mitochondrial dynamic of cisplatin-resistant and sensitive cancer cells with different experimental approaches, in order to reveal targets useful to overcome the resistance. In our laboratory we have already revealed that cisplatin resistant ovarian cancer cell line C13, as compared to sensitive line 2008, exhibits metabolic changes. Indeed, resistant clone showed a different mitochondrial and metabolic profile characterized by an increase of glucose and glutamine uptake, a decrease of the mitochondrial membrane potential and mitochondrial mass. In this scenario, we proceeded to phenotype other cancer cells that present acquired or intrinsic resistance in order to identify new targets to sensitize to cisplatin treatment. Our results pointed out alterations in mitochondrial fusion and fission in chemoresistant cancer cells. Moreover, data obtained showed that resistant clones, with an imbalance toward fission process, present a faster mitochondrial turn-over using mitophagy as a mitochondrial quality control mechanism. Furthermore, the data showed a mitochondrial network differently organized in resistant variants underlining a probable implication of dynamic process in resistance mechanisms. Having regard to the data about metabolic reprogramming, breast cancer cells that have an innate resistance to cisplatin were evaluated. The expression of c-Myc nuclear transcription factor, involved in the metabolic reprogramming of tumor cells, has been evaluated highlighting a different expression of some of its target genes involved in the glycolylisis and glutaminolysis, besides an increased dependency of glucose in cisplatin resistant cells.
Il cisplatino è uno dei più potenti agenti antitumorali utilizzati nel trattamento di vari tumori solidi. Purtroppo l'insorgenza della resistenza è il limite principale di questa terapia e compromette gravemente l'efficacia del trattamento. Anche se sono stati eseguiti numerosi studi per quanto riguarda la resistenza al cisplatino, i meccanismi molecolari non sono del tutto chiari. Classicamente, il cisplatino è studiato come agente chemioterapico che crea danno a livello del DNA, ma studi più recenti hanno dimostrato che solo il 5-10% del platino è legato al DNA nucleare, mentre la maggior parte del farmaco intracellulare può interagire con diverse componenti cellulari tra cui proteine, RNA e DNA mitocondriale. Il DNA mitocondriale, a differenza del DNA nucleare, non possiede sistemi di riparazione efficienti ed è quindi più suscettibile alla comparsa di mutazioni spesso associate allo sviluppo del cancro, alla perdita di oncosoppressori, attivazione di oncogeni e ad alterazioni della funzionalità mitocondriale correlata ad aumento dell'attività glicolitica. L'aumento della glicolisi anaerobica, anche in presenza di alte concentrazioni di ossigeno (effetto Warburg), è l'alterazione del metabolismo energetico utilizzata dalle cellule tumorali come strategia per adattarsi e crescere in modo indipendente dalla disponibilità del substrato. Queste evidenze scientifiche ci hanno suggerito di verificare l'ipotesi che una simile strategia possa essere rilevante nell'insorgenza della resistenza al cisplatino. Pertanto, lo scopo di questo studio è stato quello di indagare il metabolismo energetico e la dinamica mitocondriale delle cellule tumorali sensibili e resistenti al cisplatino con diversi approcci sperimentali, al fine di rivelare utili targets per superare questa importante forma di resistenza. Nel nostro laboratorio abbiamo già dimostrato che la linea di carcinoma ovarico resistente al cisplatino C13, rispetto alla linea sensibile del 2008, presenta cambiamenti metabolici. Infatti, il clone resistente ha mostrato un profilo mitocondriale e metabolico differente, caratterizzato da un aumento della dipendenza da glucosio e glutammina, una diminuzione del potenziale di membrana e della massa mitocondriale. In questo scenario, lo studio ha proseguito con la valutazione del meccanismo di resistenza cisplatino fenotipizzando altre cellule tumorali che presentano resistenza acquisita o intrinseca. I nostri risultati indicano una alterazione dei meccanismi di fusione e fissione mitocondriale nelle cellule tumorali chemioresistenti. I dati ottenuti hanno dimostrato che i cloni resistenti, che presentano uno squilibrio verso processo di fissione, attivano un turn-over mitocondriale più veloce, utilizzando la mitofagia come meccanismo di controllo della qualità mitocondriale. Inoltre, i dati ottenuti hanno mostrato un network mitocondriale diversamente organizzato nelle resistenti sottolineando una probabile implicazione della dinamica mitocondriale nei meccanismi di resistenza. Per quanto riguarda i dati relativi alla riprogrammazione metabolica, sono state prese in esame cellule del cancro al seno che hanno una resistenza innata al cisplatino. È stata valutata l'espressione del fattore di trascrizione c-Myc che è coinvolto nella riprogrammazione metabolica delle cellule tumorali, per di più si è evidenziata una diversa espressione di alcuni geni bersaglio di c-Myc coinvolti nella glicolisi e glutamminolisi, oltre che una maggior dipendenza dal glucosio nelle linee resistenti di carcinoma al seno.

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Medeiros, Melissa Soares. « Polimorfismo do RRE e ResistÃncia aos Antirretrovirais do VÃrus da ImunodeficiÃncia Humana e Efeito CitopÃtico e Replicativo In vitro da Enfuvirtida no CÃdon 36 do VÃrus Modificado pNL4-3 ». Universidade Federal do CearÃ, 2011. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=10351.

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nÃo hÃ
IntroduÃÃo: Rev Responsive Element (RRE) Ã uma molÃcula RNA responsÃvel pelo transporte do mRNA viral do HIV-1 do nÃcleo para o citoplasma da cÃlula CD4, atravÃs da via RRE-Rev, essencial para a replicaÃÃo viral. As mutaÃÃes de resistÃncia a Enfuvirtida sÃo primariamente localizadas no perÃmetro de 10 aminoÃcidos do HR1, regiÃo correspondente no RRE. Caracterizar o RRE poderÃ fornecer uma nova abordagem terapÃutica para a terapia do HIV. Objetivos: Sequenciar e caracterizar o RRE da gp41 para avaliar sua variabilidade e correlaÃÃo com parÃmetros laboratoriais em sequÃncias de pacientes infectados pelo HIV-1 que receberam terapia antirretroviral ou virgens. Em estudo in vitro avaliar a mutaÃÃo 36D na presenÃa de Enfuvirtida. Metodologia: 62 amostras de pacientes com HIV-1 do CearÃ foram coletadas e 35 sequÃncias de RRE foram obtidas e distribuÃdas em trÃs grupos para fins de anÃlise comparativa: N (virgens de terapia), T (uso de antirretroviral sem inibidor de fusÃo) e F (uso de antirretroviral associados a Enfuvirtida). SequÃncias obtidas foram alinhadas com o banco de dados de Los Alamos para HIV usando HIV BLAST Search. Estudo in vitro utilizou dois vÃrus de laboratÃrio pNLA-3 (36D e 36G) observando citopatogenicidade e proliferaÃÃo na presenÃa de doses crescentes de Enfuvirtida. Resultados: A anÃlise filogenÃtica demonstrou alta prevalÃncia do HIV-1 subtipo B (97,2%). Observou-se aumento da resposta imunolÃgica no grupo T (71,5%) comparado ao F (2,98%). MutaÃÃes mais comuns e polimorfismos do Grupo N foram Q32L (41,6%), N42S (8,3%), R46K (33,3%), L54M (41,6%); no grupo T: Q32R (8,3%), R46K (25%), L54M (33,3%); e no grupo F: Q32L (18,2%), G36D (9,1%), V38A (9,1%), N42S (27,3%), N42T (9,1%), R46K (27,3%), L54M (45,4%), K77R (54,5%). TrÃs amostras demonstraram mutaÃÃes de resistÃncia significativas para os inibidores de fusÃo. AnÃlise dos sÃtios primÃrios de ligaÃÃo do RRE observou presenÃa de mutaÃÃo 28A em 27,2% e 8,3% nos grupos F e N respectivamente, e 27S em 8,3% no grupo T. Houve pressÃo seletiva da regiÃo HR1 do HIV-1 de pacientes usando antirretroviral, independente da exposiÃÃo Ã Enfuvirtida. NÃo houve diferenÃa estatÃstica significativa nas curvas de p24 do vÃrus 36D comparado com 36G, independente de concentraÃÃes de T20 (p>0.05). Observou-se menor formaÃÃo de sincÃcio, com diminuiÃÃo da capacidade fusogÃnica, sem impacto na infectividade. ConclusÃo: O estudo definiu as mutaÃÃes e polimorfismos mais prevalentes no CearÃ, sugerindo alta preservaÃÃo nas regiÃes de sÃtio primÃrio de ligaÃÃo do Rev-RRE. Evidenciou baixo perfil de resistÃncia a Enfuvirtida em regimes com falha utilizando esta medicaÃÃo. Detectou-se pressÃo seletiva no HR1 do HIV-1 de pacientes em uso de Antirretroviral, independente de exposiÃÃo Ã Enfuvirtida. Evidenciado in vitro menor formaÃÃo sincicial no vÃrus 36D, com diminuiÃÃo na atividade fusogÃnica, mantendo infectividade.
Introduction: Rev Responsive Element (RRE) is a RNA molecule responsible to mRNA from HIV-1 virus nuclear transportation to cytoplasm through RRE-Rev pathway, essential to virus replication. Enfuvirtide resistance mutations are primary located in a perimeter of 10 amino acids of HR1, a corresponded region of RRE. Characterize RRE should provide a new approach for HIV therapy. Objectives: Sequence and characterize RRE from gp41 to evaluate variability and correlate with laboratory parameters in sequences from HIV-1-infected patients, which were receiving regimens including Enfuvirtide, naÃve or rescue therapy. Also evaluated mutation G to D at codon 36 in the presence of fusion inhibitor (enfuvirtide). Methods: Sixty-two samples from HIV patients in Ceara/Brazil were collected and Thirty-five RRE sequences and clinical follow-up were analyzed, distributed into three groups: N (naÃve therapy), T (treated patients with rescue regimens) and F (rescue regimens containing Enfuvirtide). Sequences obtained were aligned with Los Alamos HIV sequence database by using the HIV BLAST Search. Culture Study was performed using two different pNLA-3 (36D and 36G) with increasing amounts of enfuvirtide. Results: A phylogenetic analyses demonstrated higher prevalence of HIV-1 subtypes B (97.2%). An increased immunology response was observed in CD4 count higher on group T (71.5%) compared with F (2.98%). Group N most common mutations and polymorphisms were Q32L (41.6%), N42S (8.3%), R46K (33.3%), L54M (41.6%); group T: Q32R (8.3%), R46K (25%), L54M (33.3%); and group F: Q32L (18.2%), G36D (9.1%), V38A (9.1%), N42S (27.3%), N42T (9.1%), R46K (27.3%), L54M (45.4%), K77R (54.5%). Three samples demonstrated significant resistance mutations to fusion inhibitors. Analysis of RRE nucleotide primary sites observed mutation 28A in 27.2% and 8.3% on groups F and N respectively, and 27S in 8.3% on group T. There was selective pressure on HR1 region from HIV-1 patients using antiretroviral, independent of enfuvirtide exposure. There was no statistical difference between p24 curves of virus 36D compared with 36G, independent of T20 concentrations (p>0.05). It was observed less syncytial formation in 36D virus, with diminished fusogenic activity besides keeping infectivity. Conclusions: This study defined most prevalent RRE polymorphisms in Ceara/Brazil and suggests highly preserved regions primary sites to Rev connection. Observed a low resistance profile to enfuvirtide in failing regimens with this drug. Selective pressure on HR1 region in failed regimens with out fusion inhibitors was detected. A less syncytial formation in 36D virus with diminished fusogenic activity was detected.

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Amorós, Reboredo Patrícia. « Tractament de l'hepatitis C crònica amb agents antivirals directes en pacients majors de 65 anys ». Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671482.

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ANTECEDENTS DEL TEMA En els últims anys els agents antivirals directes aprovats pel tractament de l’hepatitis C han permès l’accés al tractament a grups de pacients que anteriorment no es tractaven pel risc que comportava l’ús d’esquemes amb interferó, incloent als pacients d’edat avançada. Es desconeix si l’alta prevalença de comorbiditats i la polimedicació dels pacients d’edat avançada poden estar associades a més esdeveniments adversos i a una disminució en l’eficàcia del tractament, ja que els pacients majors de 65 anys no es veuen representats en els assaigs clínics. És conegut que els pacients d’edat avançada acostumen a tenir un risc superior de reaccions adverses a medicaments, entre elles les que presenten efectes anticolinèrgics. Per aquest motiu diversos anticolinèrgics s’han llistat com a medicaments potencialment inadequats en pacients geriàtrics i actualment existeixen diverses escales per mesurar la càrrega anticolinèrgica que mostren diferències en els medicaments que inclouen, la puntuació i la potència anticolinèrgica que atribueixen a cada medicament. HIPÒTESI DE TREBALL Reconèixer la influència de les comorbiditats i la tolerància als possibles esdeveniments adversos dels nous tractaments amb antivirals d’acció directa pot ajudar en l’optimització del tractament de l’hepatitis C en aquests pacients. Els pacients geriàtrics que inicien tractament per l’hepatitis C poden estar rebent altres medicaments amb efectes anticolinèrgics que poden influir en la seguretat del tractament. Un enfocament multidisciplinar amb la presència d’un farmacèutic clínic a l’equip pot ajudar a millorar l’atenció als pacients geriàtrics que inicien tractament per l’hepatitis C. OBJECTIUS Avaluar l’impacte de les comorbiditats i les interaccions farmacològiques en l’eficàcia i la tolerabilitat dels antivirals d’acció directa pel tractament de l’hepatitis C crònica en la població geriàtrica. Determinar la prevalença de càrrega anticolinèrgica, relacionada amb medicació crònica, en pacients d’edat avançada tractats amb antivirals d’acció directa, i els factors de risc associats, així com analitzar les conseqüències en la seguretat dels tractaments. METODOLOGIA Estudi retrospectiu observacional en un hospital universitari de tercer nivell. Mitjançant el programa informàtic utilitzat per a la prescripció i preparació de medicaments es van identificar tots els pacients que havien estat tractats amb antivirals de acció directa associats o no a ribavirina, al centre, des d’abril de 2015 a març del 2016. Es van recollir variables clíniques i demogràfiques, incloent la presència de comorbiditats. També els factors virològics i clínics relacionats amb la infecció pel virus de l’hepatitis C. Les comorbiditats es van analitzar segons els valors de Clinical Risk Group, que classifiquen als pacients segons la presència de malalties cròniques. Es van recollir variables farmacològiques, com el nombre de medicaments concomitants, el tipus de medicament, i es va classificar als pacients segons el grau d’exposició a polifarmàcia (entesa com la presa de cinc o més medicaments), així com es va considerar el risc d’interaccions farmacològiques. La presència de càrrega anticolinèrgica es va valorar amb tres escales validades diferents: l’Anticholinergic Cognitive Burden scale (ACB), l’Anticholinergic Risk Scale (ARS) i l’Anticholinergic Drug Scale (ADS). L’eficàcia dels tractaments es va avaluar segons la resposta viral sostinguda la setmana 12 després de finalitzar el tractament, classificant els pacients en tres grups d’edat (65 a 74, 75 a 79 i ≥80 anys). L’anàlisi en seguretat va incloure dades especificades a la història clínica i les notificacions d’esdeveniments adversos registrades a la mateixa. Els resultats en seguretat es van analitzar d’acord amb la presència o no de medicaments anticolinèrgics, cirrosi, alta comorbiditat i interaccions farmacològiques. RESULTATS Es van identificar 261 pacients, amb una edat promig de 71 anys, sent el 61% dones. La prevalença de cirrosi era alta (74%) i el genotip 1b el predominant (n=232; 89%). Un elevat nombre de pacients (n=126; 48%) havia fracassat a un tractament anterior amb interferó i 22 pacients (8%) havien rebut un trasplantament de fetge prèviament. El 90% dels pacients prenien medicació concomitant. La resposta viral sostinguda global va ser del 96.9%, sense diferències entre les cohorts d’edat. Tampoc es van observar diferències en eficàcia tenint en compte la presència de cirrosi o haver rebut un tractament previ. El 86% dels pacients va presentar algun esdeveniment advers, majoritàriament fatiga i símptomes gastrointestinals o dèrmics. Tots els pacients que van presentar algun esdeveniment advers greu (6.5%) tenien una alta comorbiditat associada. Entre els pacients que prenien almenys un medicament crònic (90%), es va observar una elevada presència de polifarmàcia, amb un 47% d’aquests prenent cinc o més fàrmacs concomitants. Els fàrmacs més observats com a medicació crònica van ser els diürètics i els psicolèptics. Un elevat nombre de pacients presentava risc d’interaccions farmacològiques tot i que aquestes no es van relacionar amb pitjors resultats terapèutics. Pel que fa a la prevalença de càrrega anticolinèrgica, els resultats van ser diferents segons l’escala de mesura utilitzada essent similars les escales ACB i ADS (35.2% i 34.3%) i lleugerament superiors respecte a l’escala ARS (10.6%). Per a totes les escales es va fer palesa la relació entre comorbiditat i presència de medicació amb càrrega anticolinèrgica, posant de manifest que els pacients amb més comorbiditat presenten més risc de rebre tractaments amb efectes anticolinèrgics. En la present tesi les tres escales valorades han mostrat resultats diferents i només l’escala Anticholinergic Risk Scale (ARS) ha mostrat relació significativa entre càrrega anticolinèrgica i esdeveniments adversos. CONCLUSIONS Els antivirals d’acció directa són efectius i ben tolerats en pacients majors de 65 anys. Els pacients geriàtrics que inicien tractament per l’hepatitis C són pacients complexes i amb múltiples comorbiditats que reben altres medicaments, alguns dels quals amb efectes anticolinèrgics, que poden influir en la seguretat del tractament. L’eficàcia dels tractaments no s’ha vist influenciada per l’edat, ni per la presència o absència de cirrosi, així com tampoc per haver rebut anteriorment tractament antiviral. El nombre d’esdeveniments adversos greus augmenta amb la comorbiditat i el nombre de medicaments concomitants associats. No es poden extreure conclusions pel que fa a quina mesura de càrrega anticolinèrgica representa el millor valor pronòstic. Per tal d’oferir un tractament antiviral òptim, la coordinació entre els hepatòlegs i els farmacèutics clínics recolzats en un equip multidisciplinari és necessària.

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Bonez, Pauline Cordenonsi. « ATIVIDADE DA CLOREXIDINA SOBRE BIOFILMES MICROBIANOS ». Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/5952.

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Biofilms are biological communities with a high degree of organization in which microorganisms are adhered to a surface. Microorganisms, when in biofilm, become a target of concern in the clinical field due to the low response to antimicrobial treatments and ease of colonization of surfaces such as implants, catheters and surgical instruments. Several studies have shown that antimicrobial and biocides have their effectiveness decreased against biofilms. Chlorhexidine is a powerful antiseptic widely used in hospitals especially applied in hand antisepsis, disinfection of environments, and sterilization of surgical instruments used in invasive procedures. Thus, the present study aimed to determine whether bacterial and fungal biofilms are able to resist the antimicrobial action of chlorhexidine. Disk diffusion and susceptibility tests were performed according to Clinical Laboratory Standards Institute (CLSI), 2013. To determine the Biofilm Inhibition Concentration (BIC), chlorhexidine was tested at concentrations of Minimum Inhibitory Concentration (MIC) and at higher concentrations when necessary. The plates were developed with a solution of 0.1% crystal violet and the optical density (OD) was obtained at 570 nm. Results showed that chlorhexidine has excellent antimicrobial activity against most organisms tested in its free form; however, it was less effective against biofilms of Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus resistant to Methicillin (MRSA) and Pseudomonas aeruginosa, developed in isolation for each species. Thus, chlorhexidine is likely to have its antimicrobial activity decreased when exposed to microorganisms in biofilms. This probably occurs due to resistance mechanisms attributed to the biofilm structure exopolysaccharide matrix, quorum sensing (QS), genetic diversity and to the inappropriate use of this biocide.
Biofilmes são comunidades biológicas com elevado grau de organização, onde os microrganismos se encontram aderidos a uma superfície. Os microrganismos, quando em biofilme, tornam-se alvo de preocupação na área clínica devido à baixa resposta aos tratamentos antimicrobianos e à facilidade de colonização de superfícies como próteses, cateteres e instrumentos cirúrgicos. Vários estudos demonstram que os antimicrobianos e biocidas têm sua eficácia diminuída frente aos biofilmes. A clorexidina é um poderoso antisséptico largamente empregado no ambiente hospitalar, aplicado especialmente na antissepsia de mãos, desinfecção de ambientes cirúrgicos e esterilização de instrumentos utilizados em procedimentos invasivos. Deste modo, o presente trabalho teve como objetivo verificar se biofilmes bacterianos e fúngico são capazes de resistir à atuação antimicrobiana da clorexidina. Testes de disco-difusão e de suscetibilidade foram conduzidos de acordo com CLSI (Clinical Laboratory Standards Institute), 2013. Para a determinação da CIB (Concentração de Inibição de Biofilme), a clorexidina foi testada nas concentrações da CIM (Concentração Inibitória Mínima) e em concentrações maiores, quando necessário. As placas foram reveladas com solução de 0.1% de Cristal Violeta e a densidade óptica (D.O.) obtida em 570nm. Os resultados demostraram que a clorexidina possui uma excelente atividade antimicrobiana para a maioria dos microrganismos testados em suas formas livres, porém, mostrou-se menos eficaz contra os biofilmes de Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus resistentes a Meticilina (MRSA) e Pseudomonas aeruginosa, desenvolvidos de forma isolada para cada espécie. Assim, sugere-se que a clorexidina apresenta sua atividade antimicrobiana diminuída quando exposta a microrganismos em biofilme. Provavelmente isso ocorra devido aos mecanismos de resistência atribuídos à estrutura do biofilme - matriz exopolissacarídica, quorum sensing (QS), diversidade genética - e também ao uso inadequado deste biocida.

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Ferraresi, C. « PRUDENT USE OF FLUOROQUINOLONES IN AVIAN SPECIES : PHARMACOKINETICS OF FLUMEQUINE AND ENROFLOXACIN FOR PK/PD MODELLING IN TURKEY ». Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/260640.

