Dissertations / Theses on the topic 'In vitro biological evaluation'

The first part of this thesis deals with the evaluation of in vitro and in vivo pharmacokinetics of novel broad-spectrum antiviral compounds active against enveloped viruses. The design and synthesis of new series of antiviral compounds with 1H-pyrrol-methylene thioxodihydropyrimidine structure, has been realized by Professor Maurizio Botta's research group, at the University of Siena. Compounds’ antiviral activity has been evaluated on several enveloped viruses, such as ZIKAV, DENV-2 and five influenza strains including the pandemic strain H7N9. The selectivity against enveloped viruses, time of addition and binding experiments confirmed their ability to intercalate in the viral envelope membrane, oxidize phospholipids and alter the fluidity of the lipid bilayer, compromising the efficacy of the virus-cell fusion step and preventing viral entry. With the aim of investigating the in vitro ADME properties, the most active compounds were selected to assay their chemical-physical properties and early select the most promising lead candidate. Thus, membrane permeability, binding to human serum albumin, and stability in human plasma and microsomes have been assayed. Finally, the lead candidate was selected to evaluate preliminary in vivo pharmacokinetic parameters; after formulation studies, the compound was administrated intravenously (iv) at the dose of 25 mg/kg and 12.5 mg/kg. The second chapter of this Ph.D. thesis concerns the investigation of the in vitro biological profile of nitric oxide-donor largazole prodrugs. Two hybrid analogues of largazole, as dual HDAC inhibitor and nitric oxide (NO) donors potentially useful as anticancer agents, have been designed and synthesized thanks to the collaboration between Professor Maurizio Botta’s research group and IRBM. Largazole is a natural product identified as the most potent and selective Class-I deacetylase (HDAC) inhibitor, that showed a broad-spectrum growth-inhibitory activity against epithelial and fibroblastic tumor cell lines and a low cytotoxicity profile. Over the last decades, dual nitric oxide (NO) donors/HDAC inhibitors have been developed as novel anticancer chemical entities, potentially more efficacious than selective HDAC inhibitors, owing to the capability of NO to specifically modulate the function of some HDAC isoforms and to overcome tumor cell resistance to conventional treatments. Thus, after the synthesis, the characterization of derivatives compounds and the in vitro NO release assay performed by Professor Maria Frosini using the Griess method, biological evaluation of their antiproliferative activities against U-2OS, Caco-2, and IMR-32 cell lines have been conducted. To further explain the additive antiproliferative effect of NO-donor compounds vs largazole, their stabilities both in human plasma and in cell culture medium were assessed. The third and last chapter of this Ph.D. thesis deals with the project I participated in during my exchange period at the research group of Professor Per Artursson, who hosted me for 5 months, at Uppsala University. In collaboration with AstraZeneca, a series of antisense oligonucleotide (ASO) conjugates, targeting MALAT1 chosen as a model target, were used to assess and validate their silencing efficiency and enhance/overcome endosomal escape. The MALAT1 silencing efficiencies of lipophilic ASO-conjugates and a peptide-ASO have been determined in the presence and absence of a cyclic cell permeation peptide (CPP) in two human embryonic kidney (HEK293) cell lines of which one overexpressing the target G-protein coupled receptor selected for the study. For this purpose, the expression levels of the MALAT1 gene mRNA were measured using qPCR in a time (0-24-48 hrs) and concentration (0.5-2.5-5 µM of ASOs and 5-10-15 µM of CPP)-dependent manner.