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The importance of prudent and rational use of antimicrobial is important, not only to safeguard the efficacy of these drugs in humans and veterinary medicine but, even more so, to prevent the emergence and spread of undesirable resistance phenotypes in zoonotic pathogens as well as in commensal bacteria that can be transmitted between animals and humans. Even more importance and attention is now given to the prudent use of medically important antimicrobial drugs, referring to those drugs for human therapeutic use. The fluoroquinolones belong to this category. These are very potent antimicrobials and active against a wide range of pathogenic organisms and are well distributed in the body after administration. This class of antimicrobials has a therapeutic effect on most infections in different organs or tissues. Although it is rare that fluoroquinolones are the only available agent for treatment of a specific infectious disease, fluoroquinolones are important alternative medicinal products for a veterinarian to have as option for treatment. Fluoroquinolones have a unique mechanism of action not related to conventional antimicrobials, and therefore their efficacy should be retained as long as possible. The avian production increased enormously in the last 50 years and the European Union (EU) is one of the world's top producers in poultry meat and a net exporter of poultry products. In this production, turkeys that is considered a “minor species”, but it is important in the livestock production of Italy. Scarce data exist about the usage of antimicrobial drugs in turkey and even less is known about their efficacy. As the limited number of medicinal products authorized in this species, antimicrobial therapy is frequently carried out with the few products authorized or with drugs “extra-label” used with the consequence of increases of selective pressure and also with the possibility of cross-resistance within the same pharmacological group of compounds. The studies reported in this thesis aimed to revise the use of fluoroquinolones in turkey to maintain the efficacy and reduce the spread of resistance against E.Coli, the most common zoonotic avian pathogen. Pharmacokinetic(PK)/pharmacodynamics (PD) models are the best tool in order to select optimal dosage regimen. To confirm dosages used at farms and allow the integration of PK/PD data, the plasma concentrations in blood from healthy animals collected during treatment with flumequine and enrofloxacin, were determined. The first step was to optimize and validate a fast, simple, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS)/MS/MS method suitable for the detection of a wide range of concentrations of fluoroquinolones as those occurring in pharmacokinetic and residue depletion studies from several matrices. The first trial presents a sensitive and reliable confirmatory method for the extraction, identification, quantification of five fluoroquinolones . For the extraction and matrix clean-up of fluoroquinolones residues from all biological matrices, the Quick Easy Cheap Effective Rugged Safe (QuEChERS) methodology was adopted; only for plasma samples acetonitrile was used. The analyses were performed by (LC-MS. LC separation was performed on a C18 Kinetex column (100x2.1 mm, 2.6 µm, Phenomenex, CA, USA) with gradient elution using ammonium acetate solution (10 mM, pH 2.5) and methanol containing 0.1% formic acid. Mass spectrometric identification was done using an LTQ XL ion trap (Thermo Fisher Scientific, CA, USA), with a heated electrospray ionization probe, in positive ion mode. The method was validated according to the European Legislation (decision 2002/657/EC) and EMA guideline (EMA/CVMP/VICH/463202/2009); selectivity, linearity response, trueness (in terms of recovery), precision (within-day repeatability and within-laboratory reproducibility), limit of detection, limit of quantification, decision limits, detection capability, absolute recovery and robustness were evaluated using turkey blank matrices. All data were within the required limits established for confirmatory methods except for flumequine which presented a recovery value slightly higher than 110% in muscle and intestinal content. For all fluoroquinolones, all the extraction rates were greater than 70% and limits of quantification ranged from 1.2 µg/kg to 118.8 µg/kg. This method was suitable for the identification and quantification of fluoroquinolones in plasma samples of turkeys treated for the purpose of second and third trials. In the second trial, the PK behavior of flumequine administered to 32 healthy turkeys as an oral bolus via gavage or as 5 days of 10-hours pulsed administration in drinking water were compared, using the EU authorized dose of 15 mg/kg and the double dose of 30 mg/kg. The MIC of 235 Escherichia coli field strains isolated from poultry were determined for PD to develop a PK/PD model. Blood samples were collected at established times over 24 h, and the obtained plasma was analyzed using the LC-MS/MS/MS method previously described. A monocompartmental model and a noncompartmental model were applied to the data to obtain the PK results. The maximum concentration (Cmax)/MIC50 and the plasma concentration-time curve from 0 to 24 hours (AUC0–24)/MIC50 ratios were, respectively, 0.67 ± 0.09 and 4.76 ± 0.48 and 1.18 ± 0.35 and 7.05 ± 2.40 for the 15 and 30 mg/kg bolus doses, respectively. After 10-hours pulsed administration of 15 mg/kg, values of Cmax/MIC50, 0.19 ± 0.02 on day 1 and 0.30 ± 0.08 on day 5 of therapy were obtained, the AUC/MIC50 ratios were 2.09 ± 0.29 and 3.22 ± 0.93 on d 1 and 5, respectively. Higher values were obtained with the doubled dose of 30 mg/kg: the Cmax/MIC50 ratios were 0.49 ± 0.11 on day 1 and 0.69 ± 0.18 on day 5; the AUC/MIC50 ratios were 5.15 ± 1.15 and 6.57 ± 1.92 on d 1 and 5, respectively. For both types of administration and both dosages, the Cmax/MIC50 and the AUC/MIC50 ratios achieved were significantly lower than the fluoroquinolones breakpoints usually considered for efficacy. The last trial involving 50 healthy turkeys, was conducted to evaluate the efficacy of enrofloxacin. As in the previous study, the effectiveness of different treatment schemes against E. coli was evaluated by a PK/PD approach, correlating the PK results with the MIC determined for 235 E. coli strains. In this study, 3 different oral treatments (a single oral gavage, 5 days of 10-hours pulsed water medication, and 5 days of 24-hours continuous water medication) and single parenteral (subcutaneous; SC) treatment using 2 different doses of enrofloxacin (i.e., the EU authorized dose, 10 mg/kg, and double the EU recommended dose, 20 mg/kg) were evaluated. Blood samples were collected at established times over 24 h. Plasma was analyzed using a LC-MS/MS/MS that was validated in house. A monocompartmental and a noncompartmental model were applied to the data to obtain the PK results. After gavage administration, the mean maximum concentration Cmax/MIC50 and area under the curve AUC0–24/MIC50 ratios were, respectively, 3.07 ± 0.62 and 7.01 ± 1.03 and 25.48± 3.04 and 57.2 ± 3.73 for the 10 and 20 mg/kg doses, respectively. After SC administration of 10 mg/kg, Cmax/MIC50 and AUC0–24/MIC50 ratios were 3.45 ± 0.75 and 33.96 ± 7.46, respectively. After the administration of 10-h pulsed or 24-h continuous medicated water at 20 mg/kg, lower values of Cmax/MIC50 (10-h pulsed: 3.45 ± 0.7; 24-h continuous: 3.05 ±0.48) and AUC0–24/MIC50 (10-h pulsed: 42.42 ± 6.17; 24-h continuous: 53.32 ± 5.55) were obtained. Based on these results, the European Union-recommended dosage of 10 mg/kg seems ineffective to achieve adequate drug plasma concentrations and even the 20 mg/kg by 10 h pulsed or continuous medicated water administration did not reach completely efficacious concentrations in plasma against colibacillosis. Although the results obtained were not completely encouraging, the medicated water should preferably be provided continuously. To conclude about the efficacy of enrofloxacin treatment against colibacillosis, target tissue concentration should be extensively considered.

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Rodrigues, Mônica de Abreu. « PREVALÊNCIA E PERFIL DE SENSIBILIDADE DE Staphylococcus aureus RESISTENTES À METICILINA (MRSA) NO HOSPITAL UNIVERSITÁRIO DE SANTA MARIA ». Universidade Federal de Santa Maria, 2013. http://repositorio.ufsm.br/handle/1/5987.

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Methicillin-resistant Staphylococcus aureus (MRSA) are reported worldwide as a high prevalence of pathogens in the etiology of infections, both nosocomial and community. This study aimed to determine the prevalence of MRSA in the University Hospital of Santa Maria (HUSM), 2007-2011, as well as evaluating the sensitivity to vancomycin front of MRSA isolates collected from May to December 2011. We analyzed retrospectively the clinical data of all patients diagnosed with MRSA infections between January 2007 and December 2011. During this period, 1,852 samples of S. aureus foram isolated in HUSM, and 616 (33.3%) were resistant to oxacillin. There was a significant reduction in the prevalence rates of this pathogen which rose from 43.4% in 2007 to 33.9% in 2008, 30.4% in 2009, 28.1% in 2010 and 27.5% in 2011. Infections were more prevalent in male patients, aged 41 to 70 years, hospitalized in Medical clinic (16.28%), Adult Intensive Care Unit (15.13%), First Aid Post (13%), Adult Emergency Care (12.67%) and Surgery Clinic (12.5%). A greater isolation of MRSA in blood samples (16.9%), followed by tracheal aspirates (16.5%), urine (10.4%), sputum (8.7%), surgery wound secretion (8.1%) and lower limb secretion (7.8%). As for the determination of susceptibility to vancomycin, 125 samples from S. aureus were collected prospectively from May to December 2011, which 31 (24.8%) were MRSA. The minimum inhibitory concentration (MIC) of vancomycin was determined using a conventional methodology manual broth microdilution. The MIC most frequent among all S. aureus were the 1μg/mL, presented by 53.6% of the strains, whereas among MRSA, there was a higher frequency of MIC of 2 mg/mL (48.4%). Therefore all isolates belonging to this study were sensitive to this antimicrobial of choice for infections caused by MRSA strains. Thus, given the high rates of morbidity and mortality associated with these infections, this study demonstrated the importance of recognizing the prevalence and profile of susceptibility to vancomycin of MRSA so that effective measures for the treatment and control of MRSA to take effect.
Staphylococcus aureus resistentes à meticilina (MRSA) são relatados mundialmente como patógenos de elevada prevalência na etiologia de infecções, tanto nosocomiais como comunitárias. Este trabalho teve por objetivo determinar a prevalência dos MRSA no Hospital Universitário de Santa Maria (HUSM), de 2007 a 2011, bem como avaliar o perfil de sensibilidade frente à vancomicina de isolados de MRSA, coletados de maio a dezembro de 2011. Analisaram-se retrospectivamente, dados clínicos de todos os pacientes diagnosticados com infecções por MRSA entre janeiro de 2007 e dezembro de 2011. Durante este período, 1.852 amostras de S. aureus foram isoladas no HUSM, sendo que 616 (33,3%) foram resistentes à oxacilina. Houve uma redução significativa nas taxas de prevalência deste patógeno que passou de 43,4% em 2007 para 33,9% em 2008, 30,4% em 2009, 28,1% em 2010 e 27,5% em 2011. As infecções foram mais prevalentes em pacientes do sexo masculino, com idades entre 41 e 70 anos, internados na Clínica Médica (16,28%), Unidade de Terapia Intensiva adulto (15,13%), Ambulatório (13%), Pronto Atendimento adulto (12,67%) e Clínica Cirúrgica (12,5%). Houve maior isolamento dos MRSA em amostras de sangue (16,9%), seguido de secreção traqueal (16,5%), urina (10,4%), escarro (8,7%), secreção de ferida operatória (8,1%) e de membro inferior (7,8%). Já para a determinação da sensibilidade à vancomicina, foram coletadas prospectivamente 125 amostras de S. aureus de maio a dezembro de 2011, das quais 31 (24,8%) foram MRSA. A concentração inibitória mínima (CIM) da vancomicina foi determinada através de metodologia convencional manual de microdiluição em caldo. A CIM mais frequente dentre todos os S. aureus foi a de 1μg/mL, apresentada por 53,6% das cepas, enquanto que dentre os MRSA, houve maior frequência da CIM de 2 μg/mL (48,4%). Portanto todos os isolados pertencentes ao presente estudo foram sensíveis a este antimicrobiano de escolha para infecções causadas por cepas MRSA. Desta forma, diante das altas taxas de morbidade e mortalidade associadas a estas infecções, este estudo demonstrou a importância do reconhecimento da prevalência e do perfil de sensibilidade à vancomicina dos MRSA, para que medidas eficazes para o tratamento e controle dos MRSA sejam efetivadas.

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Alves, Izabel Almeida. « AVALIAÇÃO DA SUSCETIBILIDADE DE Candida glabrata RESISTENTES AO FLUCONAZOL FRENTE A COMBINAÇÕES DE ANTIFÚNGICOS ». Universidade Federal de Santa Maria, 2011. http://repositorio.ufsm.br/handle/1/5928.

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In the last decades the invasive mycoses caused by opportunistic fungi has showed a significant growth. The extensive use of azoles showed that the development of resistance of Candida species to these drugs is an emergent phenomenon. C. glabrata typically presents high degree of primary resistance to fluconazole what determine additional difficulties in the therapies. Here two groups of C. glabrata isolates were evaluated: the first formed by strains primarily fluconazole-susceptible and the second generated by the first, was composed by the same isolates after induction of the fluconazole-resistance. The susceptibility tests detected even in the first group, strains azole-resistant as against itraconazole and voriconazole. The induction of resistance to fluconazole was carried out in order to compare the susceptibilities between the two groups against combined drugs. The fluconazole-resistant strains (Group 2) showed MIC ≥ 64 μg/ml and cross-resistance was either detected with fluconazole, voriconazole and itraconazole as well as to other antifungal agents as amphotericin B and caspofungine. The more frequent interaction observed was indifference, however important synergisms were showed against fluconazole-resistant isolates. The best synergism was registered with amphotericin B+ flucytosine (G1 = 61,77% and G2=76,47%). Other synergistic interactions were amphotericin B+ketoconazole (G2=73,53%), amphotericin B+voriconazole (G2=55,88%), amphotericin B+voricoonazole (G2=55,88%), all of them without antagonisms. The highest percentage of the antagonism were observed with ketoconazole+flucytosine (G1=61,77% and G2= 55,88%).
A freqüência de micoses invasivas devido a fungos oportunistas tem evidenciado significativo aumento nas duas últimas décadas. O uso prolongado e indiscriminado dos azólicos evidenciou o desenvolvimento de resistência a estes fármacos nas espécies de Candida. A espécie C. glabrata caracteriza-se por elevados percentuais de resistência primária ao fluconazol fato que determina dificuldades terapêuticas adicionais. No presente estudo, dois grupos de isolados de C. glabrata foram avaliados: o primeiro formado por isolados clínicos sensíveis ao fluconazol, e o segundo, derivado do primeiro, foi formado pelos mesmos isolados, após terem sido induzidos a resistência ao fluconazol in vitro. Mesmo neste grupo de C. glabrata sensíveis ao fluconazol, os testes de suscetibilidade já evidenciaram resistência frente a outros azólicos como ao itraconazol e voriconazol. Os derivados resistentes ao fluconazol evidenciaram concentrações inibitórias mínimas maiores ou iguais a 64 g/mL. Neste grupo de isolados fluconazol-resistentes, os testes de suscetibilidade detectaram resistência cruzada com cetoconazol, voriconazol e itraconazol bem como com outros agentes de outras classes como a anfotericina B e caspofungina. A indução da resistência foi realizada com o objetivo de se comparar a suscetibilidade entre os dois grupos, frente a fármacos combinados. As associações de antifúngicos entre os grupos sensíveis e resistentes ao fluconazol resultaram, em maior parte, interações indiferentes. No entando as associações mostraram-se mais eficazes nos isolados resistentes ao fluconazol. Em ambos os grupos, a combinação que resultou em maior sinergismo foi anfotericina B com flucitosina (.G1 = 61,77%.% e G2 = 76,47%); outras combinações sinérgicas foram anfotericina B + cetoconazol (G2 = 73,53%), anfotericina B + voriconazol (G2 = 55,88%), todos com ausência de antagonismo. Os maiores percentuais de antagonismo foram observados na combinação cetoconazol + flucitosina (G1 = 61,77% e G2 = 55,88%).

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Scheid, Liliane Alves. « COMPORTAMENTO FENOTÍPICO E PERFIL DE SUSCETIBILIDADE DE C. dubliniensis RESISTENTES AO FLUCONAZOL FRENTE A ANTIFÚNGICOS E ASSOCIAÇÕES ». Universidade Federal de Santa Maria, 2009. http://repositorio.ufsm.br/handle/1/5880.

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Widespread and prolonged usage of azoles in recent years has led to the rapid development of drug resistance in Candida species. In Candida albicans, resistance to fluconazole causes cross-resistance to other antifungals and an increase in virulence, making treatment still more difficult because of the limited therapeutical options. Nonetheless, other species has emerged as significant pathogens of clinical importance. Fluconazole resistance has been clinically described in Candida dubliniensis isolates and it is easily induced by in vitro exposure to the drug, but little is known about its consequences. In the present study, two groups of C. dubliniensis isolates were evaluated and compared in some points with the closely related species, C. albicans. One group was composed by fluconazole-susceptible clinical isolates and the other was composed by fluconazole-resistant laboratory derivatives from the former, in order to examine the changes on phenotypic characteristics and antifungal susceptibility accompanying the development of resistance to fluconazole. Resistant derivatives showed minimal inhibitory concentrations equal or higher than 64 μg/mL and proved to keep most of phenotypic characteristics that were tested before resistance was induced. However, some strains were not found to produce pseudomycelia and chlamydospores. Against killer toxins, C. dubliniensis did not present biotypes enough to permit its differentiation from C. albicans. On the other hand, when proteinase activity was evaluated, C. albicans activity was significantly higher than C. dubliniensis . Resistant derivatives of C. dubliniensis showed proteinase activity similar to fluconazole-susceptible isolates, suggesting that fluconazole resistance may not necessarily result on an increase of virulence in this species. Partial atmosphere of CO2, fluconazole at subinhibitory concentrations, or combination of both conditions, as well as addition of antiretrovirals on induction culture medium did not influence on proteinase activity of C. albicans and C. dubliniensis isolates. Finally, fluconazole-resistant isolates showed cross-resistance with ketoconazole, itraconazole, ravuconazole and terbinafine. In addition, associations of amphotericin B or terbinafine with azoles resulted mainly on indifferent interactions. On fluconazole-susceptible isolates, the most positive interaction came from association of amphotericin B with voriconazole. When resistance was induced, the best activity was found when terbinafine was combined with itraconazole.
O uso prolongado e indiscriminado dos azólicos nos últimos anos permitiu um rápido desenvolvimento de resistência aos fármacos nas espécies de Candida. Em Candida albicans, a resistência ao fluconazol causa resistência cruzada a outros antifúngicos e aumento na virulência, tornando o tratamento ainda mais complicado por causa das opções terapêuticas limitadas. Ainda assim, outras espécies emergiram como patógenos significantes de importância clínica. A resistência ao fluconazol tem sido clinicamente descrita em isolados de Candida dubliniensis e é facilmente induzida pela exposição in vitro ao azólico, mas pouco se conhece sobre suas conseqüências. No presente estudo, dois grupos de isolados de C. dubliniensis foram avaliados e comparados em alguns aspectos com C. albicans. Um grupo era composto de isolados clínicos sensíveis ao fluconazol, e o outro, derivado do primeiro, era composto de isolados resistentes, com o intuito de analisar as alterações nas características fenotípicas e na suscetibilidade aos antifúngicos que acompanham o desenvolvimento de resistência ao fluconazol. Os derivados resistentes obtidos evidenciaram concentrações inibitórias mínimas maiores ou iguais a 64 μg/mL e mantiveram grande parte das características fenotípicas avaliadas anteriormente à indução da resistência. Entretanto, apenas o Ágar Suco de Tomate permitiu a identificação dessas estruturas em 100% dos isolados resistentes. Diante das toxinas killer , C. albicans e C. dubliniensis exibiram biotipos incapazes de permitir a diferenciação entre as espécies. Por outro lado, quando avaliada a atividade de proteinase, a de Candida albicans foi significativamente maior do que a de C. dubliniensis. Os derivados resistentes de C. dubliniensis evidenciaram atividade de proteinase semelhante a dos isolados sensíveis ao fluconazol, sugerindo que a resistência ao antifúngico pode não necessariamente acarretar aumento de virulência na espécie. Microaerofilia, fluconazol a concentrações subinibitórias, ou a combinação dessas duas condições, bem como a adição de anti-retrovirais ao meio de indução da enzima não exerceram influência sobre a atividade de proteinase em C. albicans e C. dubliniensis. C. dubliniensis resistentes ao fluconazol evidenciaram resistência cruzada com cetoconazol, itraconazol, ravuconazol e terbinafina. Em adição, as associações de anfotericina B ou terbinafina com azólicos resultaram principalmente em interações indiferentes. Em isolados sensíveis, a interação mais positiva resultou da associação de anfotericina B com voriconazol. Quando a resistência foi induzida, a melhor atividade foi encontrada quando a terbinafina foi combinada com itraconazol.

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Rigatti, Fabiane. « DETECÇÃO DA RESISTÊNCIA À OXACILINA E PERFIL DE SENSIBILIDADE DE Staphylococcus COAGULASE NEGATIVOS ISOLADOS EM UM HOSPITAL ESCOLA ». Universidade Federal de Santa Maria, 2010. http://repositorio.ufsm.br/handle/1/5898.

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For many years coagulase negative Staphylococcus (CoNS), skin commensals, were regarded as mere contaminants. However, in recent decades, they emerged as important agents of nosocomial infections, particularly in immunocompromised patients and patients with biomaterials. This is attributed to increasing antimicrobial resistance by these microorganisms, and oxacillin resistance was the main mechanism presented by CoNS. Thus, this study aimed to characterize this resistance, as well as the susceptibility of 160 isolates of CoNS obtained from patients admitted to HUSM, and also compare phenotypic tests used in laboratory detection of oxacillin resistance with genotypic detection of mecA gene, considered the gold standard. The antimicrobial susceptibility tests were performed by the qualitative technique of disk diffusion (CLSI 2009), as well as by semi-quantitative method (MicroScan ®). The phenotypic detection of resistance was performed by using cefoxitin and oxacillin disk. The genotypic identification of mecA gene was performed by PCR. Together with the molecular detection of mecA gene was performed the detection of gene icaD, responsible for biofilm formation. S. epidermidis (62%) was the main species identified among the samples selected for this study, and the prevalent clinical material was blood (38%). Due to the fact that they are skin commensals, an evaluation of clinical significance of the selected samples was performed. It presented that 48% of the isolates were considered more likely to be the etiologic agents of infections. The adult and neonatal ICUs represent prevalent clinical units in isolation of CoNS, with 33% and 29% of the isolates, respectively. Regarding the antimicrobial susceptibility, a high rate of resistance among the isolates was observed. These were grouped into seven prevalent susceptibility profiles, in which three were characterized by resistance to most of the antimicrobials tested. All strains were susceptible to linezolid, teicoplanin and tigecycline, in total 59 isolates (36.8%). All isolates were susceptible to vancomycin. Through the results of phenotypic tests, 89% and 94% of the isolates were characterized as oxacillin resistant by using cefoxitin and oxacillin disks, respectively. The molecular technique revealed that 79% of isolates carried mecA gene. A susceptibility of 96% to cefoxitin disk and 95% to oxacillin disk could be found from the statistical analysis when compared to the gold standard. Biofilm formation was observed in 45% of samples tested, in which S. epidermidis has been identified as prevalent in positive biofilm samples (74%). The phenotypic methods used in this study are equivalent for detecting oxacillin resistance when compared to the gold standard, and the use of oxacillin disk together with cefoxitin disk should be encouraged, since the oxacillin disk can identify other resistance mechanisms not mediated by mecA. In relation to susceptibility of the isolates, there was a high resistance to first-choice antimicrobial used for the treatment of CoNS.
Por muitos anos os Staphylococcus coagulase negativos (SCoN), comensais da pele, foram considerados como simples contaminantes. No entanto, nas últimas décadas, emergiram como importantes agentes de infecções nosocomiais, principalmente em imunocomprometidos e portadores de biomateriais. Isso é atribuído a crescente resistência antimicrobiana apresentada por estes microrganismos, sendo a resistência à oxacilina o principal mecanismos apresentado pelos SCoN. Desta forma, este estudo objetivou caracterizar esta resistência, bem como o perfil de sensibilidade de 160 isolados de SCoN obtidos de pacientes admitidos no HUSM. Além de comparar testes fenotípicos utilizados na detecção laboratorial da resistência à oxacilina com a detecção genotípica do gene mecA, considerado padrão ouro. Os testes de sensibilidade aos antimicrobianos foram efetuados pela técnica qualitativa de difusão do disco (CLSI 2009), bem como pelo método semi-quantitativo (MicroScan®). A detecção fenotípica da resistência foi realizada com os discos de cefoxitina e oxacilina. A identificação genotípica do gene mecA foi realizada por PCR. Juntamente a detecção molecular do gene mecA foi realizado a detecção do gene icaD, responsável pela formação de biofilme. S. epidermidis (62%) foi à principal espécie identificada entre as amostras selecionadas para este estudo e sangue, o material clínico prevalente (38%). Devido a serem comensais da pele, foi realizada uma avaliação da significância clínica das amostras selecionadas, de onde evidenciamos que 48% dos isolados foram considerados os prováveis agentes etiológicos das infecções. As UTIs adulto e neonatal representam as unidades clínicas prevalentes no isolamento de SCoN, com 33% e 29% dos isolados, respectivamente. Em relação a sensibilidade aos antimicrobianos observou-se um elevado índice de resistência entre os isolados. Estes foram agrupados em 7 perfis de sensibilidade prevalentes, sendo que 3 caracterizaram-se pela resistência a maioria dos antimicrobianos testados. Todas as cepas foram sensíveis à linezolida, teicoplamina e tigeciclina, totalizando 59 (36,8%). Todos os isolados foram sensíveis à vancomicina. Através dos resultados dos testes fenotípicos, verificou-se que 89% e 94% dos isolados foram caracterizados como resistentes à oxacilina, utilizando os discos de cefoxitina e oxacilina, respectivamente. Pela técnica molecular, 79% dos isolados carreavam o gene mecA. A partir da análise estatística evidenciou-se sensibilidade de 96% para o disco de cefoxitina e de 95% para o disco de oxacilina, quando comparados ao padrão ouro. A formação de biofilme foi evidenciada em 45% das amostras testadas, onde o S. epidermidis foi identificado como prevalente nas amostras biofilme positivas (74%). Os métodos fenotípicos utilizados neste estudo mostrarm-se equivalentes na detecção da resistência à oxacilina quando comparados ao padrão ouro, sendo que o uso do disco de oxacilina em conjunto com o de cefoxitina deve ser encorajado, uma vez que, o disco de oxacilina pode identificar outros mecanismos de resistência não mediados pelo gene mecA. Em relação ao perfil de sensibilidade dos isolados houve grande resistência aos antimicrobianos de primeira escolha usados para o tratamento de SCoN. Palavras-chave: SCoN, resistência, oxacilina, cefoxitina, gene mecA.

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Mario, Débora Alves Nunes. « ATIVIDADE DAS EQUINOCANDINAS, ANFOTERICINA B E VORICONAZOL FRENTE A ISOLADOS DE Candida glabrata SENSÍVEIS E RESISTENTES AO FLUCONAZOL ». Universidade Federal de Santa Maria, 2012. http://repositorio.ufsm.br/handle/1/5906.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Widespread and prolonged usage of azoles in recent years has led to the rapid development of drug resistance in Candida species. In Candida albicans, resistance to fluconazole causes cross-resistance to other antifungals and an increase in virulence, making treatment still more difficult because of the limited therapeutical options. Nonetheless, similar phenomenon has been observed for Candida glabrata, specie that has emerged as a pathogen of clinical importance. Fluconazole resistance has been clinically described in Candida glabrata isolates and it is easily induced by in vitro exposure to the drug, but little is known about its consequences. In the present study, two groups of C. glabrata isolates were evaluated. One group was composed by fluconazole-susceptible clinical isolates (FS), and the other was composed by fluconazole-resistant (FR) laboratory derivatives from the former through an in vitro method of fluconazole resistance induction, in order to compare the activitie of the three major antifungal agent classes azoles, echinocandins and poliens. Resistant derivatives showed minimal inhibitory concentrations equal or higher than 64 μg/mL. All yeasts were tested by the broth microdilution method. Based on susceptibility parameters (MIC range, MIC50, MIC90 and geometric mean), the fluconazole-susceptible C. glabrata group (FS) was susceptible to amphotericin B (MIC90 of 0.125 μg/ml). For inhibition, the fluconazole-resistant C. glabrata group (FR) required higher concentrations of amphotericin B (MIC90 of 2 μg/ml). C. glabrata FR group showed cross-resistance with voriconazole (MIC90 of 16 μg/ml). Echinocandins showed the lowest MICs against to both group, flucoanzole-susceptible and resistant. Micafungin demostrated the lowest MIC values among all antifungal agents evaluated (MIC90 of 0.008 μg/ml in FS and 0.015 μg/ml in FR). Our results showed that resistance to fluconazole affected voriconazole susceptibility but not the echinocandin susceptibility, which demonstrated excellent activitie against tested C. glabrata groups.
O uso prolongado e indiscriminado dos azólicos nos últimos anos permitiu um rápido desenvolvimento de resistência aos fármacos nas espécies de Candida. Em Candida albicans, a resistência ao fluconazol causa resistência cruzada a outros antifúngicos e aumento na virulência, tornando o tratamento ainda mais complicado por causa das opções terapêuticas limitadas. Ainda assim, fenômeno semelhante tem sido observado para Candida glabrata, espécie que emergiu como patógenos de importância clínica. A resistência ao fluconazol tem sido clinicamente descrita em isolados de Candida glabrata, e é facilmente induzida pela exposição in vitro ao azólico, mas pouco se conhece sobre suas conseqüências. No presente estudo, dois grupos de isolados de C. glabrata foram avaliados. Um grupo era composto de isolados clínicos sensíveis ao fluconazol (FS), e o outro, derivado do primeiro através de técnica de indução de resistência, era composto de isolados resistentes ao fluconazol (FR), com o intuito de comparar a atividade das três maiores classes de agentes antifúngicos azóis, equinocandinas e poliênicos. Os derivados resistentes obtidos evidenciaram concentrações inibitórias mínimas (CIMs) maiores ou iguais a 64 μg/mL. Foram realizados testes de suscetibilidade aos antifúngicos através da técnica de microdiluição em caldo. Com base nos parâmetros de suscetibilidade (faixa de CIM, CIM50, CIM90 e média geométrica), o grupo FS foi suscetível à anfotericina B (CIM90 de 0,125 μg/ml). Entretanto, o grupo FR requereu doses maiores do antifúngico para ser inibido (CIM90 de 2 μg/ml). O grupo de C. glabrata FR evidenciou resistência cruzada com voriconazol (CIM90 de 16 μg/ml). As equinocandinas mostraram os melhores resultados frente a ambos os grupos, sensíveis e resistentes. Micafungina demonstrou os menores valores de CIM entre todos os agentes antifúngicos estudados (CIM90 de 0.008 μg/ml para FS e 0.015 μg/ml para FR). Os resultados deste estudo mostraram que a resistência ao fluconazol afeta a suscetibilidade do voriconazol, mas não das equinocandinas, as quais mostraram excelente atividade frente aos grupos de C. glabrata testados.

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Nunes, Melise Silveira. « AVALIAÇÃO DA ATIVIDADE CITOTÓXICA E ANTIBACTERIANA DE UM COMPOSTO TRIAZENIDO COMPLEXADO COM ÍON OURO (I) ». Universidade Federal de Santa Maria, 2015. http://repositorio.ufsm.br/handle/1/6029.

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Cancer represents one of the leading causes of death worldwide and efforts to discover more effective anticancer therapies have led to the synthesis of a wide diversity of molecular species. Another major challenge is the anti-infective treatment, because despite the large arsenal of active substances available, many show inefficiency due to the rapid emergence of resistant bacterial strains. As a result, arises the necessity of finding new active substances, more effective and targeted properties in both treatments of neoplasms as microbial resistance. Notably, the Triazenes compounds (TZCs) have been asserting itself as a promising class of metallodrugs with relevant antimicrobial and antiproliferative activity. Moreover, the association of pharmacophoric radical TZC with metal ions, such as gold, leads to a significant increase in biological activity. Because of this wide pharmacological versatility, this study aimed the evaluating in vitro of the biological activity of a compound TZC complexed with ion gold (I). The antibacterial activity was carried out by the conventional method of broth microdilution, through the technique of the minimum inhibitory concentration (MIC), against bacterial strains reference standard American Type Culture Collection (ATCC), clinical isolates with multidrug resistance (MDR) and clinical isolates biofilm producers. Cytotoxicity was evaluated by colorimetric assay based on the reduction of bromide of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) against the standard cell line K562 (Chronic Myeloid Leukemia). The results obtained demonstrate that the compound in study present a narrow spectrum of action, being active only against microorganisms classified as Gram positive, moreover, proved to be active against all isolates producing biofilm when compared to non-producing strains of biofilm. Also showed remarkable cytotoxic activity, with IC50 4.96 μM. Thus, these results demonstrate an alternative to the design of a new class of antibacterial and antitumor metallodrugs with activity.
O câncer representa uma das principais causas de óbito no mundo e esforços para descobrir terapias antineoplásicas mais eficazes têm conduzido à síntese de inúmeras moléculas. Outro grande desafio é o tratamento anti-infeccioso, pois apesar do grande arsenal de substâncias ativas disponíveis, muitas mostram-se ineficazes devido ao rápido aparecimento de estirpes bacterianas resistentes. Em razão disso, surge a necessidade da descoberta de novas substâncias ativas, com propriedades mais eficazes e direcionadas, tanto nos tratamentos de neoplasias como de resistência microbiana. Notoriamente, os compostos Triazenos (TZCs) vêm afirmando-se como uma classe promissora de metalofármacos com relevante atividade antimicrobiana e antiproliferativa. Além disso, a associação do radical farmacofórico TZC com íons metálicos, como o ouro, leva a um aumento significativo na atividade biológica. Em virtude dessa ampla versatilidade farmacológica, este estudo teve como objetivo a avaliação in vitro da atividade biológica de um composto TZC complexados com íon ouro (I). A atividade antibacteriana foi realizada pelo método convencional da microdiluição em caldo, através da técnica da concentração inibitória mínima (CIM), frente às cepas bacterianas padrão de referência American Type Culture Collection (ATCC), isolados clínicos com resistência a múltiplas drogas (RMD) e isolados clínicos produtores de biofilme. A citotoxicidade foi analisada através do ensaio colorimétrico baseado na redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5 difeniltetrazólio) (MTT), frente às células da linhagem padrão K562 (Leucemia Mieloide Crônica). Os resultados obtidos demonstram que o composto em estudo apresentou estreito espectro de ação, sendo ativo somente frente aos microrganismos classificados como Gram positivos, além disso, mostrou-se ativo frente a todos os isolados produtores de biofilme, quando comparado às cepas não produtoras de biofilme. Também demonstrou notável atividade citotóxica, tendo a IC50 4.96 μM. Sendo assim, esses resultados demonstram uma alternativa para a concepção de uma nova classe de metalofármacos com atividade antibacteriana e antitumoral.

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Medeiros, Melissa Soares. « Genotyping and antiretroviral resistance profile test from HIV-1 samples in patients with therapeutic failure from CearÃ ». Universidade Federal do CearÃ, 2006. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=39.

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Faculdade Christus
Introduction: Genotypic testing for HIV-1 drug resistance is useful for selecting antiretroviral drugs for patients developing treatment failure. O melhor entendimento da sua interpretaÃÃo facilitarÃ sua utilizaÃÃo como ferramenta mÃdica na terapÃutica do HIV. The optimal understanding of its interpretation will give an important tool for HIV treatment. Objective: To identify common combinations of resistance mutations and antiretroviral resistance profile. Methods: Between April 2002 and March 2004, 101 protease and reverse transcriptase (RT) sequences were determined for HIV-1 isolates from patients who were failing antiretroviral therapy. Resistance profile was obtained by Stanford program. Results: male were 76.2%, median age 38 years, CD4 media was 279.21 cells/mm3 and Viral load 4.49 log. Total of 31 mutational patterns were detected to protease inhibitor (IP), 49 to nucleoside RT inhibitor (NRTI), and 17 to nonnucleoside RT inhibitor (NNRTI). K65R was detected in 5.9% isolates. The most frequent mutations were L90M, M184V and K103N to IP, NRTI and NNRTI respectively. The main mutational patterns accounted for 49% of mutant sequences to IP, 38.5% to ITRN accounted and 40,9% to NNRTI. Patients with three or more therapeutic failure had worst resistance profile to all IP except for Lopinavir, and NRTI except for Tenofovir. High resistance to Lamivudine and NNRTI were independent of failure quantity. Conclusion: The best susceptibility was found to Lopinavir at IPâs class and to Tenofovir at ITRNâs. The main mutational patterns to IP, ITRN and NNRTI represented almost half of all patterns found.
A Genotipagem estÃ sendo usada como mÃtodo para guiar a seleÃÃo de antiretrovirais em pacientes com falha terapÃutica. O melhor entendimento da sua interpretaÃÃo facilitarÃ sua utilizaÃÃo como ferramenta mÃdica na terapÃutica do HIV. Objetivo: Avaliar o perfil de resistÃncia aos antiretrovirais e identificar padrÃes mutacionais das seqÃÃncias de protease e TR do HIV-1. MÃtodos: Foram estudadas as sequÃncias de genes da protease e TR isoladas de 101 amostras de pacientes com HIV-1 em falha terapÃutica, entre abril/2002 a marÃo/2004, atravÃs de Genotipagem realizadas no CearÃ. O Banco de dados de Stanford foi utilizado para avaliaÃÃo de resistÃncia e SPSS versÃo 11 e Epi Info versÃo 6 para anÃlise estatÃstica. Resultados: Sexo masculino 76,2%, mediana de idade 38 anos, CD4 mÃdio de 279,21 cells/mm3 e Carga Viral 4.49 log. Na classe de Inibidores de Protease (IP) 31 padrÃes mutacionais foram encontrados, nos inibidores da transcriptase reversa anÃlogos de nucleosÃdeos (ITRN) 49 e para inibidores da transcriptase reversa nÃo anÃlogos de nucleosÃdeos (ITRNN) 17. As mutaÃÃes mais frequentes foram L90M, M184V e K103N para IP, ITRN e ITRNN espectivamente. A K65R foi detectada em 5,9% dos isolados. TrÃs ou mais falhas terapÃuticas apresentaram maior perfil de resistÃncia para todos os IPs exceto para Lopinavir, e para todos os ITRNs exceto para Tenofovir. Os seis principais padrÃes mutacionais para IPs equivaleram a 49% das sequÃncias, para ITRNs a 38,5%, e para ITRNNs os dois principais padrÃes corresponderam a 40,9%. Foram encontrados altos Ãndices de resistÃncia para ITRNNs independente da quantidade de falhas terapÃuticas. ConclusÃo: Nos IPs a menor resistÃncia encontrada foi ao Lopinavir e nos ITRNs ao Tenofovir. Os principais padrÃes mutacionais para IPs, ITRNs e ITRNNs representaram quase metade de todos os padrÃes de resistÃncia encontrados.

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Denardi, Laura Bedin. « SUSCETIBILIDADE IN VITRO DE ISOLADOS CLÍNICOS DE Candida glabrata SENSÍVEIS E RESISTENTES AO FLUCONAZOL FRENTE À COMBINAÇÕES ENTRE FÁRMACOS ANTIFÚNGICOS E NÃO ANTIFÚNGICOS ». Universidade Federal de Santa Maria, 2013. http://repositorio.ufsm.br/handle/1/5970.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Candida species are the most common etiologic agent of opportunistic fungal infections. Candida albicans is the most frequent species in candidiasis, however species of non-albicans species have emerged increasingly in hospitals in which the use of azoles as a prophylactic or therapeutic is common. C. glabrata is the species most relevant in this scenario, causing infections with high morbidity and mortality due to high rates of resistance to antifungal azoles, especially fluconazole, and reduced sensitivity to amphotericin B and echinocandins. The remarkable resistance of these species to antifungal agents requires the search for new therapeutic options. An alternative is the combination of drugs with different mechanisms of action. This study aimed to evaluate the in vitro susceptibility of Candida glabrata to conventional antifungal agents (amphotericin B, ketoconazole, itraconazole, fluconazole and voriconazole), and these associations with non-antifungal drugs (chlorpromazine, linezolid, minocycline, sulfamethoxazole, tacrolimus and diphenyl diselenide). Two groups of clinical isolates of C. glabrata were evaluated. The first group consists of fluconazole-sensitive clinical isolates (FS) (n = 30) and the second, derivative of the first, fluconazole-resistant clinical isolates (FR) (n = 30). Based on protocol M27-A3 (CLSI, 2008), the checkerboard method. In isolation, MICs (Minimum Inhibitory Concentration) for fluconazole ranged from 0.25 to ≥ 64.00 μg/mL (FS) and 64.00 to 256.00 μg/mL (FR); for amphotericin B ranged from 0.06 to 0.50 μg/mL (FS) and 0.03 to 0.50 μg/ mL (FR); for itraconazole 0.50 to 8.00 μg/ mL (FS) and 1.00 to 16.00 μg/mL (FR); for ketoconazole 0.13 to 2.00 μg/mL (FS) and 0.50 to 16.00 μg/mL (FR); for voriconazole 0.13 to 4.00 μg/mL (FS) and 1.00 to 16.00 μg/mL (FR). The combinations of tacrolimus with azoles showed significant synergism rates; for the group FS tacrolimus + ketoconazole (43%), tacrolimus + itraconazole (43%), tacrolimus + voriconazole (37%); for the group FR ketoconazole + tacrolimus (77% ) tacrolimus + itraconazole (73%), tacrolimus + voriconazole (63%). Linezolid combined with ketoconazole and voriconazole showed high rates of synergism against the FR group, 76.67% and 70%, respectively. Minocycline + amphotericin B obtained 46.67% (FS) and 36.67% (FR) of synergism; chlorpromazine + amphotericin B was synergistic for 40% of the FR. Chlorpromazine combined to azoles showed high rates of antagonism for resistant group, 76.67%, 70% and 80% for ketoconazole, itraconazole and voriconazole, respectively, for the sensitive group combinations had higher rates of indifference. Sulfamethoxazole evidenced a higher percentage of indifferent interactions associated with all tested antifungals, 70% (FS), 73.34% (FR) associated to ketoconazole, 60% (FS), 43.34% (FR) associated to itraconazole, 56.67% (FS), 66.67% (FR) ins association with voriconazole, 86.66% (FS), 80% (FR) associated to amphotericin B. Synergistic (76.66%) and indifferent (23.34%) interactions were observed for diphenyl diselenide + amphotericin B combination for the sensitive group. Diphenyl diselenide + fluconazole combination demonstrated indifferent (50%) and antagonistic (40%) interactions for sensitive group, whereas synergistic interactions were observed in 10% of the isolates. The most significant associations deserve in vivo evaluation in order to verify their potential in the treatment of infections caused by C. glabrata.
Candida spp é o mais frequente agente etiológico de infecções fúngicas oportunistas. Candida albicans é a espécie mais isolada nas candidíases, no entanto espécies de Candida não-albicans têm emergido de forma crescente nas unidades hospitalares em que o uso de azólicos de forma profilática ou terapêutica é frequente. C. glabrata é uma das espécies mais relevantes neste cenário, causando infecções com alta morbidade e mortalidade, devido às altas taxas de resistência frente aos antifúngicos azólicos, em especial ao fluconazol, e a reduzida sensibilidade a anfotericina B e equinocandinas. A marcante resistência desta espécie aos antifúngicos impõe a busca por novas possibilidades terapêuticas. Uma alternativa é a combinação entre fármacos com distintos mecanismos de ação. Este estudo objetivou avaliar a suscetibilidade in vitro de C. glabrata a antifúngicos convencionais (anfotericina B, fluconazol, cetoconazol, itraconazol e voriconazol) e associações destes, com fármacos e compostos não-antifúngicos (clorpromazina, linezolida, minociclina, sulfametoxazol, tacrolimus e disseleneto de difenila). Foram avaliados dois grupos de isolados clínicos de C. glabrata. O primeiro grupo composto de isolados sensíveis ao fluconazol (FS) (n=30) e o segundo, derivado do primeiro, isolados clínicos resistentes ao fluconazol (FR) (n=30). Com base no protocolo M27-A3 (CLSI, 2008), pelo método de checkerboard. Isoladamente, as CIMs (Concentrações Inibitórias Mínimas) para o fluconazol variaram de 0,25 - ≥64,00 μg/mL (FS) e 64,00 - 256,00 μg/mL (FR); para anfotericina B variaram de 0,06 - 0,50 μg/mL (FS) e 0,03 - 0,50 μg/mL (FR); para o itraconazol 0,50 - 8,00 μg/mL (FS) e 1,00 - 16,00 μg/mL (FR); para cetoconazol 0,13 - 2,00 μg/mL (FS) e 0,50 - 16,00 μg/mL (FR); para voriconazol 0,13 - 4,00 μg/mL (FS) e 1.00 - 16.00 μg/mL (FR). As combinações de tacrolimus com azólicos apresentaram consideráveis taxas de sinergismo; para o grupo FS tacrolimus + cetoconazol (43%), tacrolimus + itraconazol (43%), tacrolimus + voriconazol (37%). Para o grupo FR tacrolimus + cetoconazol (77%), tacrolimus + itraconazol (73%), tacrolimus + voriconazol (63%). Linezolida associada ao cetoconazol e ao voriconazol apresentou altas taxas de sinergismo frente ao grupo FR, 76,67% e 70%, respectivamente. Minociclina + anfotericina B obteve 46,67% (FS) e 36,67% (FR) de sinergismo; clorpromazina + anfotericina B foi sinérgica para 40% do grupo FR. Clorpromazina combinada aos azólicos apresentou altas taxas de antagonismo para o grupo resistente, 76,67%, 70% e 80% para cetoconazol, itraconazol e voriconazol, respectivamente; para o grupo sensível as combinações com clorpromazina tiveram maiores taxas de indiferença. Interações sinérgicas (76,66%) e indiferentes (23,34%) foram observadas na combinação de anfotericina B e disseleneto de difenila para o grupo sensível. A combinação de disseleneto de difenila com fluconazol apresentou indiferença (50%) e antagonismo (40%) para o grupo sensível, 10% dos isolados apresentaram sinergismo. Sulfametoxazol apresentou um maior porcentual de interações indiferentes, associado a todos os antifúngicos testados frente aos dois grupos, 70% (FS), 73,34% (FR) associado ao cetoconazol, 60% (FS), 43,34% (FR) associado ao itraconazol, 56,67% (FS), 66,67% (FR) na associação com voriconazol, 86,66% (FS), 80% (FR) associado à anfotericina B. As associações de maior relevância merecem avaliação in vivo, a fim de verificar o potencial das mesmas no tratamento de infecções por Candida glabrata.

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Bandeira, Laissa Arévalo. « SUSCETIBILIDADE DE ISOLADOS DE Candida dubliniensis SENSÍVEIS E RESISTENTES AO FLUCONAZOL FRENTE A AZÓLICOS E EQUINOCANDINAS ». Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/5996.

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Candida spp. is the most common etiologic agent of opportunistic fungal infections and Candida albicans species still remains the most frequently isolated species in candidiasis. However, the rate of Candida non-albicans isolates has increased in hospitals, where the use of azole antifungals for prophylaxis or therapy is frequent. The prolonged and indiscriminate use of antifungals has favored the development of resistant yeasts. In this context, other species have emerged as clinically important pathogens. Candida dubliniensis species is one that has become important because the easy way that acquires resistance to fluconazole, and may become a relatively more resistant pathogen and therefore more difficult to treat. This framework requires that measures of surveillance the susceptibility or investigations about the magnitude of the resistance are taken. This study aimed to evaluate and interpret the results of in vitro susceptibility of Candida dubliniensis sensitive and resistant to fluconazol and Candida albicans to echinocandins, voriconazole, itraconazole and posaconazole in the light of official documents and also by using proposed points of epidemiological cuts. Where evalued thre groups of isolates: a group of isolates of C. albicans against fluconazole sensitive (Group I), the other two isolates of C. dubliniensis, a compound of isolates sensitive to fluconazole (Group II) and other derived from the first fluconazole resistant (Group III). Groups I and II tests with fluconazole were shown to be susceptible to this antifungal and group III after exposure to increasing concentrations of fluconazole was classified as resistant based on document M27-S3 (2008) and in epidemiological cutoffs. The group I was considered 100% sensitive to antifungal agents based on documents M27-S4 (2012) and M27-S3 (2008). Group II was regarded as 100% sensitive to echinocandins, voriconazole, itraconazole, based on M27-S3 and 100% sensitive to posaconazole based on epidemiological cutoff. However, when interpreting the epidemiological cutoffs MICs, was observed 9.09% for non-sensitive isolates to micafungin. Finally, the group III was 100% sensitive to echinocandins, voriconazole, 100% itraconazole resistant to based on the M27-S3. Based on the epidemiological cutoffs, 4.54% were resistant to anidulafungin, 31.8% resistant to micafungin, 22.7% resistant or susceptible to voriconazole and 100% posaconazole sensitive. Evaluation of antifungal susceptibility is a complex issue and, at the time re-definitions of MICs defining susceptibility or resistance are under intense review, note difficulties in the interpretation of such tests.
Candida spp. é o agente etiológico mais frequentemente causador de infecções fúngicas oportunistas e a espécie Candida albicans ainda permanece como a espécie mais frequentemente isolada nas candidíases. No entanto, a taxa de isolamentos de espécies de Candida não-albicans tem aumentado nas unidades hospitalares, onde o uso de antifúngicos azólicos de forma profilática ou terapêutica é frequente. O uso prolongado e indiscriminado dos antifúngicos tem favorecido o desenvolvimento de leveduras resistentes. Neste contexto, outras espécies emergiram como patógenos de importância clínica. Candida dubliniensis é uma das espécies que tornou-se relevante, por causa da facilidade com que adquire resistência ao fluconazol, podendo tornar-se um agente patogênico relativamente mais resistente e, consequentemente, mais difícil de tratar. Este quadro impõe que medidas de vigilância da suscetibilidade ou de investigações sobre a magnitude da resistência sejam tomadas. Este estudo objetivou avaliar e interpretar os resultados da suscetibilidade in vitro de Candida dubliniensis sensível e resistente ao fluconazol e Candida albicans às equinocandinas, voriconazol, itraconazol e posaconazol à luz dos documentos oficiais e também pelas propostas utilizando pontos de corte epidemiológicos. Foram avaliados três grupos de isolados: um grupo de isolados de C. albicans sensíveis ao fluconazol (Grupo I), os outros dois de isolados de C. dubliniensis, um composto por isolados sensíveis ao fluconazol (Grupo II) e outro derivado do primeiro resistente ao fluconazol (Grupo III). Os grupos I e II nos testes com o fluconazol foram evidenciados como sensíveis a este antifúngico e o grupo III após exposições a concentrações crescentes de fluconazol foi classificado como resistente, com base no documento M27-S3 (2008) e nos cutoffs epidemiológicos. O grupo I foi considerado 100% sensível aos antifúngicos com base nos documentos M27-S4 (2012) e M27-S3 (2008). O grupo II foi considerado 100% sensível às equinocandinas, voriconazol, itraconazol, com base no M27-S3 e 100% sensível ao posaconazol com base no cutoff epidemiológico. No entanto, ao interpretar as CIMs pelos cutoffs epidemiológicos, foi evidenciada 9,09% de isolados não sensíveis a micafungina. E, por fim, o grupo III que foi 100% sensível às equinocandinas, voriconazol, 100% resistente ao itraconazol com base no M27-S3. Com base nos cutoffs epidemiológicos, 4,54% foram considerados resistentes a anidulafungina, 31,8% resistente a micafungina, 22,7% resistente ou não sensíveis ao voriconazol e 100% sensível ao posaconazol. A avaliação da suscetibilidade aos antifúngicos é um tema complexo e, no momento em que redefinições das CIMs definidoras de sensibilidade ou resistência estão sofrendo intensas revisões, nota-se dificuldades nas interpretações de tais testes.

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Marfia, G. « ACQUIRED ALTERATIONS OF SPHINGOSINE-1-PHOSPHATE METABOLISM CONFER STEMNESS AND DRUG RESISTANCE PROPERTIES ON GLIOBLASTOMA MULTIFORME : A NEW POTENTIAL TARGET FOR A COMBINED APPROACH TO TREAT BRAIN CANCER ». Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/333056.