Today we can easily find the most diverse kind of information about a particular medicinal plant, but without scientific basis, making their use a potential risk to health. Overall, the findings on the safety and efficacy are based on precarious evaluations and popular use. There is a need for qualified professionals to access, critical analyze and assign such information in a way that it could be easily understood, not only by health professionals, but also by the users of these products. One example of these plants used in popular medicine, but without scientific evidence, is Euphorbia tirucalli L., popularly known as Dog-Stick, Pencil tree, or more commonly as Aveloz. This plant has been used for the treatment of many diseases, such as microbial diseases, immunossupression problems, and even in the cancer treatment. However, some works reveal precisely the opposite, namely that the latex of Euphorbia tirucalli can cause immunosuppression, and often is associated to the appearance of Burkitt's Lymphoma, a type of cancer. Lupeol was isolated and identificated from hexane fraction by GC-MS for the first time for the plant, among other 3 hydrocarbons, 7 long chain fat acids, 2 steroids, and 3 compounds of the vegetal metabolism. A preliminary phytochemistry screening allowed to the visualization of the principal groups in the plant. Polyphenols and condensed tannins contents were determined in the crude extract and fractions. Ethyl ether and ethyl acetate fractions showed the greatest antioxidant activity in the DPPH test. Antimicrobial activity was observed mainly against Candida albicans, Candida glabrata and Saccharomyces cereviseae, as well as for the opportunist algae Prototheca zopfii. A preliminary study of toxicity using Artemia saline and acute oral toxicity in mice, indicate the plant as low toxicity. The latex of the E. tirucalli, even in small doses (1%) can cause in vitro inhibition over the human Acetylcholinesterase enzyme. A prominent in vitro inhibitory activity over human platelets aggregation was also observed. The effects of the extract of the plant over the blood cells in a culture medium using ex-vivo blood samples of male Wistar rats were evaluated. The results demonstrated that the extract caused thrombocytopenia, leucopenia and lymphopenia.
Atualmente podemos encontrar facilmente as mais diversas informações sobre uma determinada planta medicinal, mas que carecem de fundamento científico, tornando assim seu uso um potencial risco a saúde. Em geral, as conclusões sobre segurança e eficácia são baseadas em avaliações precárias do uso popular. Portanto, há necessidade de que profissionais qualificados possam, além de acessar tais informações, analisá-las criticamente para disponibilizá-las de forma que sejam facilmente compreendidas, não só por profissionais da saúde, mas também pelos usuários destes produtos. Um exemplo destas plantas utilizadas na medicina popular, mas sem comprovação científica, é a Euphorbia tirucalli L., conhecida popularmente como Graveto-do-cão, Árvore Lápis, ou mais comumente como Aveloz. Esta planta tem sido utilizada para o tratamento de inúmeras enfermidades, como afecções microbianas, problemas de imunossupressão, cicatrização de berrugas e até mesmo no tratamento do câncer. Entretanto, alguns trabalhos revelam justamente o contrário, ou seja, que o látex da Euphorbia tirucalli pode causar imunossupressão, e freqüentemente encontra-se associado ao aparecimento do Linfoma de Burkitt, que é um tipo de câncer. Foram isolados e identificados por cromatografia gasosa acoplada a espectrometria de massas, 03 hidrocarbonetos, 07 ácidos graxos de cadeia longa, 02 esteróides, 03 compostos do metabolismo vegetal e 01 triterpeno, não relatado até o momento, o qual foi isolado da fração hexânica e identificado como sendo o lupeol. Foi realizada uma análise fitoquímica preliminar, o que permitiu a visualização dos grupos de compostos presentes na planta. A quantidade de polifenóis e taninos condensados foi determinada na planta e suas frações. Realizou-se o teste de atividade antioxidante e com ele verificamos uma excelente atividade das frações éter etílico e acetato de etila. Analisou-se a atividade antimicrobiana da planta e obtivemos resultados excelentes para os fungos Candida albicans, Candida glabrata e Saccharomyces cereviseae, bem como para a alga oportunista Prototheca zopfii. Realizou-se um estudo de toxicidade sobre a Artemia salina e estudo de toxicidade oral aguda. Os resultados apontam a espécie como sendo não tóxica. O látex da E. tirucalli, mesmo em doses pequenas (1%) pode causar inibição (in vitro) a enzima Acetilcolinesterase Humana. Uma acentuada atividade inibitória sobre a agregação plaquetária foi observada. O extrato da planta sobre cultura de células sanguíneas de ratos Wistar (exvivo) causou diminuição do número de leucocitos, linfócito e plaquetas.