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The eradication of glioblastoma multiforme (GBM) (WHO grade 4) remains a tremendous clinical challenge in human oncology. Indeed, this tumor accounts for the most common, aggressive and lethal primary brain cancer in adults, exhibiting a dismal prognosis, despite extensive surgical resection and adjuvant radio- and chemo-therapy. The finding that GBM contains functional subsets of cells with stem-like properties named glioblastoma stem cells (GSCs) has opened up novel opportunities and promises for the development of new therapies for this devastating cancer. GSCs are self-renewing, multipotent cells, with the capacity to establish and maintain glioma tumors at the clonal level, leading to the hypothesis that they are tumor-initiating cells. Moreover, these rare subpopulations of cells possess an elevated proliferative potential, and intrinsic resistance to therapy, being thus considered a key determinant driving tumor growth, and relapse after resection and therapy. In serum free culture conditions, GSCs form neurospheres. free-floating cell aggregates with a spheroid morphology. These neurospheres preserve many of the important characteristics of the parent tumor, as cell heterogeneity and the ability to drive the parent tumor’s cell invasiveness, when transplanted in murine brain. In addition, GSCs possess the capacities to give rise to a heterogeneous population of cells such as endothelial cells (glioblastoma endothelial cells, GECs) which may directly participate in the vascularization, a critical step in tumors, particularly in malignant GBM, one of the most vascularized/angiogenic tumor described. Moreover, it has been observed that microvasculature structures are the regions responsible for the localization and the maintenance of GSCs. Different molecules, including lipid mediators appear to play a key role in the GBM microenvironment. Among lipid mediators involved in GSC properties, ceramide (Cer) and sphingosine-1-phosphate (S1P) has recently emerged as key signals, able to control growth, invasion, and therapy resistance in various human cancers, including GBMs. Of relevance, the presence of S1P in both glioma cell lines and human gliomas is critical for tumor cell proliferation and survival, a down-regulation of sphingosine kinase 1 (SK1) suppressed growth of human GBM cells and xenografts, and a higher expression of S1P receptor 1 (S1PR1) in GBM has been correlated with poor prognosis. However, little is known on the role of Cer and S1P in GSCs. Recent studies reported that S1P acts as an invasive signal in GSCs, and that the inhibition of SKs, or the administration of a S1PR antagonist, results in GSC death. Very recently it was reported that GSCs derived from U87GBM cells, and those isolated from a human GBM specimen can release S1P extracellularly, and that S1P acts as a first messenger to enhance GSC chemoresistance to Temozolomide. In GSC invasiveness and chemoresistance very little is known about autocrine machinery controlling GSC proliferation and particularly on the significance of S1P in these events. In order to expand previous investigation on S1P and to better understand its role on GSC activity and chemoresistance in this project I focused on the pivotal role of S1P on GSC stemness properties.

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Quatrin, Andréia. « EFEITO DA CASCA DE JABUTICABA (Myrciaria jaboticaba (Vell.) Berg.) SOBRE ESTRESSE OXIDATIVO E RESPOSTA INFLAMATÓRIA EM MODELO DE DIABETES MELLITUS TIPO 2 EM RATOS ». Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/9014.

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Type 2 diabetes mellitus (DM2) is a multifactorial disease mainly characterized by metabolic disorders related to insulin. Hyperglycemia is one of the causes of excessive generation of reactive oxygen species (ROS) leading to oxidative stress. In addition, DM2 is associated with increased inflammatory response. There is an intensive search for novel therapeutic drugs because the synthetic drugs currently used have side-effects and have their effectiveness reduced along the time of use. The jaboticaba (Myrciaria jaboticaba Vell Berg) peel powder (JAB) exhibit high antioxidant capacity and anti-inflammatory action due to the presence of polyphenols. The aim of this study was to evaluate the potential of JAB to prevent biochemical changes, oxidative stress, inflammatory response and pancreatic damage in a DM2 model induced by high-fat diet and a singular injection of streptozotocin (STZ; 35 mg/kg) in Wistar rats. After DM2 induction, instead of drinking water, the animals received vehicle (water containing 0.5% carboxymethyl cellulose) or JAB at 2.7 (JAB-I), 5.4 (JAB-II) or 10.8 (JAB-III) g/L of drinking water (equivalent to 0.07, 0.14 and 0.28 g anthocyanins/L, respectively). After 8 weeks of treatment animals were killed to evaluate the glycemia, insulinemia, oxidative stress and inflammatory markers were evaluated in the serum, besides antioxidant enzymes activities were evaluated in the blood and the histological changes in pancreatic tissue. Increased glycemia and fructosamine levels, insulin resistance and epididymal fat were observerd in the DM2 rats beyond decreased number and area of pancreatic islets. All JAB doses prevented the increase in glycemia over time, whereas JAB-III also reduced the end glycemia, fructosamine levels and the insulin resistance. JAB-I and JAB-II prevented body weight gain over the experiment, whereas JAB-I also decreased the amount of epididymal fat. Furthermore, JAB-I and JAB-III increased the area of pancreatic islets of diabetic rats. In addition, JAB-II and JAB-III treatment reduced the levels of oxidized LDL, whereas JAB-II treatment also reduced protein oxidation in diabetic rats. JAB treatment also prevented the decrease in the activities of glutathione peroxidase, catalase and thioredoxin reductase in the diabetic rats but did not change the decrease in the activity of superoxide dismutase. Only JAB-III treatment prevented the diabetes-induced increase in the inflammatory markers (IL-6, IL-1 and TNF-α). These findings suggest that JAB could have a beneficial effect against diabetes, reducing the hyperglycemia, the insulin resistance, oxidative stress and inflammatory response, besides decreasing pancreatic damage.
O diabetes mellitus tipo 2 (DM2) é uma doença multifatorial caracterizada principalmente por desordens metabólicas relacionadas à insulina. A hiperglicemia é uma das causas da geração excessiva de espécies reativas de oxigênio (EROs) que leva ao estresse oxidativo. Além disso, o DM2 está associado a aumento da resposta inflamatória. Há uma intensa busca por novos medicamentos terapêuticos, pois os medicamentos sintéticos utilizados atualmente apresentam efeitos adversos e tem sua eficácia reduzida ao longo do tempo de uso. O pó de casca de jabuticaba (JAB) (Myrciaria jaboticaba (Vell.) Berg.) apresenta alta capacidade antioxidante e ação anti-inflamatória devido à presença de polifenóis. O objetivo deste trabalho foi avaliar o potencial do JAB em prevenir alterações bioquímicas, estresse oxidativo resposta inflamatória e dano pancreático em modelo de DM2 induzido por dieta hipercalórica e administração única de estreptozotocina (STZ, 35 mg/kg) em ratos Wistar. Após indução do DM2, em vez de água potável, os animais receberam veículo (água acrescida de 0,5% de carboximetilcelulose) ou JAB em 2,7 (JAB-I), 5,4 (JAB-II) e 10,8 (JAB-III) g/L (equivalente a 0,07, 0,14 and 0,28 g antocianinas/L, respectivamente). Após 8 semanas de tratamento os animais foram mortos para avaliar a glicemia, insulinemia, estresse oxidativo e marcadores inflamatórios avaliados no soro, além da atividade das enzimas antioxidantes avaliadas no sangue total e as alterações histológicas no tecido pancreático. O aumento da glicemia e dos níveis de frutosamina, resistência à insulina e gordura epididimal foram observados em ratos com DM2, além da redução do número e área das ilhotas pancreáticas. Todas as doses do JAB preveniram o aumento da glicemia ao longo do tempo, enquanto que JAB-III também reduziu os níveis de glicemia final, frutosamina e a resistência a insulina. JAB-I e JAB-II preveniram o ganho de peso corporal ao longo do experimento, enquanto que JAB-I também reduziu a quantidade de gordura epididimal. Além disso, JAB-I e JAB-III aumentaram a área das ilhotas pancreáticas dos ratos diabéticos. Adicionalmente, os tratamentos JAB-II e JAB-III reduziram os níveis de LDL oxidada, enquanto que o tratamento JAB-II também reduziu a oxidação proteica em ratos diabéticos. O tratamento com JAB também preveniu a redução nas atividades das enzimas glutationa peroxidase, catalase e tiorredoxina redutase nos ratos diabéticos, mas não alterou a redução na atividade da superóxido dismutase. Apenas o tratamento JAB-III preveniu o aumento dos marcadores inflamatórios (IL-6, IL-1 e TNF-α) induzidos no diabetes. Estes resultados sugerem que JAB poderia ter um efeito benéfico contra o diabetes, reduzindo a hiperglicemia, a resistência à insulina, estresse oxidativo e resposta inflamatória, além de diminuir o dano pancreático.

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Pozzatti, Patrícia. « SUSCETIBILIDADE DE Candida spp. RESISTENTES E SENSÍVEIS AO FLUCONAZOL FRENTE A ÓLEOS ESSENCIAIS EXTRAÍDOS DE CONDIMENTOS ». Universidade Federal de Santa Maria, 2007. http://repositorio.ufsm.br/handle/1/5993.

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In the present study, it was assessed antifungal activity of different essential oils obtained from plants traditionally used as condiments, against Candida isolates proven to be susceptible and resistant to the antifungal agent fluconazole. In this context, partial and total minimal concentration values, MICP e MICT, respectively, as well as minimal fungicidal concentration (MFC) of essential oils against different species of Candida were determined. In addition, it was evaluated minimal concentrations of oils that could inhibit germ tube formation by fluconazole susceptible and resistant isolates of C. albicans and C. dubliniensis. Candida species used in this study were: Candida albicans, C. dubliniensis, C. tropicalis, C. glabrata and C. krusei. The essential oils were obtained from: Cinnamomum zeylanicum Breyn (cinnamon), Lippia graveolens HBK (Mexican oregano), Ocimum basilicum L. (basil), Origanum vulgare L. (oregano), Rosmarinus officinalis L. (rosemary), Salvia officinalis L. (sage), Thymus vulgaris L. (thyme) and Zingiber sp. (ginger). The methodology used was broth microdilution, according to M27-A2 document provided by National Committee for Clinical Laboratory Standards (NCCLS, 2002). In order to assess inhibition of germ tube formation by essential oils, it was used the synthetic medium Phase M. Chemical composition of essential oils was obtained through gas chromatography-mass spectroscopy (GC-MS) and through calculation of retention index (RI). Results of antifungal activity tests were analyzed through Mann Whitney statistic test. Subsequently, it was noticed that basil, rosemary and sage essential oils did not show antifungal activity against Candida isolates on tested concentrations. However, cinnamon, Mexican oregano, oregano, thyme and ginger essential oils showed different levels of antifungal activity, being oregano oil the most potent and ginger oil the least efficient. Considering the extended spectrum of antifungal activity presented by these essential oils, it was noticed that similar concentrations could inhibit fungal growth or be fungicidal to isolates originally sensible and resistant to fluconazole. Besides, some oils also demonstrated moderate activity against Candida species which naturally show high fluconazole MICs, such as C. glabrata e C. krusei. All the essential oils inhibited germ tube formation, being oregano oil the most active and rosemary oil the least one. The majoritary constituents in the essential oils were: Z-isoeugenol (93,3%) for cinnamon; carvacrol (56,8%) and o-cymene (32,2%) for Mexican oregano; linalool (32,22%) and 1,8-cineole (23,61%) for basil; carvacrol (92,6%) for oregano; 1,8-cineole (28,59%) and camphor (26,31%) for rosemary; cis-thujone (40,61%) for sage; g-terpinene (64%) for thyme; and zingiberene (20,81%) for ginger essential oil. In conclusion, these results made possible evidencing that Candida spp isolates which are resistant to fluconazole, except for some particularities, were sensible to essential oils that demonstrated antifungal activity.
No presente estudo avaliou-se a atividade antifúngica de diferentes óleos essenciais obtidos de plantas tradicionalmente utilizadas como condimentos, frente a isolados de Candida comprovadamente sensíveis e resistentes ao fluconazol. Para tanto, determinou-se a concentração inibitória mínima parcial e total, CIMP e CIMT, respectivamente, bem como a concentração fungicida mínima (CFM) frente as diferentes espécies de Candida. Também foi avaliada a concentração mínima de óleo capaz de inibir a formação de tubos germinativos (CIMTG) em C. albicans e C. dubliniensis sensíveis e resistentes ao fluconazol. As espécies de Candida utilizadas na pesquisa incluíram: Candida albicans, C. dubliniensis, C. tropicalis, C. glabrata e C. krusei. Os óleos essenciais testados foram obtidos de: Cinnamomum zeylanicum Breyn (canela), Lippia graveolens HBK (orégano mexicano), Ocimum basilicum L. (manjericão), Origanum vulgare L. (orégano), Rosmarinus officinalis L. (alecrim), Salvia officinalis L. (sálvia), Thymus vulgaris L. (tomilho) e Zingiber sp. (gengibre). A metodologia empregada foi a microdiluição em caldo, de acordo com o documento M27-A2 do National Committee for Clinical Laboratory Standards (NCCLS, 2002). Para avaliar a inibição da formação de tubos germinativos pelos óleos essenciais, utilizou-se o meio sintético Fase M. A composição química dos óleos essenciais foi obtida através de cromatografia gasosa acoplada a um espectrômetro de massas (CG-EM) e através do cálculo do índice de retenção (IR). Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico de Mann Whitney. Diante disto, observou-se que os óleos essenciais de manjericão, alecrim e sálvia não evidenciaram atividade antifúngica frente aos isolados de Candida nas concentrações testadas. Entretanto, os óleos essenciais de canela, orégano mexicano, orégano, tomilho e gengibre demonstraram diferentes níveis de atividade antifúngica, sendo o óleo de orégano o mais potente, e o de gengibre, o de menor atividade. Em adição ao amplo espectro de atividade antifúngica dos óleos essenciais citados, observou-se que concentrações semelhantes de óleo essencial foram capazes de inibir o crescimento fúngico ou de serem fungicidas para isolados originalmente sensíveis e resistentes ao fluconazol. Além disso, alguns dos óleos também demonstraram moderada atividade contra espécies de Candida que naturalmente apresentam elevadas CIMs ao fluconazol, tais como C. glabrata e C. krusei. Todos os óleos essenciais inibiram a formação de tubos germinativos, havendo destaque para o de orégano, e menor inibição pelo óleo de alecrim. Os constituintes majoritários presentes nos óleos essenciais foram: Z-isoeugenol (93,3%) para a canela; carvacrol (56,8%) e o-cimeno (32,2%) para orégano mexicano; linalol (32,22%) e 1,8-cineol (23,61%) para o manjericão; carvacrol (92,6%) para o orégano; 1,8-cineol (28,59%) e cânfora (26,31%) para o alecrim; cis-tujona (40,61%) para a sálvia; g-terpineno (64%) para o tomilho; e zingibereno (20,81%) para o óleo essencial de gengibre. Em conclusão, os resultados permitiram constatar que Candida spp resistentes ao fluconazol, respeitadas algumas particularidades, foram sensíveis aos óleos essenciais que evidenciaram atividade antifúngica.

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Faustino, Symonara Karina Medeiros. « PrevalÃncia dos subtipos do vÃrus da imunodeficiÃncia humana do tipo 1 (HIV-1) no estado do PiauÃ - Brasil e o perfil de resistÃncia das cepas identificadas ». Universidade Federal do CearÃ, 2011. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=7482.

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nÃo hÃ
A variabilidade genÃtica do HIV-1 Ã reconhecida como um problema em potencial para o diagnÃstico e tratamento do HIV/AIDS, assim como para a transmissÃo, progressÃo da doenÃa e desenvolvimento de vacinas globalmente efetivas, o que torna importante o monitoramento da distribuiÃÃo global dos diferentes subtipos de HIV-1 e formas recombinantes circulantes (CRFs) no Brasil. O presente trabalho teve como objetivo descrever a prevalÃncia dos subtipos do HIV-1 circulantes no Estado do PiauÃ e o perfil de resistÃncia das cepas identificadas aos ARV, assim como verificar possÃveis associaÃÃes entre os subtipos virais e as informaÃÃes epidemiolÃgicas e laboratoriais da populaÃÃo estudada. As amostras de sangue de 60 pacientes portadores do HIV-1/AIDS foram coletadas no LaboratÃrio Central de SaÃde PÃblica da cidade de Teresina/PI, no perÃodo de maio a abril de 2009. ApÃs a extraÃÃo do DNA proviral, foi realizada a tÃcnica de Nested PCR, para amplificaÃÃo de duas regiÃes genÃmicas (pro e tr), sendo 37 amostras seqÃenciadas posteriormente. Em relaÃÃo Ã anÃlise do segmento do gene pro, 32 (86,5%) foram do subtipo B e 2 (5,4%) do subtipo D, jÃ em relaÃÃo ao segmento do gene da tr, todas amostras pertenceram ao subtipo B (20). Na anÃlise estatÃstica foram encontradas associaÃÃes significantes (p < 0,05) entre os subtipos virais com usuÃrios de drogas endovenosas, transfusÃo sanguÃnea e DST. AlÃm disso, verificou-se uma baixa prevalÃncia de cepas com mutaÃÃes resistentes aos inibidores de protease (IP) (2,9%; 1/34), inibidores de transcriptase reversa nucleosÃdicos (ITRN) (15%; 3/20) e nÃo nucleosÃdicos (ITRNN) (10%; 2/20), sugerindo que hÃ baixa circulaÃÃo de cepas do HIV-1 resistentes aos ARV no estado do PiauÃ.
HIV-1 genetic variability is a wellkown problem that makes diagnosis and treatment difficult to manage. Also, this variability is a problem for trasmission, disease progression and for the development of vaccines, being important to know the subtypes presented in each population worldwilde and in Brazil to provide more effective treatment. The present study have the aim to describe the subtype prevalent in PiauÃ state, the resistance profile of thouse to antiviral treatment, laboratory exams and the epidemiologic point of view of HIV-1 in PiauÃ. Samples of blood from 60 patients (HIV-1 positive) were colect at Central Public Laboratory in Teresina/PI, from maio to april 2009. Proviral DNA were isolated and Nested PCR were performed for two genomic regions (pro e tr), being sequenced 37 samples. Analysis to the segment for gene pro, 32 (86,5%) were subtype B and 2 (5,4%) subtype D. For tr segment all samples were subtype B (20). Statistics analysis found a significant association between vÃrus subtype and drug users, blood transfusion and STD (p < 0,05). Furthermore, the study reveal that even with two subtypes of HIV-1 detected (B e D), a low prevalence of drug resistance was observed to the protease inhibitors (PI) (2,9%; 1/34), nucleosidic reverse transcriptase inhibitor (NRTI) (15%; 3/20) and non nucleosidic (NNRTI) (10%; 2/20).

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Gindri, Lívia. « COMPARAÇÃO DE METODOLOGIAS PARA DETECÇÃO DA RESISTÊNCIA À METICILINA EM Staphylococcus aureus ». Universidade Federal de Santa Maria, 2013. http://repositorio.ufsm.br/handle/1/6005.

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Staphylococcus aureus is considered as a microorganism belonging to the normal flora of humans, being both a settler as an infectious pathogen Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major cause of nosocomial infections and community. The large increase in resistance to antimicrobials has limited therapeutic options. Thus the proper conduct of testing the susceptibility to drugs is vital to ensure the proper treatment of these infections, and the use of methods capable of detecting resistance quickly and accurately, it is essential in clinical diagnosis . The aim of this study was to compare manual and automated phenotypic methods with genotypic based on Polymerase Chain Reaction (PCR) to detect MRSA strains. A total of 139 samples of S. aureus were analyzed using automated and manual methods phenotypic and compared with the determination of the mecA gene by PCR. A total of 139 samples, 37 (26.6 %) had the gene searched. The oxacillin had a sensitivity of 62.2% and specificity of 95.1 %, while cefoxitin disk for these parameters were 67.6% and 94.1 %, respectively. The automated method was the one that had the highest percentages of sensitivity (67,6 %) and specificity (96.1 %). Some differences were observed when comparing the phenotypic methods with PCR. A combination of methods can be considered as an efficient alternative for the diagnosis of infections caused by MRSA.
Staphylococcus aureus é considerado como um microrganismo pertencente à microbiota normal dos seres humanos, podendo ser tanto um colonizador como um patógeno infeccioso. Staphylococcus aureus resistente à meticilina (MRSA) emergiu como uma das principais causas de infecções nosocomiais e comunitárias. O elevado aumento na resistência a diferentes antimicrobianos tem limitado as opções terapêuticas. Assim, a realização adequada de testes de sensibilidade a essas drogas é vital para garantir o adequado tratamento dessas infecções, e o emprego de metodologias capazes de detectar esta resistência de forma rápida e precisa, torna-se imprescindível no diagnóstico clínico. O objetivo desse estudo foi comparar métodos fenotípicos manual e automatizado com o genotípico, baseado na Reação em Cadeia da Polimerase (PCR), para detectar cepas MRSA. Um total de 139 amostras de S. aureus foram analisadas por meio de métodos fenotípicos manuais e automatizado e comparadas com a determinação do gene mecA por PCR. De um total de 139 amostras, 37 (26,6%) apresentaram o gene pesquisado. O disco de oxacilina apresentou sensibilidade de 62,2% e especificidade de 95,1%, enquanto que para o disco de cefoxitina estes parâmetros foram 67,6% e 94,1%, respectivamente. O método automatizado foi o que obteve as maiores percentagens de sensibilidade (67,6%) e especificidade (96,1%). Algumas divergências foram observadas quando comparamos os métodos fenotípicos com a PCR. A combinação de metodologias pode ser considerada como uma alternativa eficiente no diagnóstico de infecções causadas por MRSA.

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Waller, Stefanie Bressan. « Potencial anti-Sporothrix spp. de plantas da família lamiaceae ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/118276.

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As propriedades terapêuticas das plantas medicinais são cada vez mais estudadas, principalmente devido aos crescentes casos de resistência antimicrobiana, como observado em cepas do Complexo Sporothrix. As plantas da família Lamiaceae são conhecidas por suas propriedades antifúngicas, entretanto, são escassos seus estudos contra agentes causadores da esporotricose. Devido ao potencial promissor dessas plantas, objetivou-se (1) avaliar a atividade anti-Sporothrix spp. in vitro de Origanum vulgare L. (orégano), Origanum majorana L. (manjerona) e Rosmarinus officinalis L. (alecrim) nas formas de óleos essenciais, extratos aquosos de infusão e decocção e extratos hidroalcoólicos contra isolados clínicos de Sporothrix spp. obtidos de casos clínicos de esporotricose humana, canina e felina; (2) avaliar os principais constituintes químicos presentes nos óleos essenciais das plantas; e (3) avaliar a atividade citotóxica in vitro. Extratos aquosos e hidroalcoólicos foram preparados a partir de partes aéreas das plantas. Em óleos essenciais, produtos comerciais e produtos extraídos de partes aéreas por hidrodestilação em Clevenger foram testados. Ambos óleos foram analisados quimicamente por cromatografia gasosa. Testes in vitro foram realizados pela técnica de Microdiluição em Caldo contra isolados clínicos de Sporothrix spp. oriundos de humanos, caninos e felinos, bem como ambiental nas fases leveduriforme (CLSIM27A3) e filamentosa (CLSI-M38A2), sendo testados entre 72 a 0.07 mg/mL. Os efeitos citotóxicos foram avaliados através do ensaio MTT em células VERO (78 a 5000 μg/mL). Na fase leveduriforme, os óleos essenciais extraído e comercial de orégano apresentaram atividade anti-Sporothrix spp. nas concentrações inibitórias mínimas (CIM) e concentrações fungicidas mínimas (CFM) de 36 a ≤2.25 mg/mL e de alecrim entre 72 a≤2.25 mg/mL, não havendo diferença estatística entre os tipos de óleos. Por sua vez, óleo comercial de manjerona apresentou CIM/CFM de 18 a ≤2.25 mg/mL. Na fase micelial, 100% dos isolados felinos e caninos foram sensíveis aos óleos essenciais de orégano (0.14 a ≤0.07 mg/mL), alecrim (18 a ≤0,07 mg/mL) e manjerona (4.5 a ≤0.07 mg/mL). Extratos aquosos e hidroalcoólicos de orégano apresentaram atividade inibitória e fungicida, respectivamente, em 100% e entre 90% a 35% dos isolados animais (40 a ≤0.07 mg/mL). Nas mesmas concentrações, o extrato hidroalcoólico de manjerona inibiu de 90% a 100% dos isolados. Entretanto, os demais extratos de manjerona e de alecrim apresentaram fraca atividade anti-Sporothrix spp. sobre 40% a 5% dos isolados, não havendo atividade antifúngica sobre as infusões de alecrim preparadas em 10 e 60 minutos. Sobre itraconazol, Sporothrix spp. foi sensível na fase leveduriforme (16 a ≤0.03 μg/mL) e filamentosa (64 a ≤0.12 μg/mL), entretanto, 5 foi observada resistência antifúngica em 4% e 28% (CIM e CFM >16 μg/mL, respectivamente) dos isolados na fase leveduriforme, ao passo que, na fase filamentosa, a resistência ocorreu em 12,5% e 85% dos isolados (CIM e CFM >64 μg/mL, respectivamente). Os óleos essenciais de orégano e manjerona apresentaram maior atividade citotóxica com 80% de inviabilidade celular, ao passo que os extratos aquosos e hidroalcoólico de manjerona foram os menos citotóxicos. A análise química foi similar nos produtos das plantas Lamiaceae, diferindo a concentração dos compostos, os quais α-pineno e 1,8-cineol foram majoritários para o óleo extraído do alecrim e α- terpineno, terpineol-4 e timol para o óleo extraído de orégano, ao passo que 1,8-cineol foi majoritário para os produtos comerciais de alecrim e manjerona, e carvacrol para orégano. A boa atividade anti-Sporothrix spp. in vitro das plantas da família Lamiaceae, em especial ao Origanum vulgare L., é promissora para o tratamento da esporotricose, inclusive sobre isolados clínicos resistentes.
Therapeutics properties of medicinal plants are increasingly studied, mainly due to increasing cases of antimicrobial cases, as observed in strains of Sporothrix Complex. Plants of Lamiaceae family are known for their antifungal properties, but studies in causative agents of sporotrichosis are scarces. Due to the promising potential of these plants, aimed to (1) evaluate the in vitro anti-Sporothrix spp. activity of aqueous extracts of infusion and decoction, hydroalcoholic extract and essential oils os Origanum vulgare L. (oregano), Origanum majorana L. (marjoram) and Rosmarinus officinalis L. (rosemary); (2) evaluate the main chemical constituents present in essential oils; and (3) evaluate the in vitro cytotoxic activity. Aqueous and hydroalcoholic extracts were prepared from the aerial parts of the plants. In essential oils, commercial products and extracted products from aerial parts through hydrodistillation in Clevenger were tested. Both essential oils were chemically analyzed by gas chromatography. In vitro tests were performed by broth microdilution technique against clinical isolates of Sporothrix spp. from humans, dogs and cats with sporotrichosis, as well as environmental soil, in yeast (CLSI-M27A3) and mycelial (CLSI-M38A2) phases and tested from 72 to 0.07 mg/mL. Cytotoxic effects were assessed by MTT assay on VERO cells (78 to 5000 μg/mL). In yeast phase, the extracted and commercial essential oils of oregano showed anti-Sporothrix spp. activity in the minimum inhibitory concentrations (MIC) and minimum fungicidal concentration (MFC) of 36 to ≤2.25 mg/mL and rosemary between 72 to ≤2.25 mg/mL, with no statistical difference between the types of oils. In turn, commercial marjoram oil showed MIC/MFC of ≤2.25 to 18 mg/mL. In the mycelial phase, 100% of feline and canine isolates were sensibles to essential oils of oregano (0.14 to ≤0.07 mg/mL), rosemary (18 to ≤0,07 mg/mL) and marjoram (4.5 to ≤0.07 mg/ml). Aqueous and hydroalcoholic extracts of oregano showed inhibitory and fungicidal activity, respectively, 100% and between 90% and 35% of animal isolates (40 to ≤0.07 mg/mL). In the same concentrations, hydroalcoholic extract of marjoram inhibited 90% to 100% of the isolates. However, other extracts of marjoram and rosemary showed weak activity anti- Sporothrix spp. about 40% to 5% of the isolates, with no antifungal activity by INF10 and INF60 rosemary. About itraconazole, Sporothrix spp. was sensitive in the yeast phase (16 to ≤0.03 mg/mL) and filamentous (64 to ≤0.12 mg/mL), however, antifungal resistance was observed in 4% and 28% (MIC and MFC > 16 μg/mL, respectively) in fungal isolates in the yeast form, and in the mycelial form, the resistence occurred in 12.5% and 85% of isolates (MIC and MFC > 64 μg/mL, respectively). Essential oils of oregano and marjoram exhibited greater cytotoxic activity with 80% cell inviability, while hydroalcoholic and aqueous extracts of marjoram were less cytotoxic. The chemical analysis was similar for the products of Lamiaceae plants and differed in the concentration of the compounds, which α-pinene and 1,8-cineole were majoritary for extracted oil of rosemary and α-terpinene, terpineol-4 and thymol for extracted oil of 7 oregano, while 1,8-cineole was majoritary for commercial products of rosemaru and marjoram, and carvacrol for oregano. The good in vitro anti-Sporothrix spp. activity of the plants of Lamiaceae family, mainly to Origanum vulgare L., is promising for the treatment of sporotrichosis, including against clinical isolates resistant.

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ANGIONI, MARIA MADDALENA. « Response to treatment with chemical and biological inhibitors of c-met mutated form ». Doctoral thesis, Università degli Studi di Cagliari, 2012. http://hdl.handle.net/11584/266057.

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c-MET is a receptor tyrosine kinase that, after binding with its ligand, hepatocyte growth factor (HGF), activates many signaling pathways, driving proliferation, motility, migration and invasion. Although c-MET is important in the control of tissue homeostasis under normal physiological conditions, it has also been found to be aberrantly activated in human cancers via mutation, amplification or protein overexpression. Activating point mutations were identified in the kinase domain of MET, either in the germline of patients affected by hereditary papillary renal carcinoma (HPRC) or in spontaneously occurring tumors; in particular, nine missense mutations (defined METPRC mutations), leading to constitutive activation of MET protein, have been identified in HPRC families. Given the importance of MET as a target for cancer therapies, clinical trials aimed at inhibiting it through the use of tyrosine kinase inhibitors (TKIs) have recently been started. The aim of project was: (i) to evaluate if METPRC mutants are sensitive to PHA-665752 (a small kinase inhibitor of MET), (ii) if some mutants are insensitive to the inhibitor, to investigate the mechanisms responsible for resistance, (iii) to check if the resistant mutants are still sensitive to other chemicals inhibitors or monoclonal antibodies against MET, (iv) to identify activating point mutations in human surgically resected lung cancers. We have found that some METPRC mutants cannot be inhibited by PHA-665752. Treatment with this TKI does not alter either receptor phosphorylation or MET mutants-induced biological activities (migration, invasion, anchorage-independent growth). We showed that these mutants are insensitive also to JNJ-38877605, a multitargeted tyrosine kinase inhibitor.. When we performed the mutational analysis on lung cancer samples, in one tumor we found the presence of one of the identified“resistant” mutations. To determine whether the mutants resistant to PHA-665752 could be inhibited with other strategies, we treated the mutant-expressing cells with the monoclonal antibody DN30, directed against the extracellular portion of the receptor. Our results showed that DN30 was indeed able to inhibit all the METPRC mutants. In conclusion, we have identified some METPRC mutants which do not respond to the ATP competitive kinase inhibitors. Since the identified METPRC mutations are located in the kinase domain and alter its conformation; it is likely that the competitive inhibitors are unable to interact with the ATP binding site in the context of the mutated receptors; this would render these mutants "resistant" to the action of tyrosine kinase inhibitors. However, these mutated forms still remain responsive to treatment antibodies directed against the MET extracellular portion: This observation is important since the use of monoclonal antibodies represent a therapeutic alternative for patients with tumors carrying MET mutants resistant to TKIs.

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GutiÃrrez-adriÃnzen, Oswaldo Augusto. « AlteraÃÃes fisiolÃgicas, cardiovasculares, neuroendÃcrinas e suas correlaÃÃes com as variaÃÃes da pressÃo arterial, durante a hemodiÃlise, em pacientes com insuficiÃncia renal crÃnica ». Universidade Federal do CearÃ, 2010. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=5195.

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nÃo hÃ
IntroduÃÃo. Os mecanismos fisiopatolÃgicos da hipertensÃo arterial (HA) durante a hemodiÃlise (HD) em pacientes com insuficiÃncia renal crÃnica terminal (IRCT) ainda sÃo pouco compreendidos. Em sua patogenia, destacam-se: hipervolemia, aumento da atividade do sistema renina-angiotensina-aldosterona, aumento da atividade do sistema nervoso simpÃtico (SNS), disfunÃÃo endotelial. Objetivo. Investigar as alteraÃÃes fisiolÃgicas, cardiovasculares e neuroendÃcrinas em pacientes portadores de IRCT e suas correlaÃÃes com as mudanÃas da PA durante sessÃo de HD. MÃtodos e CasuÃstica. Estudo observacional, em pacientes com IRCT em tratamento de hemodiÃlise crÃnica (HC), no perÃodo de marÃo a julho de 2008. Foram incluÃdos 21 pacientes, selecionados atravÃs de monitorizaÃÃo da pressÃo arterial (MAPA), durante sessÃo de HD: os pacientes que apresentaram aumento da PA foram incluÃdos no grupo A (estudo) e os pacientes que apresentaram reduÃÃo da PA durante sessÃo de HD, no grupo B (controle). Cinco ecocardiogramas foram realizados durante cada sessÃo individual de HD, a intervalos de hora em hora, para avaliar dÃbito cardÃaco (DC) e resistÃncia vascular sistÃmica (RVS). A atividade do SNS foi avaliada atravÃs de anÃlise espectral da variabilidade da freqÃÃncia cardÃaca (VFC). Antes e depois da sessÃo de hemodiÃlise foram dosados o peptÃdeo natriurÃtico cerebral (BNP), catecolaminas, endotelina-1 (ET-1), Ãxido nÃtrico (ON), eletrÃlitos, hematÃcrito (Ht), albumina (Alb) e substÃncias nitrogenadas. Os dados foram armazenados atravÃs do programa SPSS. Resultados. A mÃdia de idade dos pacientes foi de 43Â4,9 anos, sendo 45,4% do sexo masculino. A RSV elevou-se, de maneira significativa, nos pacientes do grupo A, quando comparada com a dos pacientes do grupo B (p <0,001). A variabilidade da freqÃÃncia cardÃaca (VFC) no domÃnio da freqÃÃncia, atravÃs da razÃo baixa freqÃÃncia/alta freqÃÃncia (BF/AF), revelou que, a atividade do SNS, nos pacientes do grupo A, nÃo foi maior que os do grupo B. (Grupo: p=0,445);Tempo:p=0,348); Grupo.Tempo: p=0,486. O valor mÃdio de ET-1, prÃ HD, foi de 10,8Â0,74 pg/mL nos pacientes do grupo A e 11,1Â0,46 pg/mL nos do grupo B (p=0,788). O valor mÃdio de ET-1 pÃs HD, foi de 25,9Â 0,89 pg/mL nos pacientes do grupo A e 13,3Â0,54 pg/mL, nos do grupo B. (p=<0,001). O valor mÃdio do ON (nitratos e nitritos), prÃ HD, foi de 84,63Â5,42 Â5,17M nos pacientes do grupo A e 86,3Â6,01 ÂM nos do grupo B (p=0,621). O valor mÃdio de ON (nitratos e nitritos), pÃs HD, foi de 90,3Â8,82 ÂM no grupo A e 79,9Â4,39 ÂM no grupo B (p=0,692). O valor mÃdio do BNP, prÃ HD, foi de 1643Â851 pg/mL nos pacientes do grupo A e 1720Â583 pg/mL, nos do grupo B (p=0,231). O valor mÃdio do BNP, pÃs HD, foi de 1574Â815 pg/mL nos do grupo A e 1382Â495 pg/mL nos do grupo B (p=0,573). O valor mÃdio de adrenalina, prÃ HD, foi de 208,4Â63,86 pg/mL nos pacientes do grupo A e 153Â69, 28 pg/ml nos do grupo B (p=0,622). O valor mÃdio de adrenalina, pÃs HD, foi de 80,4Â11,60 pg/mL no grupo A, e de 212,1Â94,91 pg/mL no grupo B. (p=0,084). O valor mÃdio de noradrenalina, prÃ HD, foi de 337Â76,79 pg/mL nos pacientes do grupo A e de 487,8Â153,26 pg/mL nos do grupo B (p=0,672). O valor mÃdio de noradrenalina, pÃs HD, foi de 194Â32,68 pg/mL nos pacientes do grupo A e 318,5Â84,35 pg/mL nos do grupo B (p=0,481). O valor mÃdio de dopamina, prÃ HD, foi de 75,5Â5,17 pg/mL nos pacientes do grupo A, e de 71,2Â6,67 pg/mL nos do grupo B (p=0,549). O valor mÃdio de dopamina, pÃs HD, foi de 74,1Â8,32 pg/mL no grupo A e 79,5Â9,41 pg/mL no grupo B (p=0,672). ConclusÃo. Os pacientes com IRCT apresentaram padrÃes hemodinÃmicos diferentes durante a sessÃo de HD, com aumento significativo da pressÃo arterial nos pacientes do grupo A, por elevaÃÃo da RVS decorrente de disfunÃÃo endotelial, evidenciada por aumento significativo dos nÃveis plasmÃticos da ET-1.
Introduction. The pathophysiologic mechanisms of arterial hypertension (AH) during hemodialysis (HD) in patients with end-stage renal disease (ESRD) are still poor understood. In its pathogeny, multifactorial, we can emphasize: hypervolemia, increase in the activity of renin-angiotensin-aldosterone system, increase in the activity of sympatic nervous system (SNS), endothelial dysfunction. Objective. To investigate physiologic, cardiovascular and neuroendocrine abnormalities in patients with ESRD and its correlations with changes in blood pressure (BP) during HD session. Methods and Cases. Observational study, in patients with ESRD in chronic hemodialysis treatment (CH), in the period from March to July 2008. It were excluded 21 patients, selected through blood pressure monitoring, during HD session: patients who presented increase in BP were included in group A (study) and patients who presented decrease in BP during HD session in group B (control). Five echocardiograms were performed during each individual HD session, with 1 hour intervals, to evaluate cardiac output (CO) and systemic vascular resistance (SVR). The activity of SNS was evaluated through spectral analysis of cardiac frequency variability (CFV). Before and after HD session brain natriuretic peptide (BNP), catecholamines, endothelin-1 (ET-1), nitric oxide (NO), electrolytes, hematocrit (Ht), albumin (Alb) and nitrogenous substances were dosed. Data were stored through the program SPSS. Results. The mean age of the patients was 43Â4.9 years, and 45.4% were male. SVR significantly increased in patients from group A, when compared with patients from group B (p <0.001). The variability of cardiac frequency (VCF) in frequency domain, through low frequency/high frequency ratio (LF/HF), revealed that SNS activity, in patients from group A, was not higher than in patients from group B. (Group:p=0.445);Time:p=0.348); Group.Time:p=0.486. The mean value of ET-1, pre HD, was 10.8Â0.74 pg/mL in patients from group A and 11.1Â0.46 pg/mL in group B (p=0.788). The mean value of ET-1 post HD, was 25.9Â 0.89 pg/mL in patients from group A and 13.3Â0.54 pg/mL, in group B. (p=<0.001). The mean value of NO (nitrate+nitrite), pre HD, was 84.63Â5.42 ÂM in patients from group A and 86.3Â6.01 ÂM in group B (p=0.621). The mean value of NO (nitrate+nitrite) post HD, was 90.3Â8.82 ÂM in group A and 79.9Â4.39 ÂM in group B (p=0.692). The mean value of BNP, pre HD, was 1643Â851 pg/mL in patients from group A and 1720Â583 pg/mL, in group B (p=0.231). The mean value of BNP, post HD, was 1574Â815 pg/mL in group A and 1382Â495 pg/mL in group B (p=0.573). The mean value of adrenalin, pre HD was 208.4Â63.86 pg/mL in patients from goup A and 153Â69.28 pg/ml in group B (p=0.622). The mean value of adrenalin, post HD, was 80.4Â11.60 pg/mL in group A and 212.1Â94,91 pg/mL in group B (p=0.084). The mean value of noradrenalin, pre HD, was 337Â76.79 pg/mL in patients from group A and 487.8Â153.26 pg/mL in group B (p=0.672). The mean value of noradrenalin, post HD, was 194Â32.68 pg/mL in patients from group A and 318.5Â84.35 pg/mL in group B (p=0.481). The mean value of dopamin, pre HD, was 75.5Â5.17 pg/mL in patients from group A, and 71.2Â6.67 pg/mL in group B (p=0.549). The mean value of dopamin, post HD, was 74.1Â8.32 pg/mL in group A and 79.5Â9.41 pg/mL in group B (p=0.672). Conclusion. Patients with ESRD presented different hemodynamic patterns during HD session, with significantly increase in blood pressure in patients from group A, due to elevation in SVR secondary to endothelial dysfunction, evidenced by a significantly increase in serum levels of ET-1.

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Oliveira, Isis Gabrielli Barbieri de. « Efeitos cardiovasculares e metabólicos em animais submetidos à dessincronização circadiana ». Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/7609.

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The presence of circadian rhythms in organisms is a biological event, indispensable to the process of adaptation to the environment and fundamental for the survival of the species. The loss of the phase relationship between the biological rhythms and the behavior leads impairment of the body function. Thus, the aim of this study was to evaluate the effects of the forced locomotor activity desynchronization on the cardiovascular and metabolic parameters. Male Wistar rats, aged approximately 60 days, were distributed into two experimental groups: control group (CTR) submitted to the light/dark cycle (LD) of 24h-T24 (n=12) and desynchronized group (DSC) submitted to the LD cycle of 22h -T22 (n = 18) for eight weeks. Records of the locomotor activity were made daily to both groups. Food ingestion, water intake and body weight were monitored weekly. Following of this period the animals were allocated into two experimental protocols. In the first protocol the CTR and DSC animals were submitted to records of mean arterial pressure (MAP) and heart rate (HR), as well as pharmacological tests of baroreceptor reflex function and cardiac autonomic control, such as intrinsic pacemaker heart rate (IPHR); vagal and sympathetic effects and sympathetic-vagal index (SVI). In the second protocol, CTR and DSC animals were fasted for at least 12 hours, anesthetized and euthanized by decapitation to collect blood, liver and adrenal glands for posterior analysis of plasma glucose, triglycerides, total cholesterol, VLDL, HDL, LDL; the expression of proteins involved in the insulin signaling cascade (IR-β; IRS-2; PI3K; AKT), the gluconeogenic enzyme - PEPCK, SOD-1 and CAT; and morphometry. Although there was no significant difference in feed and water intake between groups, a higher weight gain of DSC animals was observed from week 7 onwards. In relation to the pattern of locomotor activity, DSC animals presented lower levels on total locomotor activity with a predominance of its activity during the light phase of the LD cycle. Although we did not observe significant alterations in the basal MAP and HR levels between the groups, the DSC animals showed reduction of the baroreflex sensitivity by the bradycardic index, as well as a higher cardiac sympathetic effect. No differences were observed between the groups for the other parameters of cardiac autonomic control. Regarding the biochemical parameters, we observed in the DSC animals, a reduction in HDL cholesterol associated with an increase in LDL cholesterol, which promoted a high index of Castelli I and II, compared to the CTR. There were no differences in plasma levels of glucose, triglycerides, total cholesterol and VLDL. DSC animals also showed an increase in weight and in the total area of the adrenal glands. In addition, we observed decreased IRβ, IRS-2, PI3-K and Akt proteins, as well as increased expression of the gluconeogenic enzyme PEPCK in DSC animals, suggesting hepatic insulin resistance. Finally, we observed a reduction in the expression of the SOD-1 and CAT enzymes in the DSC animals in relation to the CTR animals. Summarizing, our results suggest that a 22h symmetrical photoperiod (T-22) promotes lower baroreflex sensitivityand increased sympathetic effect on the heart. In addition, metabolic disorders such as hepatic insulin resistance and increased hepatic oxidative stress were observed in the desynchronized rats.
A presença de ritmos circadianos nos organismos é um evento biológico, indispensável ao processo de adaptação ao meio e fundamental para a sobrevivência das espécies. A perda de relação de fase entre os ritmos biológicos e o comportamento promovem prejuízo às funções orgânicas. Diante disso, o objetivo deste estudo foi avaliar os efeitos de uma dessincronização forçada da atividade locomotora nos parâmetros cardiovasculares e metabólicos. Ratos Wistar machos, com idade aproximada de 60 dias, foram distribuídos em dois grupos experimentais: grupo controle (CTR) submetido ao ciclo claro/escuro (CE) de 24h - T24 (n=12) e grupo dessincronizado (DSC) submetido ao ciclo claro/escuro (CE) de 22h - T22 (n=18) por oito semanas. Registros da atividade locomotora foram feitos diariamente para ambos os grupos. O consumo alimentar, ingestão de água e o peso corporal foram monitorados semanalmente. Ao final deste período os animais foram alocados em dois protocolos experimentais. No primeiro protocolo os animais CTR e DSC foram submetidos a registros da pressão arterial média (PAM) e frequência cardíaca (FC), bem como testes farmacológicos da função do reflexo barorreceptor e controle autonômico cardíaco, tais como frequência cardíaca intrínseca de marcapasso (FCIM); efeitos vagal e simpático e índice simpático-vagal (ISV). No segundo protocolo os animais CTR e DSC foram deixados em jejum de no mínimo 12 horas, anestesiados e eutanasiados por decapitação para coleta de sangue, fígado e glândulas adrenais para posterior análise dos níveis plasmáticos de glicose, triglicerídios, colesterol total, VLDL, HDL, LDL; da expressão de proteínas envolvidas na cascata de sinalização da insulina (IR-β; IRS-2; PI3K; AKT), enzima gliconeogênica – PEPCK, SOD-1 e CAT; e da morfometria. Embora não houve diferença significativa no consumo de ração e água entre os grupos, observou-se maior ganho de peso dos animais DSC a partir da 7ª semana. Em relação ao padrão da atividade locomotora os animais DSC apresentaram menor atividade locomotora total e um predomínio da mesma durante a fase clara do ciclo CE. Apesar de não observamos alterações significativas nos níveis de PAM e FC basais entre os grupos, os animas DSC apresentaram redução da sensibilidade barorreflexa pelo índice bradicárdico, assim como maior efeito simpático cardíaco. Não foram observadas diferenças entre os grupos para os demais parâmetros de controle autonômico cardíaco. Quanto aos parâmetros bioquímicos, observamos, nos animais DSC, redução do colesterol HDL associado a uma elevação do colesterol LDL, o que promoveu elevados índice de Castelli I e II comparados aos CTR. Não houve diferenças nos níveis plasmáticos de glicose, triglicerídios, colesterol total e VLDL. Os animais DSC também apresentaram aumento no peso e na área total das glândulas adrenais. Além disso, observamos diminuição das proteínas IRβ, IRS-2, PI3-K e Akt, bem como aumento da expressão da enzima gliconeogênica PEPCK nos animais DSC, sugerindo resistência hepática à insulina. Por fim observamos redução na expressão das enzimas SOD-1 e CAT nos animais DSC em relação aos animais CTR. Sumarizando, nossos resultados sugerem que um fotoperíodo simétrico de 22h (T-22) promove menor sensibilidade barorreflexa, maior efeito simpático sobre o coração. Além disso, foram observados distúrbios metabólicos, como resistência à insulina hepática e aumento do estresse oxidativo hepático, nos ratos dessincronizados.

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Jesus, Francielli Pantella Kunz de. « SUSCEPTIBILIDADE DE ISOLADOS DE Malassezia pachydermatis SENSÍVEIS E RESISTENTES AO FLUCONAZOL FRENTE A ANTIFÚNGICOS E ÓLEOS ESSENCIAIS ». Universidade Federal de Santa Maria, 2011. http://repositorio.ufsm.br/handle/1/8963.

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Malassezia pachydermatis is an opportunistic fungus associated to dermatomycoses and otopathies in domestic and wild animals. The aim of this study was to compare the susceptibility profile of clinical isolates of M. pachydermatis against topic and systemic antifungals, through two standardized CLSI techniques: broth microdilution (M27-A3, 2008) and disk diffusion (M44-A, 2004). A standard Candida albicans strain (ATCC 28367) was used as quality control. M. pachydermatis isolates assayed through the broth microdilution method showed susceptibility to anfotericina-B (100%), fluconazole (97,83%), ketoconazole (95.66%), itraconazole (93,48%), followed by clotrimazole and miconazole (86.96%). The disk diffusion method showed susceptibility of 97,83% to nystatin, 95,66% to itraconazole and to amphotericin B, 91,32% to ketoconazole, 89,14% to fluconazole and of 86.96% to miconazole and clotrimazole. Through the in vitro induction of resistance to fluconazole (100%), cross-resistance was observed through the broth microdilution method among the 30 M. pachydermatis isolates against the azoles itraconazole (93%), ketoconazole (97%) and voriconazole (100%). The study of the activities of the essential oils obtained through the broth microdilution, based on geometric means of the minimum fungicidal concentration, showed that concentrations above 195.42 μg/mL, 332.49 μg/mL and 448.8 μg/mL of oregano, Mexican oregano and cinnamon essential oils, respectively, have fungicidal action against M. pachydermatis, independently of the sensitivity or resistance to fluconazole. The fungicidal activity of the essential oils against fluconazole-resistant M. pachydermatis strains is important for further therapeutic studies of this mycosis. Despite the susceptibility observed among sensitive M. pachydermatis strains suggesting fluconazole as a safe drug for the treatment of most cases of superficial and invasive malasseziosis, it is essential continuous surveillance studies to detect changes in the microbiological profile due to therapeutic practices.
Malassezia pachydermatis é um fungo oportunista, associado à dermatomicoses e otopatias em animais domésticos e selvagens. O objetivo deste estudo foi comparar o perfil de susceptibilidade de isolados clínicos de M. pachydermatis frente antifúngicos tópicos e sistêmicos, através de duas técnicas padronizadas pelo CLSI: microdiluição em caldo (M27- A3, 2008) e disco-difusão (M44-A, 2004). Para todos os testes foi utilizada uma cepa padrão de Candida albicans (ATCC 28367) como controle de qualidade. Isolados de M. pachydermatis testados através do método de microdiluição em caldo apresentaram em ordem decrescente de susceptibilidade: anfotericina-B (100%), fluconazol (97,83%), cetoconazol (95,66%), itraconazol (93.48%), seguido de clotrimazol e miconazol (86,96%). Pelo método de disco-difusão 97,83% dos isolados foram suscetíveis a nistatina, 95,66% ao itraconazol e a anfotericina-B; 91,32% ao cetoconazol, 89,14% ao fluconazol; e 86,96% ao miconazol e clotrimazol. Através da indução de resistência in vitro, de 30 isolados de M. pachydermatis, ao fluconazol (100%) foi possível evidenciar resistência cruzada pelo método de microdiluição em caldo frente aos antifúngicos azólicos, itraconazol (93%), cetoconazol (97%) e voriconazol (100%). O estudo da atividade de óleos essenciais gerados pelo método de microdiluição em caldo, baseado nas médias geométricas das CFMs, evidenciou-se que concentrações acima de 195.42 μg/mL de óleo essencial de orégano, 332.49 μg/mL de óleo essencial de orégano mexicano e 448.8 μg/mL de óleo essencial de canela, possuem ação fungicida in vitro sobre M. pachydermatis, independente da sensibilidade ou resistência ao fluconazol. A atividade de óleos essenciais frente M. pachydermatis fluconazol resistente, é um achado que consolida a importância destes para estudos futuros voltados a terapêutica desta micose. Apesar dos dados de susceptibilidade apresentados pelas cepas sensíveis de M. pachydermatis sugerirem que o fluconazol ainda é uma droga segura para terapêutica da maioria dos casos de malasseziose superficial ou invasiva, é fundamental que estudos de vigilância epidemiológica sejam realizados continuamente para evidenciar mudanças no perfil microbiológico em função de práticas terapêuticas.