Infectious diseases are responsible for an overwhelming number of deaths and morbidity worldwide. In tropical regions of the world, in particular, developing countries like Zambia, poor health is prevalent and diseases such as malaria, meningitis, pneumonia, tuberculosis and gastrointestinal infections strongly persist. Folkloric medicines are still actively used against some of these infections as primary care before seeking conventional treatment at hospitals. Members of the genus Ficus (Moraceae) are traditionally used in Zambia against many diseases caused by bacterial, fungal and protozoal infections. Thus according to the plant parts used traditionally for herbal preparations, aerial and root parts of eight Ficus species namely; F. ingens, F. lutea, F. natalensis, F. ovata, F. sansibarica subsp. macrosperma, F. sycomorus subsp. gnaphalocarpa, F. sycomorus subsp. sycomorus and F. wakefieldii were collected from different parts of Zambia. The main aim of this thesis was to evaluate the medicinal potential of members of the genus Ficus. This was achieved by three objectives, which involved the phytochemical profiling of the crude extracts and subextracts of the Ficus for their constituents using chromatographic methods such as thin layer chromatography (TLC), proton Nuclear Magnetic Resonance (1H NMR) and high performance liquid chromatography (HPLC). Secondly, the extracts were screened for various biological activities after which they were evaluated against recombinant FAS-II elongation enzymes, FabG, FabI and FabZ as potential targets in liver stage malaria parasites. In this case, finely ground dried plant material was extracted with methanol (MeOH) to yield the crude methanol extracts (CR-MeOH) which, were further partitioned to provide a coarse separation of the crude extracts according to polarity. The three subextracts obtained included n-hexane, chloroform (CHCl3) and aqueous methanol (aq-MeOH). The obtained extracts and subextracts were screened for biological activities such as: antifungal and antibacterial activities using the broth dilution and agar disc diffusion assays, antitubercular activity using the MTT assay, antischistosomal activity using the microscopic in vitro assay. In addition, antiprotozoal activitites which included antileshmanial activity using an assay against amastigotes of L. donovani strain MHOM/ET/67/L82, trypanocidal activity against T. cruzi and T. brucei rhodesiense STIB 900 strain, and antiplasmodial activity by modified [3H]-hypoxanthine incorporation assay, using the chloroquine/pyrimethamine resistant K1 strain were performed. Cytotoxity activity was also performed using rat skeletal myoblasts L6-cells. The chemical profiling was done by TLC, NMR and RP-HPLC. Meanwhile the chemical compound isolation for F. sansibarica was attempted by different chromatographic techniques and characterization by spectroscopic methods. The phytochemical profiling revealed the presence of closely related polyphenolic compounds to which some of the biological activities were attributed to. For instance, the antibacterial and the FAS-II enzyme inhibition activities were mostly retained in the aq-MeOH subextracts, which were composed of very polar metabolites including flavonoids. Antiplasmodial activity was observed mostly in the less polar metabolites which were retained in the hexane and CHCl3 subextracts of the stem barks. This pattern was similar with antitrypanosomal and antileishmanial activities, though with lesser sensitivity. The same subextracts including those of the root barks showed the most activity against M. tuberculosis with MIC values of 256 and 128 μg/ml, and against Schistosoma, for both larval and adult worms. The extracts did not exert any antifungal activity by the agar disc diffusion method we used. Detailed phytochemical investigation of the leaves of F. sansibarica was performed, and led to the isolation of two compounds; epicatechin and apigenin-6-C-glucoside from the chloroform and aq. MeOH subextracts respectively. The predominant constituent of the CR-MeOH extract of F. sansibarica was identified as having a molecular weight of 432 g/mol by LC-MS analysis which could be set as an identification chemical marker for F. sansibarica. The results highlight the potential that Ficus species could have as a valuable source for potent compounds which can be identified as scaffolds for the development of novel liver stage antimalarial drugs. Our results support previous research on the antimicrobial activity of Ficus species and they also provide an in vitro scientific basis supporting the use of Ficus species in traditional herbal preparations against some bacterial and parasitic infections.