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FaÃanha, MÃnica Cardoso. « Barreira funcional intestinal, absorÃÃo e biodisponibilidade de Rifampicina, Isoniazida e Pirazinamida em pacientes com tuberculose pulmonar ativa ». Universidade Federal do CearÃ, 2007. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=870.

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FundaÃÃo de Amparo Ã Pesquisa do Estado do CearÃ
Baixas concentraÃÃes sÃricas de drogas antituberculose podem ser causa da resistÃncia de Mycobacterium tuberculosis Ãs drogas utilizadas para tratamento, a qual Ã mais freqÃente em pacientes em tratamento irregular, mas pode ser verificada em pacientes em tratamento diretamente observado. Os objetivos desse estudo foram avaliar a permeabilidade intestinal e a biodisponibilidade de rifampicina, isoniazida e pirazinamida em pacientes com tuberculose pulmonar ativa. Realizou-se estudo transversal controlado. No perÃodo entre julho de 2004 e dezembro de 2005 foram selecionados 56 pacientes consecutivos com tuberculose pulmonar ativa, atendidos no Centro de SaÃde Carlos Ribeiro em Fortaleza, CearÃ e 29 controles sadios recrutados entre profissionais de saÃde, seus familiares e vizinhos para avaliaÃÃo da permeabilidade intestinal. Foram coletadas informaÃÃes sociodemogrÃficas e exames bioquÃmicos e realizou-se o teste de lactulose/manitol de todos. Os primeiros 30 casos de tuberculose e os 29 controles dessa amostra foram selecionados para a avaliaÃÃo da biodisponibilidade sÃrica de rifampicina, isoniazida e pirazinamida em amostras coletadas duas horas e seis horas depois da ingestÃo. A mÃdia de idade dos casos foi de 39,2 Â 15 anos e a dos controles 34 Â 11,0 (p=0,61). Eram do sexo masculino 71 % dos casos e 66% dos controles (p=0,57). O peso mÃdio dos casos (52,4 Â 6,3 kg) foi significativamente menor do que o dos controles (71,1 Â 14,0 kg) (p=0,014). Verificou-se menor excreÃÃo de lactulose entre os casos (mediana de 0,1721%; variando de 0,0-5.0139%) do que entre os controles (0,4301%; variando de 0,0-2,064) (p=0,049%); a excreÃÃo de manitol entre os casos foi 17,9910% (1,9567-71,5446%) e entre controles foi de 24,3899% (1,3883-63,0539%) (p=0,147); a relaÃÃo lactulose/manitol foi de 0,0095 (0,0-0,0759%) entre os casos e 0,0136 (0,0-0,0136%) entre os controles (p=0,018). A concentraÃÃo sÃrica mÃxima de rifampicina teve mÃdia de 1,46 Â 0,72 Âg/ml nos casos e de 6,69 Â 3,07 Âg/ml nos controles (p<0,001); a de isoniazida foi 2,62 Â 1,53 Âg/ml entre os casos e 1,98 Â 0,76 Âg/ml entre os controles (p=0,057) e a de pirazinamda foi de 44,10 Â 10,40 Âg/ml entre os casos e 36,32 Â 12,02 Âg/ml entre os controles (p=0,007). Quatro (13,3%) casos nÃo chegaram a alcanÃar os limites mÃnimos das concentraÃÃes normais esperadas de nenhuma das drogas de primeira linha para o tratamento da tuberculose; 21 (70,0%) alcanÃaram essa concentraÃÃo sÃrica apenas para uma droga (pirazinamida) e cinco (16,7%) apenas para duas drogas (pirazinamida e isoniazida). Nenhum caso alcanÃou as concentraÃÃes sÃricas normais esperadas para as trÃs drogas, simultaneamente. Em conclusÃo, observou-se reduÃÃo da absorÃÃo paracelular entre os pacientes com tuberculose bem como mÃ absorÃÃo intestinal de rifampicina e isoniazida. Estes resultados sugerem a necessidade da avaliaÃÃo de medidas para reduzir a mÃ absorÃÃo intestinal de drogas e evitar o retardo ou a impossibilidade de cura da doenÃa, bem como o risco de multirresistÃncia
Reduced antituberculosis drugs concentrations are associated with Mycobacterium tuberculosis resistance, mostly in patients in irregular but also in directly observed treatment. This study aims to evaluate intestinal permeability and bioavailability of rifampin (R), isoniazid (I) and pyrazinamide (P) in patients with active pulmonary tuberculosis. A controlled cross sectional study evaluated intestinal permeability from 56 consecutive active pulmonary tuberculosis (TB) patients who attended Carlos Ribeiro Health Unit in Fortaleza, CearÃ, northeast of Brazil, from July 2004 to December 2005. The Thirty who first came were selected to have R, I and P serum concentration dosed. Twenty nine healthy controls were select among health professionals, their relatives and neighboring. To access intestinal permeability lactulose/manitol (L/M) test was performed by HPLC in urine samples collected during five hours. To access bioavailability two blood samples were collect at 2 and 6 hours after drug ingestion. Demographic information was recorded from all volunteers. Mean age was 39.2 Â 15 years in cases and 34 Â 11.0 in controls (p=0.61); 71.4% cases and 65.5% controls were men (p=0.57). Lactulose urinary excretion was significantly lower in TB patients (median 0.1721%; range 0.0-5.0139) than in controls (median 0.4301%; range 0.2125-2.064) (p=0.0194); median manitol excretion in cases was 17.9910% (1.9567-71.5446) and in controls, 24.39894% (range 1.3883-63.0539) (p=0.147) and the lactulose/manitol ratio was of 0.0095 (range 0.0-0.0759) in cases and 0.0153 (0.0-0.0136) in controls (p=0.0698). Maximum means seric rifampin concentration in cases was1.46 Â 0.72 Âg/ml and in controls, 3.07 Â 6.69Âg/ml (p<0.001); maximum means seric isoniazid concentration was 2.62 Â 1.53 Âg/ml in cases and 0.76 Â 1.98 Âg/ml in controls (p=0.057); maximum means seric pirazinamide concentratio was 44.10 Â 10.40 Âg/ml in cases and 12.02 Â 36.32 Âg/ml in controls (p=0.007). Four cases (13.3%) had all tested drugs serum concentrations under expected normal; 21/30 (70.0%) had expected normal concentrations only for one drug (pyrazinamide) and 5/30 (16.7%) for 2 drugs only (pirazinamide and isoniazid). None of the cases had expected concentration levels for all 3 drugs, simultaneously. In conclusion, there was lesion in the functional intestinal barrier and malabsorption for rifampin and isoniazid in active pulmonary TB patients suggesting it is necessary a deeper evaluation of measures to reduce malabsorption, since only these drugs are used during last the four months of treatment

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Dalmolin, Tanise Vendruscolo. « AVALIAÇÃO DE MÚLTIPLOS MECANISMOS DE RESISTÊNCIA ASSOCIADOS EM ISOLADOS CLÍNICOS DE Klebsiella pneumoniae RESISTENTE AOS CARBAPENÊMICOS ». Universidade Federal de Santa Maria, 2015. http://repositorio.ufsm.br/handle/1/6033.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Antimicrobial resistance is considered a serious public health problem worldwide and complicates the treatment of infections caused by resistant microorganisms. The carbapenems are antimicrobial agents considered the last resource for treatment of severe infections caused by Klebsiella pneumoniae and the resistance to β-lactams can result in the accumulation of different resistance mechanisms (carbapenemases, efflux pump and loss of porins). This study aimed to evaluate multiple resistance mechanisms in 27 clinical isolates of K. pneumoniae resistant to carbapenems coming from the University Hospital of Santa Maria-RS from July 2013 to August 2014. These isolates were evaluated the susceptibility profiles through broth microdilution against ciprofloxacin, imipenem, ertapenem, meropenem, cefepime, ceftazidime and cefoxitin. Carbapenemase detection was performed through phenotypic tests with combined disc test with phenylboronic acid (AFB) and ethylenediaminetetraacetic acid (EDTA) and Blue-Carba test. In addition, genotypic tests to detect genes enconding carbapenemase were performed. Efflux pump was evaluated by broth microdilution together with efflux pump inhibitor and loss of porins were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). High levels of resistance verified by the minimum inhibitory concentration (MIC) 50 and 90 for ciprofloxacin (64 and 128μg/mL), imipenem (32 to >128μg/mL), ertapenem (>128 and >128μg/mL), meropenem (128 and >128 μg/mL), cefepime (>128 and >128 μg/mL), ceftazidime (64 and 128μg/mL) and cefoxitin (128 and >128 μg/mL), respectively. In the resistance through carbapenemases production, 89% of the clinical isolates showed blaKPC gene and no clinical isolated showed genes encoding the metallo- β-lactamases. It was observed that the Blue-Carba test and combined disc test with AFB showed 100% concordance, while the combined disc test with EDTA showed high number of false positive (48%) when compared with genotypic test. Four isolates showed phenotypic profile consistent with the presence of efflux pump and all clinical isolates had lost one or both porins, being that in three isolated, this was the only resistance mechanism found. In 14% of the isolates can observe simultaneously observe the presence of three resistance mechanisms. Consequently, it is of fundamental scientific interest that studies are conducted in order to investigate and understand the mechanisms involved in resistance to carbapenems in order to assist strategies of prevention and infection control.
A resistência aos antimicrobianos é considerada um grave problema de saúde pública em âmbito mundial e dificulta o tratamento de infecções causadas por microrganismos resistentes. Os carbapenêmicos são os antibacterianos considerados último recurso para o tratamento de infecções graves causadas por Klebsiella pneumoniae e a resistência a esse grupo de β-lactâmicos pode resultar da acumulação de diferentes mecanismos de resistência (carbapenemases, bomba de efluxo e perdas de porinas). Este trabalho teve como objetivo avaliar os múltiplos mecanismos de resistência de 27 isolados clínicos de K. pneumoniae resistente a carbapenêmicos oriundos do Hospital Universitário de Santa Maria-RS no período de julho de 2013 a agosto de 2014. Foram avaliados os perfis de suscetibilidade desses isolados através de microdiluição em caldo frente aos antimicrobianos ciprofloxacino, imipenem, ertapenem, meropenem, cefepima, ceftazidima e cefoxetina. A detecção de carbapenemases foi realizada através de testes fenotípicos com a utilização do disco de antimicrobiano associado com os inibidores ácido fenilborônico (AFB) e ácido etilenodiamino tetra-acético (EDTA) e através do teste Blue-Carba. Também foram realizados testes genotípicos para detectar os genes que codificam as carbapenemases. Bombas de efluxo foram avaliadas através de microdiluição em caldo juntamente com inibidor de bomba de efluxo e a perda de porinas foi avaliada pela eletroforese em gel de sulfato de dodecilo de sódio-poliacrilamida (SDS-PAGE). Altos níveis de resistência foram verificados nesses isolados pela concentração inibitória mínima (CIM) 50 e 90 para ciprofloxacino (64 e 128μg/mL), imipenem (32 e >128μg/mL), ertapenem (>128 e >128μg/mL), meropenem (128 e >128 μg/mL), cefepima (>128 e >128μg/mL), ceftazidima (64 e 128μg/mL) e cefoxitina (128 e >128 μg/mL), respectivamente. Na resistência através da produção de carbapenemases, 89% dos isolados apresentaram o gene blaKPC e nenhum isolado apresentou genes que codificam as metalo-β-lactamases. Foi observado que os testes Blue-Carba e disco combinado com AFB apresentaram 100% de concordância, enquanto o teste de disco combinado com EDTA apresentou elevado número de falso-positivos (48%) quando comparados com o teste genotípico. Quatro isolados apresentaram perfil fenotípico compatível com a presença de bomba de efluxo e todos os isolados apresentaram perda de uma ou ambas porinas, sendo que em três isolados, esse foi o único mecanismo de resistência encontrado. Em 14% dos isolados pode-se observar concomitantemente a presença dos três mecanismos de resistência. Devido ao exposto, é de fundamental interesse científico que estudos sejam realizados para investigar e compreender os mecanismos envolvidos na resistência aos carbapenêmicos, a fim de auxiliar em estratégias de prevenção e controle de infecção.

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Speretta, Guilherme Fleury Fina. « Alterações cardiovasculares induzidas pela obesidade : envolvimento do sistema reninaangiotensina no núcleo do trato solitário ». Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/7095.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
In the last decades, obesity has become a worldwide epidemy. Excess of adipose tissue favors the development of associated diseases such as hypertension, obstructive sleep apnea and type II diabetes. Data from the literature have shown that obesity activates the reninangiotensin system (RAS), increases sympathetic nerve activity and pro-inflammatory cytokines levels, including in the central nervous system. However, the pathways and neural mechanisms involved in these responses are not yet fully elucidated. Therefore, the aim of this study was to evaluate the cardiovascular and metabolic responses in high-fat diet (HFD) feeding rats. We also study the possible participation of the RAS and the immune system in the nucleus of the solitary tract (NTS) in the obesity-induced cardiovascular changes. Finally, we tested if the resistance training (RT) performed at moderate intensity would be able to prevent obesityinduced cardiovascular changes. To achieve these goals, adult Holtzman rats (300-320 g) were fed with HFD (3.82 kcal/g and 26.4% total fat) or standard chow diet (SD; 2.25 kcal/g and 5.4% total fat) for 6 weeks. We observed higher blood levels of total cholesterol, triglycerides, leptin and glucose and decreased insulin sensitivity after 6 weeks of HFD. There was an increase in mean arterial pressure (MAP), the sympathetic modulation of systolic blood pressure (SBP), heart rate (HR) and sympathovagal balance of pulse interval (PI), and an impairment in the bradycardic response of the baroreflex in HFD feeding animals. After 6 weeks of HFD, there was an increase in the mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukine-6 (IL-6), an increase in the mRNA expression of the angiotensin converting enzyme (ACE) and AT1 receptor, and a decrease in the expression of AT2 and Mas receptors. In agreement with the increased expression of cytokines in the NTS we have demonstrated higher GFAP immunoreactivity (specific marker of astrocytes) and higher number of positive cells for Iba-1 (specific marker for microglia) in the NTS in rats fed with HFD. The blockade of AT1 receptor in the NTS in animals fed with HFD promoted a decrease in MAP, reduced the sympathetic modulation in SBP, reduced the sympathovagal balance of PI and restored the bradycardic response of the baroreflex. The overexpression of the AT2 receptors in the NTS in rats fed with HFD reestablished HR, baroreflex sensitivity and sympathovagal balance of PI, and partially attenuated the increase in sympathetic modulation of SBP. These responses were associated with increased Mas receptor mRNA expression and reduction in the TNF-α mRNA expression in the NTS. However, these effects were not sufficient to restore the MAP in HFD feeding rats with AT2 receptors overexpression in the NTS. Finally, the RT prevented cardiovascular changes induced by HFD, including increases in MAP, sympathetic modulation of SBP, HR, sympathovagal balance of PI, and reduced baroreflex sensitivity. These responses were associated with increased expression of mRNA for components of protective RAS axis (AT2 and Mas receptors and ACE2), and the antiinflammatory cytokine IL-10 as well as the reduction of pro-inflammatory cytokines (TNF-α and IL -1β) in the NTS. Together, our data suggest that HFD promotes increases in plasma levels of glucose and leptin, with dysfunction in insulin sensitivity. HFD also promotes increases in blood pressure associated with increased sympathetic modulation in SBP, sympathovagal balance of PI, HR and baroreflex dysfunction. The neuroinflammatory process and an imbalance between the pressor and protective RAS axis in the NTS seem to be involved with the development/maintenance of the cardiovascular alterations induced by HFD. The TF performed at moderate intensity seems to be an important tool in preventing cardiovascular changes induced by HFD.
Nas últimas décadas a obesidade se tornou uma epidemia mundial. O excesso de tecido adiposo favorece o desenvolvimento de doenças associadas como a hipertensão arterial, apnéia obstrutiva do sono e diabetes tipo II. Dados da literatura mostram que a obesidade ativa o sistema renina-angiotensina (SRA), e aumenta a atividade do sistema nervoso simpáticoe os níveis de citocinas pró-inflamatórias, inclusive no sistema nervoso central. Entretanto, as vias e mecanismos neurais envolvidos nessas respostas ainda não estão completamente elucidados. Portanto, o objetivo do presente estudo foi avaliar as respostas cardiovasculares e metabólicas em ratos alimentados com dieta hiperlipídica (DH). Estudamos também a possível participação do SRA e do sistema imune no núcleo do trato solitário (NTS) nas alterações cardiovasculares induzidas pela DH. Por fim, testamos se o treinamento de força (TF) realizado em intensidade moderada seria capaz de prevenir as alterações cardiovasculares induzidas pela DH. Para tanto, foram utilizados ratos Holtzman adultos (300 a 320 g) alimentados com DH (3,82 kcal/g e 26,4% de gorduras) ou dieta padrão (DP; 2,25 kcal/g e 5,4% de gorduras) por 6 semanas. Nossos resultados demonstraram maiores níveis de colesterol total, triacilgliceróis, leptina e glicose no sangue e menor sensibilidade à insulina após 6 semanas de DH. Houve um aumento da pressão arterial média (PAM), da modulação simpática da pressão arterial sistólica (PAS), da frequência cardíaca (FC) e do balanço simpatovagal do intervalo de pulso (IP) e menor resposta bradicárdica do barorreflexo em animais alimentados com DH. Após 6 semanas de DH, houve maior expressão do RNAm das citocinas pró-inflamatórias, fator de necrose tumoral-α (TNF- α) e interleucina-6 (IL-6), maior expressão do receptor AT1 e da enzima conversora de angiotensina (ECA) e menor expressão dos receptores AT2 e Mas. Corroborando com a maior expressão de citocinas no NTS, foi demonstrado maior imunoreatividade para GFAP (marcador especifico de astrócitos) e maior número de células positivas para Iba-1 (marcador especifico para micróglias) no NTS de ratos alimentados com DH. O bloqueio do receptor AT1 no NTS de animais alimentados com DH promoveu queda na PAM, reduziu a modulação simpática na PAS, reduziu o balanço simpatovagal do IP e restabeleceu a resposta de bradicardia reflexa. A super-expressão de receptores AT2 no NTS de ratos alimentados com DH reestabeleceu a FC, a sensibilidade do barorreflexo e o balanço simpatovagal do IP, além de uma atenuação parcial na modulação simpática da PAS. Essas respostas foram associadas com o aumento da expressão do RNAm do receptor Mas e a redução na expressão do TNF-α no NTS. Porém, esses efeitos não foram suficientes para atenuar a PAM nos ratos com DH e super-expressão de receptores AT2 no NTS. Por fim, o TF preveniu as alterações cardiovasculares induzidas pela DH, incluindo o aumento da PAM, da modulação simpática da PAS, da FC, do balanço simpatovagal do IP, e a redução da sensibilidade do barorreflexo. Essas respostas foram associadas com um aumento na expressão do RNAm de componentes da via protetora do SRA (receptores AT2 e Mas e ECA2) e da citocina anti-inflamatória IL-10, bem como a redução de citocinas próinflamatórias (TNF-α e IL-1β) no NTS. Em conjunto, nossos dados sugerem que a DH promove aumentos na leptina plasmática e na glicemia, com disfunção na sensibilidade à insulina e no perfil lipídico. A DH também promove aumento na pressão arterial associada aos aumentos na modulação simpática na PAS, balanço simpatovagal do IP, FC e disfunção no barorreflexo. O processo neuroinflamatório e um desequilíbrio entre as vias pressora e protetora do SRA no NTS parecem estar envolvidos com o desenvolvimento/manutenção das alterações cardiovasculares encontradas em ratos alimentados com DH. O TF realizado em intensidade moderada parece ser uma importante ferramenta na prevenção das alterações cardiovasculares induzidas pela DH.
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Serrano, Marco Lucía. « PPAR β/δ, inflamació i resistència a la insulina en adipòcits ». Doctoral thesis, Universitat de Barcelona, 2011. http://hdl.handle.net/10803/96332.

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Des de fa un parell de dècades s’ha establert una relació causa efecte entre l’estat inflamatori crònic de baixa intensitat que es presenta en obesitat i l’aparició de resistència a insulina i Diabetis Mellitus tipus 2 associades a aquesta patologia. Aquest estat inflamatori es caracteritza per la sobreproducció de citocines proinflamatòries com el TNF-α(factor de necrosi tumoral-α) o la interleucina 6 (IL-6), que són produïdes en gran mesura pel teixit adipós. Els Receptors Activats per proliferadors Peroxisòmics (PPAR) s'han proposat com a possibles dianes terapèutiques per al tractament i la prevenció del procés inflamatori i de la resistència a la insulina. D’aquests receptors nuclears el subtipus PPARβ/δ juga un paper important en la prevenció del procés inflamatori, ja que diversos estudis han demostrat que pot evitar la producció de IL-6 induïda per lipopolisacárid en el teixit adipós, i a més pot inhibir l’activació del factor de transcripció proinflamatori NF-κB. Per això, els objectiu d’aquesta tesi doctoral han estat, d'una banda, avaluar els efectes de l’obesitat i la inflamació sobre PPARβ/δ, i d’altra banda, conèixer si l’activació d’aquest receptor nuclear per GW501516 és capaç de prevenir la resistència a la insulina induïda per la via IL-6/STAT3/SOCS3 i els mecanismes implicats en adipòcits. Els resultats que es van obtenir a partir de mostres de teixit adipós humà indiquen que en presència d’obesitat hi ha un increment de citocines proinflamatòries (IL-6 i TNF-α) i de NF-κB, ambdós factors afavoreixen el procés inflamatori. Aquesta situació s’acompanyà d'una disminució de l’activitat de PPARβ/δ, la qual es va veure reflectida en la disminució dels gens diana d'aquest. Però l'activació de PPARβ/δ i la inhibició de NF-κB en estudis in vitro amb cèl•lules adiposes SGBS van ser capaços de revertir aquest efecte (Serrano-Marc L, BBA of Lipids, 2012). D'altra banda, estudis en ratolins i en cèl•lules 3T3-L1 van demostrar que l’activació de PPARβ/δ per GW501516 podria evitar l’aparició de resistència a la insulina inhibint l’activació de la via IL-6/STAT3/SOCS3 mitjançant dos mecanismes. L’activació del receptor nuclear va impedir la fosforilació en el residu de Tyr705 de STAT3 (Signal Transductor and Activator of transcription 3, un factor de transcripció) i d’aquesta manera va inhibir la unió de Hsp90 amb aquest (necessària per a queSTAT3 s’activi). D’altra banda, l’activació per GW501516 va impedir la fosforilació de la ERK1 / 2 (Extracellular Receptor Kinase 1/2) i d’aquesta manera es va evitar la fosforilació en el residu de Ser727 de STAT3, necessària per a la seva activació transcripcional. La inhibició del factor de transcripció STAT3 va implicar una disminució en els seus gens diana, entre ells SOCS3 (Supressor of Citokine Signaling 3), que és el responsable de la degradació d'IRS-1 (Insulin Receptro Substrate-1), proteïna essencial per donar resposta a la insulina (Serrano-Marco, L, Diabetis, 2011).
It has been established a causal link between the chronic inflammatory state of low intensity that occurs in obesity and the onset of insulin resistance and type 2 diabetes. This inflammatory condition is characterized by the overproduction of proinflammatory citokines such TNF-α (Tumor Necrosis Factor-α) or interleukin 6 (IL-6), which are largely produced by the adipose tissue. The Peroxisomic Proliferator Activated Receptors (PPAR) have been proposed as potential therapeutic targets for the treatment and prevention of inflammation and insulin resistance. Of these nuclear receptors the subtype PPARβ/δ plays an important role in preventing the inflammatory process. Therefore, the objective of this work is, one hand, to evaluate the effects of inflammation in obesity and in PPARβ/δ; and secondly, to determine whether the activation of PPARβ/δ with GW501516 in adipocytes is capable of preventing insulin resistance induced through the IL-6/STAT3/SOCS3 pathway, and also the mechanisms involved. The results obtained from human adipose tissue samples show that in the presence of obesity there is an increase of proinflammatory cytokines (IL-6 and TNF-α) and of NF-κB. This was accompanied by a decrease in the PPARβ/δ activity. But the activation of PPARβ/δ and the inhibition of NF-κB in in vitro studies with SGBS adipose cells were able to reverse these effects (Serrano-Marco L, BBA of Lipids, 2012). Furthermore, studies in mice and in 3T3-L1 cells demonstrated that activation of PPARβ/δ by GW501516 could prevent the development of insulin resistance by inhibiting the activation of the IL-6/STAT3/SOCS3 pathway (Serrano-Marco L, Diabetes, 2011). Taking all these data together the final conclusion of this work is: PPARβ/δ is involved in the regulation of the chronic inflammatory state that links obesity and insulin resistance. Therefore, the activation of this nuclear receptor could prevent IL6 induced insulin resistance.