L’attività di ricerca svolta durante i tre anni di dottorato ha avuto come obiettivo la progettazione, la sintesi e l’ottimizzazione di nuovi potenziali agenti antitumorali dotati di attività antiproliferativa e antivascolare che hanno come target biologico i microtubuli, strutture dinamiche cellulari generate dalla polimerizzazione di eterodimeri di α,β tubulina. Il sistema di microtubuli è essenziale per la divisione cellulare, essendo coinvolto nella formazione del fuso mitotico, ed è importante per diversi processi cellulari quali la regolazione della motilità, segnalazione cellulare, secrezione e trasporto intracellulare. Tra le molecole di derivazione naturale, il cis-stilbene Combretastatina A-4 (CA-4), legando la tubulina a livello del sito di legame della colchicina, ne inibisce la polimerizzazione mostrando una potente attività antiproliferativa contro diverse linee cellulari tumorali. L’attività di ricerca relativa al primo anno di dottorato ha riguardato la sintesi di una nuova serie di composti a struttura 1-(3',4',5' trimetossibenzoil)-3-arilamino-5-amino-1,2,4-triazolica per i quali si è andati a valutare l’attività antiproliferativa in vitro, l’interazione con la tubulina e gli effetti prodotti sul ciclo cellulare. Per il derivato più attivo della serie, 1-(3,4,5-trimetossibenzoil)-3-(p-toluidino)-5-ammino-1,2,4-triazolo 3c è stata valutata l’attività antitumorale in vivo. I migliori risultati in termini di inibizione della proliferazione di linnee cellulari tumorali sono stati ottenuti con i derivati p-Me, m,p-diMe and p-Et fenil 3c, 3e e 3f, rispettivamente, i quali sono risultati essere equipotenti rispetto al composto di riferimento Combretastatina A-4 (CA-4). Proseguendo nel nostro lavoro di ricerca, nel corso del secondo anno di dottorato, una nuova serie di composti caratterizzati dalla presenza di un gruppo 2-metossi/etossicarbonile sono stati valutati per l’attività antiproliferativa su linee cellulari tumorali e per i composti più attivi della serie si è andati a valutare l’inibizione della polimerizzazione della tubulina, gli effetti sul ciclo cellulare e l’attività antitumorale in vivo. I migliori risultati in termini di attività antiproliferativa si sono ottenuti introducendo il sostituente metossilico in posizione C-6. Il composto più attivo della serie è risultato essere il derivato 2-metossicarbonil-3-(3’,4’,5’-trimetossianilino)-6-metossi-benzo[b]furano 3g, il quale ha prodotto una inibizione della proliferazione di linee cellulari tumorali a concentrazioni nanomolari (IC50’s, 0.3-27 nM), lega la tubulina a livello del sito di legame della colchicina, induce l’apoptosi e ha mostrato, sia in vitro che in vivo, una potente attività antivascolare su cellule endoteliali vascolari. Infine durante il terzo anno di dottorato è stata messa a punto la sintesi di una nuova serie di inibitori della polimerizzazione della tubulina a struttura 1- (3’,4’,5- trimetossiifenil) -2-aril-1H-imidazolica e progettati come cis-analoghi della combretastatina A-4, con l’obiettivo di valutare l’effetto sull’attività biologica prodotto dall’introduzione di diversi gruppi sostituenti a livello dell’anello fenilico in posizione C-2 dell’eterociclo imidazolico. L’introduzione di un atomo di cloro e di un gruppo etossilico nelle posizioni meta- e para-, rispettivamente, ha prodotto il composto più attivo della serie 1-(3’,4’ ,5’ -trimetossifenil)-2-(3’-Cl, 4’-OEt fenil)-1H-imidazolo 4o, con un valori di IC50 di 0.4-3.8 nM su un pannello di sette linee cellulari tumorali. Esperimenti condotti su un modello di topo singenico hanno dimostrato una potente attività antitumorale del composto 4o, il quale ha prodotto una significativa riduzione della massa tumorale a dosi trenta volte più basse rispetto a quelle richieste per la CA-4P usato come composto di riferimento.
During these three years of PhD our research work has been focused on the design, synthesis and optimization of novel potential anticancer agents with antivascular and antiproliferative activities which target microtubules, dynamic tubular proteins that are assembled from α tubulin/β tubulin (αβ-tubulin) heterodimers. The microtubule system is essential in a variety of fundamental cellular processes, including mitosis, formation and maintenance of cell shape, regulation of motility, cell signaling, secretion and intracellular transport. Among natural occurring compounds, Combretastatin A-4 (CA-4), a cis-stilbene isolated from the bark of the South African bush willow tree Combretum caffrum , is one of the most potent inhibitors of colchicine binding presently known. CA-4 has been shown to possess a powerful cytotoxic activity against a panel of tumor cell line, including multi-drug resistant cells. During the first year of PhD, a new class of compounds that incorporated the structural motif of the 1-(3’,4’,5’-trimethoxtbenzoyl)-3-arylamino-5-amino-1,2,4-triazole molecular skeleton was synthesized and evaluated for their in vitro antiproliferative activity, interactions with tubulin and cell cycle effects. The most active agent,( 1-(3,4,5-trimethoxybenzoyl)-3-(p-toluyl)-5-ammino-1,2,4-triazole, 3c), was evaluated for antitumor activity in vivo. The best results for inhibition of cancer cell growth were obtained with the p-Me, m,p-diMe and p-Et phenyl derivatives 3c, 3e and 3f, respectively, and, overall, these compounds were more or less as active as CA-4. Their vascular disrupting activity was evaluated in HUVEC cells, with compound 3c showing activity comparable with that of CA-4. Compound 3c almost eliminated the growth of syngeneic hepatocellular carcinoma in Balb/c mice, suggesting that 3c could be a new antimitotic agent with clinical potential. During the second year a new series of compounds characterized by the presence of a 2-methoxy/ethoxycarbonyl group were evaluated for antiproliferative activity against cancer cells in culture, and, for selected, highly active compounds, inhibition of tubulin polymerization, cell cycle effects and in vivo potency. The greatest antiproliferative activity occurred with a methoxy group introduced at the C-6 position, the least with this substituent at C-4. Thus far, the most promising compound in this series was 2-methoxycarbonyl-3-(3’,4’,5’-trimethoxyanilino)-6-methoxybenzo[b]furan (3g), which inhibited cancer cell growth at nanomolar concentrations (IC50’s, 0.3-27 nM), induced apoptosis and showed, both in vitro and in vivo, potent vascular disrupting properties derived from the effect of this compound on vascular endothelial cells. Compound 3g had in vivo antitumor activity in a murine model comparable to the activity obtained with combretastatin A-4 phosphate. The research work of the third year of PhD has been focused on the synthesis of a novel series of tubulin polymerization inhibitors, based on the 1-(3’,4’,5’-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, with the goal of evaluating the effects of various patterns of substitution on the phenyl at the 2-position of the imidazole ring on biological activity. A chloro and ethoxy group at the meta- and para-positions, respectively, produced the most active compound in the series (1-(3’,4’ ,5’ -trimethoxyfenyl)-2-(3’-Cl, 4’-Ethoxyfenyl)-1H-imidazole ,4o), with IC50 values of 0.4-3.8 nM against a panel of seven cancer cell lines. Except in HL-60 cells, 4o had greater antiproliferative than CA-4, indicating that the 3’-chloro-4’-ethoxyphenyl moiety was a good surrogate for the CA-4 B-ring. Experiments carried out in a mouse syngenic model demonstrated high antitumor activity of 4o, which significantly reduced the tumor mass at a dose thirty times lower than that required for CA-4P, which was used as a reference compound.