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Tatsch, Etiane. « Dano ao DNA no diabetes tipo 2 e sua associação com inflamação, estresse oxidativo, disfunção endotelial, resistência à insulina e à ocorrência de complicações crônicas microvasculares ». Universidade Federal de Santa Maria, 2016. http://repositorio.ufsm.br/handle/1/3421.

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Several pathophysiological mechanisms are associated with type 2 diabetes mellitus (type 2 DM), such as glucotoxicity, insulin resistance, inflammation, oxidative stress and endothelial dysfunction, which can result in DNA strand breakage and changes in the nitrogenous bases. In this manner, DNA damage biomarkers may be useful in elucidating the pathophysiology of diabetes, as well as serving as an alternative to better evaluate its chronic complications. However, a great number of pathophysiological mechanisms related to increased DNA damage in diabetes need to be clarified. Thus, the objective of this study was to evaluate DNA damage in type 2 diabetes and its association with inflammation, oxidative stress, endothelial dysfunction, resistance towards insulin and the occurrence of chronic microvascular complications. The DNA damage was evaluated through the comet assay and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) urinary, the inflammatory process through the serum levels of interleukin (IL) 1, 6 and 10 and the alpha tumor necrosis factor (TNF-α), protein oxidation through plasma levels of advanced oxidation protein products (AOPPs), endothelial dysfunction by serum levels of NOx (nitrite/nitrate) and urinary albumin and insulin resistance through the index HOMA-IR. The present study was carried out in two phases. In the first phase, 32 patients with type 2 DM and 30 healthy individuals (control) were investigated. In the second phase, 54 patients with type 2 DM and 22 healthy individuals (control) were recruited from the University Hospital of Santa Maria (HUSM). This study found that patients with type 2 diabetes showed increased DNA fragmentation, assessed by comet assay, and increased oxidative DNA damage, assessed by 8-hydroxy-2'-deoxyguanosine (8-OhdG) urinary levels, compared to healthy control subjects. Additionally, increased DNA damage was observed in the type 2 DM group with inadequate glycemic control. When the areas obtained under the ROC curve were analyzed, 8-OHdG urinary presented higher diagnostic ability in identifying microvascular chronic complications compared with urinary albumin in the type 2 DM group. Furthermore, it was demonstrated that type 2 diabetic patients with microvascular complications had higher levels of oxidative DNA damage compared to patients without such complications. Interestingly enough, it was observed that type 2 diabetic patients with increased DNA damage had higher levels of proinflammatory cytokines such as IL-1, IL-6 and TNF-α, and a decrease in IL-10 levels, which is considered an anti-inflammatory cytokine. An association between increased DNA damage in type 2 diabetes and the index HOMA-IR, AOPPs levels and NOx levels and urinary albumin was also verified. Patients with type 2 diabetes exhibited factors that can directly contribute to the increase of DNA damage, such as insulin resistance, inflammation, endothelial dysfunction and increased generation of reactive species.
Diversos mecanismos fisiopatológicos estão associados ao Diabetes Mellitus (DM) tipo 2, como glicotoxicidade, resistência à insulina, inflamação, estresse oxidativo e disfunção endotelial, podendo resultar em quebras nos filamentos de DNA e modificações nas bases nitrogenadas. Desta maneira, biomarcadores de dano ao DNA podem ser úteis na elucidação da fisiopatologia do DM, bem como uma alternativa para a melhor avaliação de suas complicações crônicas. No entanto, muitos destes mecanismos fisiopatológicos relacionados ao aumento do dano ao DNA no diabetes precisam ser esclarecidos. Assim, o objetivo deste estudo foi avaliar o dano ao DNA no DM tipo 2 e sua associação com inflamação, oxidação proteica, disfunção endotelial, resistência à insulina e à ocorrência de complicações crônicas microvasculares. O dano ao DNA foi avaliado através do ensaio cometa e dos níveis de 8-hidroxi-2'-desoxiguanosina (8-OHdG) urinário, o processo inflamatório através dos níveis séricos das interleucinas (IL) 1, 6 e 10 e do fator de necrose tumoral alfa (TNF-α) , a oxidação proteica através dos níveis plasmáticos dos produtos proteicos de oxidação avançada (AOPPs), a disfunção endotelial através dos níveis séricos de NOx (nitrito/nitrato) e albumina urinária e a resistência insulínica através do índice HOMA-IR. O presente estudo foi conduzido em duas fases. Na primeira fase 32 pacientes com DM tipo 2 e 30 controles saudáveis foram investigados. Na segunda fase 54 pacientes com DM tipo 2 e 22 indivíduos controle foram recrutados no Hospital Universitário de Santa Maria (HUSM). Neste estudo, foi verificado que os pacientes com DM tipo 2 apresentaram aumento na fragmentação do DNA, avaliado pelo ensaio cometa, e um maior dano oxidativo ao DNA, avaliado pelos níveis urinários de 8-OHdG, comparados com indivíduos controles saudáveis. Também foi verificado no grupo DM tipo 2 com controle glicêmico inadequado um maior dano ao DNA. Quando foram analisadas as áreas sob a curva ROC obtidas, verificamos que o 8-OHdG urinário apresentou uma maior capacidade diagnóstica em identificar as complicações crônicas microvasculares, quando comparada com a albumina urinária no grupo DM tipo 2. Além disso, foi demonstrado que os pacientes diabéticos tipo 2 com complicações microvasculares apresentaram maiores níveis de dano oxidativo ao DNA, comparado aos pacientes que não apresentavam estas complicações. Interessantemente, foi observado que os pacientes DM tipo 2 com maior dano ao DNA apresentaram maiores níveis de citocinas pró-inflamatórias, como IL-1, IL-6 e TNF-α, além de um decréscimo nos níveis de IL-10, considerada um citocina anti-inflamatória. Também foi verificada uma associação entre o aumento do dano ao DNA no DM tipo 2 e o índice HOMA-IR, os níveis de AOPPs e os níveis de NOx e albumina urinária. Desta forma, nós especulamos que os pacientes com DM tipo 2 apresentaram uma cascata de eventos como, resistência à insulina, processo inflamatório, disfunção endotelial e aumento da geração de espécies reativas, fatores que podem contribuir diretamente para o aumento do dano ao DNA.

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Soares, Celina Maria Pereira de Moraes [UNIFESP]. « Prevalência de resistência transmitida do HIV-1 aos antirretrovirais no Brasil, pré- início de tratamento ». Universidade Federal de São Paulo (UNIFESP), 2011. http://repositorio.unifesp.br/handle/11600/9962.

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A seleção de mutações de resistência aos medicamentos antirretrovirais pós-falha terapêutica representam um grande desafio para a tomada de decisão de novos esquemas de tratamento na pandemia global. A elevada variabilidade genética do HIV-1 e a seleção de mutações de resistência transmitida a pacientes infectados cronicamente, sem que tenham participado de qualquer esquema terapêutico, tem sido objeto de vários estudos no mundo. Os padrões de resistência são estudados principalmente em países da Europa e Estados Unidos, que apresentam prevalência majoritária do subtipo B. Contudo, estudos que direcionam a seleção de mutações transmitidas aos medicamentos antirretrovirais e em subtipos não-B, assim como em suas formas recombinantes, tem aumentado significativamente em várias regiões do mundo. No Brasil, esses estudos são realizados esporadicamente e em regiões distintas do país e, principalmente, em pacientes recém-infectados. A alta variabilidade genética do HIV-1 no nosso país é representada de forma diversificada, com a presença do subtipo B, seguida do F, e especificamente na região Sul a importante prevalência do subtipo C. O objetivo principal deste estudo está embasado nas características de mutações de resistência transmitida aos medicamentos antirretrovirais pelo HIV-1 no gene pol, frações da transcriptase reversa e protease, com análise do perfil mutacional por grupos populacionais de pacientes cronicamente infectados pelo HIV-1 e não tratados, porém com indicação de início imediato de tratamento. Foram avaliados os pacientes representados nas regiões demográficas do Brasil. A prevalência nacional, resultou em 12,1% de mutações de resistência transmitida aos antirretrovirais pelo HIV-1 (grau intermediário de 5 a 15%) e 70,8% de subtipo B; 15,5% C; 6,4% F; 4,0% BF e 3,0% BC na classificação dos subtipos do HIV-1. Além disso, foram classificadas as prevalências de mutações transmitidas, dos subtipos do HIV-1 e características sociodemográficas, laboratoriais e os dados comportamentais na população HIV positiva pré-terapia por cidade nas cinco regiões brasileiras.
The selection of resistance mutations to antiretroviral drugs after failure of antiretroviral therapy represents a major challenge for decision-making of new therapeutic regimens in the global pandemic. The high genetic variability of HIV- 1and the selection of resistance mutations trasmitted to patients chronically infected without having participated in the regimen has been the subject of several studies in the world. Resistance patterns are studied mainly in European countries and the United States, wich have majority prevalence of subtype B. However, studies that guide the selection of transmitted mutants to antiretroviral drugs and non-B subtypes, and in their recombinant forms, has increased significantly in the several regions of the world. In Brazil, these studies are conducted sporadically and in different regions of the country and specially in newly infected patients. The high genetic variability of HIV-1 is represented in our country so diverse, with the presence of subtype B, followed by F, and specifically in the South region, the prevalence of subtype C. In addition, coexist the prevalence of recombinant forms, where the principal is the subtype BF, followed by BC. The main objective of this study estimate the characteristics of transmitted resistance mutations to antiretroviral drugs in HIV-1 ol gene, fractions of reverse transcriptase and protease, with mutational analysis of the profile by a population patients chronically infected with HIV-1 and not treated, but with indication of immediate initiation of treatment. We evaluated the patients represented in the demographic regions of Brazil. The national prevalence resulted in 12.1% of transmitted resistance mutations to antiretroviral (intermediate grade 5% to 15%) and 70.8%, 15.5% C, 6.4% F, 4.0% BF and 3.0% BC in the classification of subtypes of HIV-1. In addition, the prevalence of transmitted mutations, the subtypes of HIV-1 and sociodemographic characteristics, laboratory parameters and behavior data in population HIV-1 positive pre-treatment were classified by the cities in five Brazilian regions.
TEDE
BV UNIFESP: Teses e dissertações

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Silva, Andréa Fernandes Emiliano da. « Estudo das alterações cardiovasculares e metabólicas em modelo experimental de programação metabólica : efeito de extrato da casca de uva Vitis vinífera ». Universidade do Estado do Rio de Janeiro, 2011. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=6293.

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Estudos epidemiológicos e experimentais têm sugerido que fatores de risco cardiovasculares podem ser parcialmente atribuídos às influências do ambiente em que vive o indivíduo, e que a nutrição materna influencia na programação de alterações metabólicas e cardiovasculares no indivíduo adulto e que caracterizam a síndrome metabólica (SM). Em contrapartida, estudos prévios de nosso laboratório demonstram que o extrato da casca de uva Vitis labrusca (GSE) possui efeito vasodilatador, antihipertensivo e antioxidante. Desta forma, o objetivo deste estudo foi avaliar o efeito do tratamento oral com GSE (200mg/kg/dia), sobre as alterações cardiovasculares e metabólicas e estresse oxidativo observados na prole adulta (fêmea e machos) com 3 e 6 meses, cujas mães foram submetidas a uma dieta rica em gordura (hiperlipídica) durante a lactação. Quatro grupos de ratas foram alimentados com dietas experimentais: controle (7% de gordura); controle + GSE (7% de gordura + GSE), hiperlipídica (24% de gordura); hiperlipídica + GSE (24% de gordura + GSE) durante a lactação. Após o desmame, todos os filhotes passaram a ser alimentados com uma dieta controle e foram sacrificados aos 3 ou 6 meses de idade. A pressão arterial sistólica (PAS) foi medida por pletismografia de cauda e o efeito vasodilatador da acetilcolina (ACh) foi avaliado em leito arterial mesentérico (LAM) perfundido. Foram avaliados o peso corporal, adiposidade (intra-abdominal e gonadal), níveis plasmáticos de colesterol total, triglicerídeos, glicose e insulina, e a resistência à insulina (RI) foi calculada pelo índice de HOMA IR. As expressões do IRS-1, Akt e GLUT-4 foram determinadas em músculo soleus. O dano oxidativo, níveis de nitritos e a atividade das enzimas antioxidantes: superóxido dismutase, catalase e glutationa peroxidase foram dosados no plasma e homogenato de LAM. A PAS e tecido adiposo foram aumentados nas proles adultas de ambos os sexos e idades do grupo hiperlipídico e revertidos pelo tratamento com o GSE. A resposta vasodilatadora à ACh em LAM não foi diferente entre os grupos de ambos os sexos, mas foram reduzidas com o envelhecimento. Nas proles fêmeas e machos do grupo hiperlipídico também foram observados o aumento dos níveis de triglicerídeos, de glicose e RI em ambas as idades e foram reduzidos pelo GSE. No grupo hiperlipídico houve redução nas expressões de IRS-1, Akt e GLUT-4 e o GSE reverteu estas expressões. Os níveis plasmáticos de malondialdeído estavam aumentados e os níveis de nitrito diminuídos no grupo hiperlipídico, de ambos os sexos e idades e foram revertidos pelo GSE. As atividades das enzimas antioxidantes no plasma e no mesentério foram reduzidas no grupo hiperlipídico e restauradas pelo GSE. Em conclusão, O GSE parece proteger as proles fêmeas e machos, cujas mães foram expostas a uma dieta hiperlipídica durante a lactação, dos fatores de riscos cardiovasculares, proporcionando uma fonte alternativa nutricional para a prevenção da SM.
Epidemiological and experimental studies have suggested that cardiovascular risk factors can be partly attributed to the influences of the environment in which the individual lives, and that maternal nutrition influences the programming of metabolic and cardiovascular diseases in adults characterizing the metabolic syndrome. On the other hand, previous studies from our laboratory show that the skin extract of grape Vitis labrusca (GSE) has antihypertensive, antioxidant and vasodilator effects. Thus, the aim of this study was to evaluate the effect of oral treatment with GSE (200mg/kg/day) on the cardiovascular and metabolic disorders and oxidative stress observed in adult offspring (female and male) at 3 or 6 months whose mothers were fed a high fat diet (HF) during lactation. Four groups of rats were fed experimental diets: control (7% fat), control + GSE (7% fat + GSE), diet (24% fat), HF + GSE (24% fat + GSE) during lactation. After weaning, all pups have become fed a control diet and were sacrificed at 3 or 6 months of age. Systolic blood pressure (SBP) was measured by plethysmography and the vasodilator effect of acetylcholine (ACh) was studied in perfused mesenteric arterial bed (MAB). We determined the body weight, adiposity, plasma total cholesterol, triglycerides, glucose, insulin, and insulin resistance was calculated by HOMA IR. The expression of IRS-1, Akt and GLUT-4 were determined in soleus muscle. Oxidative damage, nitrite levels and antioxidant enzyme activity: superoxide dismutase, catalase and glutathione peroxidase were measured in plasma and homogenate of mesentery bed. The SBP and adipose tissue were increased in adult offspring of both sex and ages of the HF group and reversed by treatment with GSE. The vasodilator response to ACh was not different between groups of both sex, but was reduced by aging. In offspring females and males of the HF group were also observed increased levels of triglycerides, glucose and insulin resistance in both ages and those changes were reduced by GSE. HF group showed a reduction in expression of IRS-1, Akt and GLUT-4 which was reversed by GSE. The malondialdehyde levels were increased and nitrite levels were decreased in the HF group of both sex and ages and those changes were reversed by GSE. The activities of antioxidant enzymes in plasma and mesentery were lower in HF group and restored by the GSE. In conclusion, GSE appears to protect the female and male offspring whose mothers were exposed to a high fat diet during lactation, against the cardiovascular risk factors, providing an alternative source of food for the prevention of metabolic syndrome.

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Frantz, Eliete Dalla Corte. « Bloqueadores do sistema renina-angiotensina em ilhotas pancreáticas e fígado na obesidade induzida por dieta em camundongos ». Universidade do Estado do Rio de Janeiro, 2014. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=7221.

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As associações entre obesidade, doença hepática gordurosa não alcoólica (NAFLD) e diabetes mellitus tipo 2 (DM2) são bem estabelecidas, e o sistema renina-angiotensina (SRA) pode proporcionar uma ligação entre eles. O bloqueio do SRA em diferentes níveis pode estar relacionado a respostas na resistência à insulina, remodelagem do pâncreas e do fígado em um modelo de obesidade induzida por dieta. Camundongos C57BL/6 foram alimentados com uma dieta hiperlipídica (HF) durante oito semanas e depois tratados com alisquireno (50 mg/kg/dia), enalapril (30 mg/kg/dia) ou losartana (10 mg/kg/dia) por um período adicional de seis semanas. As drogas foram incorporadas na dieta. Avaliou-se a massa corporal (MC), pressão arterial, consumo e gasto energético (GE), metabolismo da glicose e lipídico, histopatologia pancreática e hepática, análise hormonal, imunohistoquímica, perfil gênico e/ou proteico do SRA no pâncreas, gliconeogênese hepática, sinalização da insulina, oxidação e acúmulo lipídico. Todos os inibidores do SRA reduziram significativamente o aumento da pressão arterial nos camundongos alimentados com dieta HF. O tratamento com enalapril, mas não alisquireno ou losartana, reduziu o ganho de MC e a ingestão alimentar; aumentou o GE; amenizou a intolerância à glicose e resistência à insulina; melhorou a massa de células alfa e beta; impediu a redução da adiponectina plasmática e restaurou a sensibilidade à leptina. Além disso, o tratamento com enalapril melhorou a expressão proteica nas ilhotas pancreáticas de Pdx1, GLUT2, ECA2 e do receptor Mas. O tratamento com losartana apresentou uma elevação na expressão proteica de AT2R no pâncreas. No fígado, a administração de enalapril atenuou a esteatose hepática, o acúmulo de triglicerídeos e preveniu o aumento dos níveis de PEPCK, G6Pase e do GLUT2. Do mesmo modo, o enalapril melhorou a transdução dos sinais da insulina através da via IRS-1/Akt, bem como reduziu os níveis de expressão gênica e/ou proteica de PPAR-gama, SREBP-1c e FAS. Esses resultados sugerem que a inibição da ECA com enalapril atenuou muitos efeitos deletérios provocados pelo consumo da dieta HF, incluindo: normalização da morfologia e função das ilhotas pancreáticas, proteção contra a resistência à insulina e acúmulo de lipídios no fígado. Estes efeitos protetores do enalapril podem ser atribuídos, principalmente, à redução no ganho de MC e ingestão alimentar, aumento do GE, ativação do eixo ECA2/Ang(1-7)/receptor Mas e dos níveis de adiponectina, o que promove uma melhora na ação hepática da insulina e leptina, normalização da gliconeogênese, amenizando a NAFLD.
The associations between obesity, NAFLD (non-alcoholic fatty liver disease) and diabetes are well established, and the reninangiotensin system (RAS) may provide a link among them. . The blocking of the RAS at different levels may be related to responses on insulin resistance, remodeling of the pancreas and liver in a model of diet-induced obesity. Mice (C57BL/6) were fed on a high-fat (HF) diet for 8 weeks and then treated with aliskiren (50 mg/kg/day), enalapril (30 mg/kg/day) or losartan (10 mg/kg/day) for an additional 6 weeks. The drugs were incorporated into the diet. We assessed body mass (BM), blood pressure, energy intake and expenditure (EE), glucose and lipid metabolism, pancreatic and hepatic histopathology, hormonal analysis, immunohistochemistry, the expression profile of genes and/or proteins affecting pancreas RAS, hepatic gluconeogenesis, insulin signaling and lipid oxidation and accumulation. All RAS inhibitors significantly attenuated the increased blood pressure in mice fed a HF diet. Treatment with enalapril, but not aliskiren or losartan, significantly attenuated BM gain, increased EE, enhanced the glucose intolerance and insulin resistance; improved the alpha and beta cell mass; prevented the reduction of plasma adiponectin and restored leptin sensibility. Furthermore, enalapril treatment improved the protein expression of the pancreatic islet Pdx1, GLUT2, ACE2 and Mas receptors. Losartan treatment showed the greatest AT2R expression in the pancreas. In the liver, the enalapril administration improved hepatic steatosis, triglycerides and prevented the increase hepatic protein levels of PEPCK, G6Pase and GLUT2. Additionally, enalapril improved the deleterious effects on the HF diet by upregulating the signal transduction through the IRS-1/Akt pathway, as well as downregulatin the protein levels and mRNA expression of PPAR-gamma, SREBP-1c and FAS. Our findings indicate that ACE inhibition with enalapril attenuated several of the deleterious effects of the HF diet. In summary, enalapril appears to be responsible for the normalization of islet morphology and function, a protective role against hepatic insulin resistance and lipid accumulation in the liver. These protective effects of enalapril were attributed, primarily, to the reduction in body mass gain and food intake, increasing EE, the enhancement of the ACE2/Ang (1-7)/Mas receptor axis and adiponectin levels, enhancing hepatic insulin action, leptin and gluconeogenesis, and attenuating NAFLD.

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Baena, Muñoz Miguel. « Mecanismos moleculares implicados en las alteraciones metabólicas inducidas por la suplementación subcrónica de fructosa líquida : estrés de retículo, autofagia y resistencia a la insulina ». Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/300894.