The cost of losses and control measures attributed to late blight of potatoes caused by Phytophthora infestans, are estimated to exceed $5 billion annually. Breeding for resistance is difficult owing to the tetraploid genotype of potato and current strains of the pathogen have developed resistance to chemical control. Consequently the search for biological control has assumed greater importance. In this investigation an in vivo bioassay was used to select soils antagonistic to late blight of potatoes, caused by Phytophthora infestans. Four out of eight samples demonstrated reproducible antagonism as determined by a reduction in the volume of tuber tissue rotted. A total of 292 bacterial and yeast samples and 20 fungal samples were recovered from suppressive soils using a variety of non-selective and selective media. When these organisms were tested individually against P. infestans in the assay, 15 isolates suppressed tuber rotting by >85% in at least three out of four assays. The antagonists were characterised as Pseudomonas spp. (3 strains), Enterobacter spp. (4 strains), Bacillus spp. (1 strain), Pantoea spp. (2 strains), Citrobacter spp. (1 strain), Buttiauxella spp. (1 strain), Trichosporon spp. (2 strains) and Geotrichum spp. (1 strain) by routine bacteriological tests, fatty acid profiling and partial sequencing of the gene encoding 16S or 18S (where appropriate) ribosomal RNA. Subsequently the possible mechanisms by which the potential biocontrol agents inhibited the disease were examined. Nine isolates showed some evidence of antibiotic production with a Pantoea spp. producing a compound that caused the hyphae of P. infestans to kink and permanently cease growth. Three isolates colonised hyphae of the pathogen and eleven produced siderophores in liquid culture. Hydrogen cyanide, proteolytic, cellulolytic and beta-1,3-glucanase activity was also evident in some species. Significant promotion of axenically grown tomato seedlings, as determined by increased stem and main root elongation, was achieved by ten of the isolates. Three population levels of the isolates were retested for disease inhibition at the end of the investigation. Isolates 3, 7 and 14 exhibited the highest levels of consistent inhibition at the lowest population levels and were therefore tested in combination. This achieved disease suppression that, at an antagonist concentration of 25 cfu/nL, was more consistent than isolate 3 alone and was over 30% greater than either isolate 7 or 14.

Autologous platelet concentrates are widely used in a variety of medical application with the aim of enhancing the regeneration of hard and soft tissue. The rationale of this clinical use lies in their enriched content of growth factors and other key molecules involved in promoting tissue healing. This thesis is composed of two different studies having as a common objective the evaluation of the biological properties of a platelet concentrate, Pure-Platelet Rich Plasma. The first part of the thesis was a pre-clinical in vitro study focused on evaluating the stimulating activity of P-PRP on human osteoblasts (hObs) and human dermal fibroblasts (hDFs). hObs and hDFs were grown in a serum-free medium supplemented by P-PRP obtained from three different donors. hObs and hDFs proliferation was assessed by cell counting and vitality through MTT assays up to 12 days of incubation. hObs osteo-differentiation was tested after 7- and 14- days of incubation by alkaline phosphatase assay. Results showed that cells maintained in the presence of P-PRP display an increased proliferation rate at 12 days of culture, compared to the standard condition. The increased vitality of hObs, induced by P-PRP, noticed after 12 days of culture, was comparable of that of control. In contrast, an increased vitality of hDFs, in comparison to the control, was observed at 12 days of culture. The addition of P-PRP did not further stimulate the enzyme activity either at day 7 and 14. The second part of the thesis was a randomized clinical trial that focused on clinical and radiographic evaluation of the adjunct of P-PRP in the management of edentulous posterior maxillae with a reduced height needing an implant rehabilitation. Clinical and radiographic outcomes of two different approaches were compared up to 3 years after loading: fixed prosthesis supported by 5 to 8.5 mm-long implants which were humidified with P-PRP versus fixed prosthesis supported by 10-mm or longer implants bioactivated with P-PRP and placed following maxillary sinus augmentation with deproteinized bovine bone mixed with P-PRP. Results showed that both procedures were safe and successful, with comparable outcomes. The use of P-PRP did not shift the balance toward one technique over the other one, but it may have contributed to make these procedures similar in term of clinical and radiographic outcomes. Since similar outcomes were reported for both approaches, the most cost-effective treatment appears the appropriate and should be advocated. Therefore, when there is an alternative for restoring the lost dentition, avoidance of a demanding surgical procedure like maxillary sinus augmentation should be considered and recommended. In conclusion, results coming from the in vitro study and the randomized clinical trial may support the clinical use of P-PRP. It may be beneficial in those situations requiring a successful bone and soft tissue regeneration at the site of surgery.

La technique dite de transmission axiale permet une évaluation ultrasonore de l'os cortical, à partir d'une mesure de vitesse. Cette technique est étudiée dans la présente thèse d'une part en modélisant par simulation numérique les phénomènes de propagation mis en jeu, d'autre part à travers une approche expérimentale. Un code de simulation de la propagation tridimensionnelle dans les milieux élastiques anisotropes et hétérogènes, reposant sur un schéma aux différences finies, a été implémenté pour répondre aux contraintes spécifiques de la propagation dans l'os cortical. A l'aide d'un tel code, la nature de l'onde se propageant le long de la corticale des os long est étudiée, en fonction de paramètres osseux tels que la géométrie de l'os (épaisseur corticale, courbure) ou la microporosité intracorticale. Parallèlement, un dispositif expérimental a été développé et adapté au contexte de mesures cliniques. En particulier, un nouveau de type de sonde permettant une mesure reproductible sur l'os (in vivo et in vitro) a été conçu et validé expérimentalement. L'originalité du dispositif tient à la possibilité d'une transmission bidirectionnelle implémentée pour s'affranchir de l'effet des tissus mous. Une étude in vitro a été réalisée sur une cinquantaine de radius humain, combinant les mesures ultrasonores de vitesse en transmission axiale à des mesures par rayons X (scanner médical conventionnel et microtomographie Synchrotron). Pour la première fois, nous avons évalué l'effet respectif de la densité minérale matérielle osseuse et de la microporosité sur la mesure de vitesse ultrasonore. Une campagne de mesure clinique est actuellement en cours, 150 patients et témoins ayant été mesurés à ce jour. La reproductibilité des mesures s'est avérée satisfaisante in vivo, grâce au concept de transmission bidirectionnelle. Nous présentons des résultats préliminaires concernant l'effet de l'âge.