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En las últimas décadas se ha observado un aumento muy notable de la prevalencia de enfermedades metabólicas como la obesidad, el síndrome metabólico y la resistencia a la insulina. Por ejemplo, en Estados Unidos, casi tres cuartas partes de la población adulta presentan sobrepeso u obesidad. De éstos, casi una tercera parte padecen el síndrome metabólico. En España, un estudio de la Sociedad Española para el Estudio de la Obesidad (SEEDO) realizado en el año 2000, muestra una prevalencia de la obesidad del 14.5% entre individuos de 25 a 60 años (Aranceta y col., 2003). Por otro lado, la prevalencia mundial estimada para la diabetes en la población adulta era de un 6.4% en el año 2010 y se prevé que este porcentaje aumente hasta un 7.7% en el año 2030, constituyendo la diabetes mellitus de tipo 2 el 90% de los casos (Nolan y col., 2011). Los malos hábitos alimenticios, así como un estilo de vida sedentario, son factores que promueven la aparición de todas estas alteraciones (Johnson y col., 2007). En los últimos años se ha observado un incremento por parte de la población en la ingesta calórica total, a la vez que se ha producido un cambio en la proporción en la que algunos nutrientes forman parte de la dieta humana. En este aspecto, cabe destacar el aumento en el consumo de fructosa debido, principalmente, a su utilización como edulcorante en bebidas refrescantes (Elliot y col., 2002; Lim y col., 2010). De hecho, son muchos los estudios que relacionan este aumento en el consumo de fructosa con la aparición de enfermedades metabólicas (Gross y col., 2004; Welsh y col., 2010). Debido a que la administración de fructosa en ratas produce unos desequilibrios metabólicos similares a los que sufren los humanos con síndrome metabólico, se ha aceptado a la rata alimentada con fructosa como un buen modelo para el estudio de este síndrome (Nagai y col., 2002). Mediante este modelo experimental, nuestro grupo de investigación demostró cómo la administración de fructosa al 10% (peso/volumen, p/v) en el agua de bebida en ratas Sprague-Dawley durante 14 días provocaba una serie de alteraciones metabólicas, entre las que se incluyen esteatosis hepática, hipertrigliceridemia, hiperleptinemia, resistencia a la insulina e intolerancia a la glucosa (Roglans y col., 2007; Vilà y col., 2008; Rebollo y col., 2014b). En el presente trabajo se ha profundizado en el estudio de estas alteraciones metabólicas inducidas por una ingesta de fructosa, así como los mecanismos moleculares implicados. Se ha valorado, además, el efecto que una suplementación de fructosa puede producir sobre otras vías de interés, como son el estrés de retículo endoplasmático, la inflamación o la autofagia hepática. Mediante un estudio comparativo entre una ingesta de fructosa y una de glucosa, se ha analizado el efecto exclusivo que presenta el consumo de fructosa sobre la aparición de dichas alteraciones metabólicas.
In recent decades the prevalence of metabolic diseases such as obesity, metabolic syndrome and type 2 diabetes mellitus has notably increased. Bad eating habits and sedentary lifestyle are factors that promote the occurrence of these disorders (Johnson et al., 2007). During the lasts years total caloric intake has increased, and also the rate at which certain nutrients are part of the human diet has changed. In this sense, the increase in fructose consumption has an important role, mainly due to its use as a sweetener in soft drinks (Elliot et al., 2002; Lim et al, 2010). In fact, many studies link the increase in fructose consumption with the onset of metabolic disorders (Gross et al., 2004. Welsh et al, 2010). Since fructose administration in rats produces metabolic imbalances similar to those suffering in humans with metabolic syndrome, it has been accepted that rat fed with fructose as a good model for the study of this syndrome (Nagai et al., 2002). Using this experimental model, our research group demonstrated previously how the administration of fructose at 10% (w/v) in drinking water in Sprague-Dawley rats for 14 days caused several metabolic disorders, including hepatic steatosis, hypertriglyceridemia, elevated plasmatic leptin levels, insulin resistance and glucose intolerance (Roglans et al., 2007; Vilà et al, 2008;. Rebollo et al, 2014b). In this work we studied the metabolic alterations induced by fructose intake during 56 days in female Sprague-Dawley rats, as well as the molecular mechanisms involved. Also we assessed the effect of fructose supplementation on other pathways of interest, such as endoplasmic reticulum stress, inflammation or liver autophagy. Through a comparative study between fructose and glucose intake, we have analyzed the specific effect of the consumption of fructose on the occurrence of these metabolic alterations.

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Salvadó, Serra Laia. « Estrès del reticle endoplasmàtic, inflamació i resistència a la insulina en cèl•lules musculars esquelètiques ». Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/283719.

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L’objectiu d’aquesta tesi doctoral ha estat elucidar nous mecanismes moleculars que expliquessin els efectes antiinflamatoris i antidiabètics de l’oleat. A més, també s’ha volgut aprofundir en l’estudi dels mecanismes moleculars mitjançant els quals l’activació del PPARß/d evita processos inflamatoris i de resistència a la insulina. Així doncs, el primer objectiu d’aquesta tesi doctoral ha estat estudiar si l’oleat és capaç d’exercir els seus efectes antiinflamatoris i antidiabètics mitjançant mecanismes que modulin l’estrès del RE en cèl•lules musculars esquelètiques. A més, tenint en compte que l’AMPK inhibeix l’estrès del RE, en aquest estudi es va voler elucidar si l’oleat, mitjançant l’activació de l’AMPK, evitava l’estrès del RE responsable de l’aparició de la inflamació i RI induïda pel palmitat. Els resultats obtinguts mostren que l’oleat evita l’estrès del RE induït pel palmitat en miotubs murins C2C12 i humans LHCN-M2. Els efectes de l’oleat sobre l’estrès del RE, així com els seus efectes antiinflamatoris i antidiabètics semblen involucrar la cinasa AMPK, atès que la inhibició farmacològica i molecular d’aquesta proteïna, va revertir els efectes de l’oleat sobre els nivells d’expressió gènica dels marcadors d’estrès del RE, d’inflamació i de RI. En resum, les dades d’aquest estudi indiquen que l’oleat evita l’estrès del RE, la inflamació i la RI produïda pel palmitat en cèl•lules musculars esquelètiques, mitjançant mecanismes que involucren l’activació de l’AMPK. El segon objectiu d’aquesta tesi doctoral ha estat determinar si l’activació de PPARß/d és capaç d’evitar la inflamació i la RI en múscul esquelètic mitjançant mecanismes que involucren l’estrès del RE, així com elucidar si la cinasa AMPK està involucrada en aquest procés. Els resultats obtinguts evidencien que l’activació farmacològica de PPARß/d redueix l’estrès del RE induït per lípids en cèl•lules musculars esquelètiques. Tanmateix, l’activació farmacològica de PPARß/d també va evitar l’estrès del RE, la inflamació i la RI induïda per coneguts activadors d’estrès del RE en cèl•lules musculars esquelètiques mitjançant mecanismes que involucraven l’AMPK. Com a resum, podem concloure que l’activació de PPARß/d evita la inflamació i la RI induïdes per l’estrès del RE en cèl•lules musculars esquelètiques mitjançant mecanismes que involucren l’activació de l’AMPK. Per tant, els resultats obtinguts en aquesta tesi doctoral destaquen el paper de l’AMPK com a proteïna clau a l’hora d’inhibir la inflamació i la RI secundària a l’estrès del RE. Així, podem proposar que el seguiment d’una dieta mediterrània, la qual conté l’oli d’oliva com a greix essencial, juntament amb la pràctica d’exercici físic, podria ser una bona estratègia per a la prevenció de la DM2. De la mateixa manera que l’oleat, l’activació farmacològica de PPARß/d inhibeix l’estrès del RE responsable de la inflamació i la RI en múscul esquelètic mitjançant l’activació de l’AMPK. Així doncs, els resultats d’aquesta tesi doctoral aporten nous mecanismes antidiabètics de l’oleat i dels activadors de PPARß/d que podrien ser bones estratègies futures per a la prevenció de la RI i de la DM2.
Although it is known that oleate has many beneficial effects for the prevention of type 2 diabetes mellitus, the mechanisms by which oleate improves insulin resistance are not completely known. The first objective of this thesis was to examine whether oleate could prevent palmitate-induced endoplasmic reticulum (ER) stress, which is involved in the link between lipid-induced inflammation and insulin resistance. The studies were conducted in mouse C2C12 and human LHCN-M2 myotubes. Palmitate increased the levels of ER stress markers, whereas oleate did not. In palmitate-exposed cells incubated with a lower concentration of oleate, the effects of palmitate were prevented. Moreover, the ability of oleate to prevent palmitate-induced ER stress, inflammation and insulin resistance was reversed in the presence of the AMP-activated protein kinase (AMPK) inhibitor, compound C, or by overexpression of a dominant negative AMPK construct. Overall, these findings indicate that oleate prevents ER stress, inflammation and insulin resistance in palmitate-exposed skeletal muscle cells by activating AMPK. The second objective of this thesis was to examine whether peroxisome proliferator-activated receptor (PPAR)ß/d could prevent ER stress-associated inflammation and insulin resistance in skeletal muscle cells. Studies were conducted in mouse C2C12 and human LHCN-M2 myotubes, and in skeletal muscle from wild-type and PPARß/d-deficient mice and mice exposed to a high-fat diet. The PPARß/d agonist GW501516 prevented lipid-induced ER stress in mouse and human myotubes and in skeletal muscle of mice fed a high-fat diet. PPARß/d activation also prevented thapsigargin- and tunicamycin-induced ER stress in human and murine skeletal muscle cells. In agreement with this, PPARß/d activation prevented ER stress-associated inflammation and insulin resistance, and glucose-intolerant PPARß/d-deficient mice showed increased phosphorylated levels of inositol-requiring 1 transmembrane kinase/endonuclease-1a in skeletal muscle. Our findings demonstrate that PPARß/d activation prevents ER stress through the activation of AMPK, and the subsequent inhibition of extracellular-signal-regulated kinase (ERK)1/2 due to the inhibitory crosstalk between AMPK and ERK1/2, since overexpression of a dominant negative AMPK construct (K45R) reversed the effects attained by PPARß/d activation. These findings indicate that PPARß/d prevents ER stress, inflammation and insulin resistance in skeletal muscle cells by activating AMPK.

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Ruzzolini, Jessica. « Un aiuto dalla natura nella lotta contro i tumori : proprietà antineoplastiche di prodotti derivanti da piante di olivo ». Doctoral thesis, 2021. http://hdl.handle.net/2158/1239183.

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Valutazione dell'attività antineoplastica di prodotti derivanti da piante di olivo su vari tipi di tumore. Studio del trattamento combinato tra agenti naturali e chemioterapici. Evaluation of antineoplastic activity of olive derivatives towards several types of cancers. Study of combinatory treatment based on the use of natural compounds in association with chemotherapy

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FLORIDDIA, ELISA. « Progettazione, sintesi e valutazione farmacologica preliminare di modulatori delle proteine di trasporto coinvolte nella multidrug resistance (MDR) ». Doctoral thesis, 2016. http://hdl.handle.net/2158/1029613.

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Questa tesi di Dottorato si sviluppa nell’ambito della Multidrug Resistance, ovvero la resistenza multifarmaco, molto frequente in ambito oncologico. Infatti, una delle cause principali del fallimento della chemioterapia antitumorale è lo sviluppo da parte delle cellule tumorali, durante la terapia stessa, di resistenza verso uno o più farmaci caratterizzati da struttura chimica e meccanismo di azione molto differenti tra loro. Questo fenomeno è chiamato Multidrug Resistance ed è dovuto ad una sovraespressione di proteine di trasporto transmembrana appartenenti alla famiglia di trasportatori ABC. Queste proteine funzionano da pompe di estrusione utilizzando l’energia fornita dall’idrolisi dell’ATP per espellere dalla cellula tumorale contro gradiente di concentrazione il chemioterapico che così non riuscirà più a mantenere o raggiungere la concentrazione terapeutica efficace. La Pgp è la proteina di trasporto più studiata. E’ una glicoproteina di membrana ed è normalmente espressa nelle cellule sane dove ha una funzione fisiologica protettiva, essendo deputata all’espulsione di molecole lipofile e xenobiotici tossici. Sfortunatamente è sovraespressa nelle cellule tumorali in seguito ad una alterazione dell’espressione del gene MDR1, e ciò sfocia nella MDR. Il sito di legame di questa proteina è molto largo e contiene amminoacidi in grado di stabilire col substrato sia legami elettrostatici che idrofobici. E’ capace quindi di ospitare simultaneamente anche più ligandi con strutture molto diverse. La Pgp può essere quindi considerata come un possibile target terapeutico per superare la MDR. Il concetto è quello di cosomministrare il farmaco antitumorale con un modulatore della pompa (chiamato chemiosensibilizzante) che ne blocchi l’attività, in modo da ripristinare all’interno della cellula tumorale la giusta concentrazione di farmaco. Negli ultimi anni sono stati individuati diversi modulatori della Pgp ed alcuni di essi hanno raggiunto le prove cliniche anche se ancora nessun composto è stato approvato in terapia. E’ molto difficile stabilire relazioni struttura-attività per modulatori della Pgp perchè, così come i substrati, anche essi presentano le strutture più svariate. Comunque sono stati individuati dei requisiti strutturali importanti per conferire alle molecole buona attività anti-MDR, ovvero: elevata lipofilia, la presenza di gruppi accettori di legami ad idrogeno, gruppi aromatici e uno o più atomi di azoto protonabili a pH fisiologico. Inoltre, confrontando la struttura del Verapamile, primo composto riconosciuto attivo contro l’MDR, e della Pervilleina A, alcaloide tropanico con marcata attività anti-MDR appare evidente che queste molecole hanno una struttura comune in cui un atomo di azoto basico è legato, tramite linker, a due gruppi idrofobici aromatici. Sulla base di tutte queste considerazioni è da diversi anni che il gruppo di ricerca dove ho svolto il mio progetto di dottorato è coinvolto nella progettazione e sintesi di modulatori della Pgp con lo scopo di ottenere un ligande potente e selettivo, in grado di contrastare la MDR. Sono state quindi progettate diverse serie di molecole mantenendo tale motivo strutturale, ma variando la natura dei linker e dei residui aromatici. Nello specifico nel mio progetto di dottorato mi sono occupata della sintesi di molecole appartenenti • serie contenenti due funzioni esteree nelle quali variano la lunghezza e la flessibilità del linker utilizzato, oltre alla natura dei gruppi aromatici; • serie prive di residui esterei, ovvero analoghi ammidici isosterici e derivati alchilpiperazinici. L’attività anti MDR dei composti sintetizzati viene valutata tramite un test preliminare basato su un metodo spettrofluorimetrico, che misura l’uptake nucleare del chemioterapico fluorescente Pirarubicina (cosomministrata con varie concentrazioni della molecola in esame) in una linea cellulare eritroleucemica antraciclina-resistente che esprime solo la Pgp. Si ottiengono così una misura della potenza e dell’efficacia del composto. Come ulteriore approfondimento abbiamo ritenuto interessante valutare la stabilità in vitro di un set di molecole (data la presenza di due gruppi esterei nella struttura) in condizioni fisiologiche, cioè in PBS, e all’azione degli enzimi idrolitici del plasma. Inoltre la Pgp non è l’unica pompa di efflusso coinvolta nella resistenza crociata ai farmaci antitumorali. Hanno un ruolo molto importante anche altre due pompe appartenenti alla stessa famiglia di trasportatori ABC, ovvero MRP1 e BCRP. Quindi abbiamo ritenuto interessante valutare la selettività rispetto alle tre pompe coinvolte nella MDR. I risultati ottenuti indicano che i modulatori della Pgp sintetizzati nel corso degli anni a seguito di successive ottimizzazioni molecolari sono dotati di elevata potenza e efficacia, confermando indubbiamente che la presenza di un atomo di azoto basico collegato a due residui aromatici con linker di diversa natura ma di opportuna lunghezza sono caratteristiche importanti per avere inibizione della Pgp. Inoltre abbiamo trovato stereoselettività ed enantioselettività nell’interazione con la Pgp, caratteristica piuttosto rara in questo tipo di interazione.

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MAUGERI, Alessandro. « Anti-cancer activity of Citrus bergamia essential oil and its constituents in in vitro experimental models of paediatric neoplasms ». Doctoral thesis, 2021. http://hdl.handle.net/11570/3209268.

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Cancer represents one of the leading causes of death worldwide. In paediatric subjects, leukaemia and brain tumours showed the highest incidence in 2020. The plant kingdom has always been a great source for bioactive compounds for treating several illnesses, including cancer, and Citrus fruits stand out among the others. For my PhD project, I focused on the anti-proliferative activity of the essential oil (BEO) of Citrus bergamia (bergamot) Risso & Poiteau and its constituents in in vitro experimental models of paediatric neoplasms. The first step of the project was performing the quali-quantitative characterization of BEO and its furocoumarin-free fraction (BEO-FF). The chemical profile of these plant matrices showed that they are composed of a volatile part (< 95%) rich in monoterpenes, and a non-volatile one, containing coumarins, results in line with those present in literature. The second step of my PhD project originated from a previous assumption by which bergamottin (BRG) and 5-geranyloxy-7-methoxycoumarin (5-G-7-MOC), two coumarins found in BEO, have been claimed to be relevant in the anti-proliferative effects exerted by BEO in the SH-SY5Y human neuroblastoma cells. Therefore, this step was designed to verify this and to assess the mechanisms underlying the anti-proliferative effect of both compounds. The results of this step demonstrate that BRG and 5-G-7-MOC are able to reduce the proliferation of SH-SY5Y cells, inducing apoptosis and increasing sub-G0/G1 phase cell population. Furthermore, the pro-oxidant activity of the two coumarins was demonstrated, which increased reactive oxygen species and consequently reduced mitochondrial membrane potential. Moreover, BRG and 5-G-7-MOC were able to modulate apoptosis-related factors both at protein and gene level. As a final point, we evaluated the synergistic effect of their combination, finding that the highest synergy was observed at a concentration ratio similar to observed in the whole BEO, supporting our initial hypothesis. These results deepen the knowledge on the effect of BRG and 5-G-7-MOC in SH-SY5Y cells, stressing the relevance of their cooperation in achieving this effect. Acknowledged the clinical relevance of multi-drug resistance (MDR) in cancer treatment, the last step of my PhD project was to evaluate the effectiveness of BEO and BEO-FF in acute leukemic lymphoblasts and in their doxorubicin-resistant counterpart (i.e., CCRF-CEM and CEM/ADR5000, respectively). Both fractions showed similar antiproliferative activity in both cell lines, as showed by cell viability assays. Flow cytometric analyses indicated that this effect was achieved by inducing apoptosis, although with different extent for BEO and BEO-FF, and confirmed by the increase of population in sub-G0/G1 phase, meaning hypodiploidy. To assess their role against MDR, likewise for coumarins present in BEO, both bergamot fractions were combined with doxorubicin at different ratios. If in CCRF-CEM cells neither BEO nor BEO-FF elicited any relevant effect, in CEM/ADR5000 cells, the whole BEO brought a strong synergistic effect, restoring the sensitivity of resistant cells to doxorubicin. Therefore, these results corroborate the hypothesis that a phytocomplex, like BEO, can be an appropriate weapon to fight MDR exploiting their collateral sensitivity, a phenomenon by which cells resistant to a specific antineoplastic agent may be particularly sensitive to another compound or a multitude of them.

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DI, GIACOMO SILVIA. « Natural and naturally-derived compounds as new chemopreventive agents ». Doctoral thesis, 2014. http://hdl.handle.net/11573/538397.

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Chemoprevention is an approach based on the use of natural or synthetic compounds to inhibit, suppress or reverse the development and progression of cancer. In order to overcome the cancer disease, the identification of chemopreventive compounds is of particular interest. Among them, antimutagens prevent the mutagen-induced DNA-injury or promote the repair and/or the reversion of damage. In addition to antimutagenicity, some agents also act as chemosensitizers, by increasing the effectiveness of cancer chemotherapy and radiotherapy, when used in combination with chemotherapeutical agents. This approach is very interesting to prevent the development of multidrug resistance (MDR), which makes cancer cells not-sensitive to a broad range of drugs. In this context, present study was aimed at evaluating the potential chemopreventive properties of some natural and naturally-derived compounds, particularly the sesquiterpenes β-caryophyllene (CRY) and β-caryophyllene oxide (CRYO), and the aldehyde α-hexylcinnamal (HCA). The antimutagenic activity was evaluated by the reverse bacterial mutation assay (Ames test), on different strains of Salmonella typhimurium and Escherichia coli, both in absence and presence of the S9-metabolic activation system. As mutagens, 2- nitrofluorene (2NF), sodium azide (SA), methyl methanesulfonate (MMS), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP), 4-nitroquinoline N-oxide (4NQO), 1-nitropyrene (1NP), 1,8-dinitropyrene (1,8-DNP) and a sample of condensed smoke (CSC) from standard 3R4F cigarette were used. In addition to antimutagenicity studies, the potential chemosensitizing properties of CRY, CRYO and HCA and their ability to interfere with ABC-transporter function were evaluated, in Caco-2, CEM/ADR5000 and CCRF/CEM human cancer cells. For each compound, low concentrations (IC10 and IC20) were assayed in order to verify their potential additive, synergistic or antagonistic effects with the anticancer doxorubicin. The nature and the extent of the interaction were evaluated by the combination index (CI) and the isobologram analysis, respectively; conversely, the potential enhancement of drug effectiveness was quantified by cytotoxicity enhancement ratio (RR). The interaction between test compounds and ABC-transporters was studied by the rhodamine 123 assay. HCA exhibited an antimutagenic activity against different nitro-compounds and in various experimental protocols, suggesting the involvement of both desmutagenic and bioantimutagenic mechanisms. The sesquiterpenes CRY and CRYO resulted able to inhibit the mutagenicity of CSC, although with different potency and specificity: CRYO was the most potent compound, acting at concentrations about ten-times lower than CRY. The antimutagenicity was highlighted in different strains and in all experimental protocols, suggesting the overlapping of various protective mechanisms; the inhibition of CSC-induced oxidative stress seems to be likely and deserves further investigations. In human cancer cells, the substances produced cytotoxic effects at high concentrations both in resistant and in sensitive cell lines: HCA was the most effective substance, especially in the sensitive CCRF-CEM cells. All the compounds synergistically acted with doxorubicin, although HCA was the most potent: IC20 HCA increased the doxorubicin cytotoxicity of about six, seven and fourthy-seven folds, in Caco-2, CEM/ADR5000, and CCRF-CEM, respectively. In addition, a remarkable inhibition of ABCtrasporter was produced by HCA in the cancer cells tested: the effect was higher than that of the standard inhibitior verapamil. Also CRY and CRYO inhibited the ABC transporters but with lower potency than verapamil. The antimutagenic and chemosensitizing properties of β-caryophyllene, β-caryophyllene oxide and the α-hexylcinnamaldehyde deserves attention and represent a starting point to better evaluate their potential applications in the field of chemoprevention.

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MOLONIA, MARIA SOFIA. « Protective effects of cyanidin-3-O-glucoside against lipotoxicity in hypertrophic adipocytes ». Doctoral thesis, 2018. http://hdl.handle.net/11570/3130792.

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Obesity is a metabolic disorder of multifactorial origin correlated with an elevated morbidity and mortality rates. It predisposes to the metabolic syndrome and is characterized by excess adipose tissue, altered levels of circulating proinflammatory adipokines, imbalances of the adaptive immune system and local and systemic chronic inflammation. In particular, obesity is associated with a state of "chronic low-grade inflammation", which plays an important pathophysiological role in the development and progression of many chronic pathologies, such as insulin resistance, endothelial dysfunction and dyslipidemia. Furthermore lipotoxicity, common in the adipose tissue, contributes to exacerbate the problems associated with these pathological conditions. FFA, including palmitic acid (PA), are in fact considered among the main causes of the onset of inflammation and insulin resistance in the adipose tissue. In recent years, epidemiological evidences have shown that anthocyanins, natural phenols commonly present in food and vegetables from Mediterranean Diet possesses not only a high antioxidant and anti-inflammatory activity, but also a marked anti-obesity and insulin sensitizing effect. Therefore, the aim of this work was to evaluate the potential beneficial effects of cyanidin-3-O-glucoside (C3G) in counteracting the inflammatory condition and the insulin resistance induced by high concentrations of PA at the adipose tissue level, through the use of an in vitro experimental models on murine (3T3-L1) and human (SGBS) adipocytes. In all experiments fully differentiated 3T3-L1 and SGBS adipocytes were pretreated with different concentrations of C3G for 24 h and then exposed to high concentrations of PA for further 24 h in order to induce cellular hypertrophy. To evaluate the insulin resistance condition, cells were subsequently treated with insulin. In particular, to characterize the effect of PA on the inflammatory process and the insulin resistance at molecular level and to demonstrate the protective effect of C3G in such conditions, for 3T3-L1 cells, we evaluated cellular signal pathways involved in adipogenesis (PPAR-γ pathway), inflammatory process (NF-kB pathway) and insulin resistance (IRS-1/PI3K/Akt pathway). Instead, in order to confirm the effects on human SGBS cells we assessed the mRNA levels of the main cytokines modulated by NF-kB (TNF-α, IL-6, IL-8, and MCP-1), and GLUT-1, GLUT-4, hexokinase and adiponectin as markers of insulin sensitivity. Data reported in this thesis demonstrate that C3G ameliorates inflammation and insulin resistance conditions induced by PA, thus suggesting new potential roles for this natural compound in the prevention and treatment of pathological conditions linked to obesity.

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Castanheira, Bruno Alexandre Martins Guerreiro. « Mecanismos de resistência a antibióticos ». Master's thesis, 2013. http://hdl.handle.net/10437/4632.

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Orientador: Maria João Simões
Os antibióticos são usados com o intuíto de prevenir ou tratar uma infeção bacteriana. No entanto, o seu uso abusivo favorece a seleção de estirpes resistentes, contribuindo para o aumento das populações resistentes. Os mecanismos de resistência bacterianos são geralmente adquiridos, por transferência de genes entre estirpes bacterianas promovida por plasmídeos, fagos ou elementos transponíveis. Também podem ocorrer por mutação do ADN, no entanto este mecanismo genético é pouco representativo. A aquisição de novos mecanismos de resistência diminui ou inibe a acção do antibiótico. Por outro lado a bactéria também apresenta uma resistência intrínseca a algumas bacterias ou seja uma resistência natural, onde os genes de resistência existem no genoma da estirpe selvagem e são transmitidos às gerações seguintes no código genético. São exemplos destas resistências a alteração da permeabilidade na membrana da bactéria, as bombas de efluxo, a produção enzimática e a alteração do local de acção na bactéria. A resistência das bactérias aos antibioticos é um problema mundial sério, que coloca em risco a saúde pública. Organizações internacionais como a OMS têm criado nos últimos anos guidelines com o objectivo de diminuir as resistências das bactérias aos antibióticos. É fundamental uma participação activa de todos os profissionais de saúde no sentido de melhorar a prescrição e dispensa de antibióticos.

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Thèses sur le sujet « Farmacologic resistance »

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