The recent progresses offered by nanotechnology in the manipulation of matter lead to the development of several nanoparticles (NPs) and nanodevices for medical applications. In oncology, nanosized objects are particularly attractive as drug delivery systems since it is expected that engineered nanovehicles of appropriate size and functionalised with specific ligands/antibodies will improve the efficacy and selectivity of cancer therapies by exploiting both the passive and active mechanism of tumour targeting. The use of delivery systems is particularly appealing in those therapies in which the administration of the drug in aqueous formulations leads to drug aggregation with decreased activity or scarce bioavailability and tumour selectivity. This is the case of most of the photosensitizers used in photodynamic therapy (PDT), which display hydrophobicity and poor selective accumulation in malignant tissues. In the last decades, PDT is emerging as a promising cancer treatment modality in alternative to conventional therapies, which often demonstrate systemic drug toxicity and multidrug-resistance phenomena. PDT is based on the administration of a photosensitizer (PS) that accumulates in the tumour and after activation with light of appropriate wavelengths, reacts with surrounding molecular oxygen leading to the formation of cytotoxic reactive oxygen species (ROS) with consequent cellular and vasculature damages. In this PhD thesis, three different nanosystems, namely, liposomes, poly-(D,L-lactide-co-glycolide) nanoparticles (PLGA NPs) and ORganically Modified SILica nanoparticles (ORMOSIL NPs) were considered for the delivery of the second generation PS meta-tetra(hydroxyphenyl)chlorin (m-THPC, Temoporfin) to cancer cells in vitro. In particular, drug delivery efficiency, dark and phototoxicity of the m-THPC nanoparticle-based formulations were evaluated. To improve m-THPC bioavailability and tumour selectivity, in the design of the nanovehicles PEGylation and targeting of NPs were considered as essential strategies in order to prolong NP circulation in the bloodstream and exploit active mechanisms of tumour targeting. For the delivery of m-THPC using unilamellar liposomes, four different PEGylated liposomal formulations (trade name Fospeg®, provided by Biolitec Research) in which the length (PEG750, PEG2000, PEG5000) and the density (2%, 8%) of PEG were varied, were tested in vitro in normal lung fibroblasts CCD-34Lu and in cancer A549 lung epithelial cells. Compared to drug delivered in the standard solvent (Foscan®, ethanol/PEG 400/water (20:30:50, by vol.)), liposomal m-THPC showed a decreased intracellular uptake in both cell lines, but the presence of the delivery system highly reduced the dark cytotoxicity of the drug. The reduction of the PS dark toxicity increased with the increasing of PEG density on liposome surface, while the length of PEG chains did not affect significantly the toxic effect of m-THPC in the dark. However, photo-toxicity measured in A549 cells was only slightly affected by the reduced uptake of m-THPC delivered by Fospeg®, and the efficiency of PDT-induced cell killing was comparable among the different liposomal formulations. Interestingly, the intracellular localization of m-THPC delivered as Fospeg® or Foscan® was the same (Golgi apparatus and endoplasmic reticulum) suggesting drug release from liposomes, especially in the presence of the serum proteins, being m-THPC only physically entrapped within liposomes. m-THPC release was confirmed by the fact that liposomes covalently labelled with rhodamine were effectively were taken up by cells but, differently from m-THPC, localized in the acidic compartments of the cells. In spite of m-THPC release from liposomes, the Fospeg® formulation was exploited to target actively cancer cells by liposome conjugation with folic acid (FA), being FA-receptors (FRs) over-expressed in several human tumours. Thus, specific uptake and photo-toxicity of FA-targeted liposomes (FA-Fospeg) with respect to liposomes of the same composition but lacking FA (un-targeted Fospeg) was evaluated in KB (FR-positive) and in A549 (FR-negative) cells. The uptake of m-THPC delivered as FA-Fospeg was twice that of un-targeted Fospeg in KB cells; however only a modest fraction (~ 15%) of the targeted vehicle was effectively internalized by FR-mediated endocytosis while nonspecific internalization remained the prevailing mechanism of liposomes uptake in both cell lines. The improved m-THPC uptake obtained with FA-Fospeg in FR over-expressing cells translated into a 1.5 higher photo-induced toxicity. A novel formulation of bare and PEGylated PLGA NPs in which m-THPC was physically entrapped were synthesized (Prof J. Kos, University of Ljubljana) and evaluated in vitro and in vivo for phototherapy and fluorescence-based tumour imaging applications. In vitro studies carried out on A549, MCF10A neo T (breast cancer cells) and U937 (lymphoma derived pro-monocytic cells) cell lines, showed reduced uptake of PEGylated NPs with respect to non PEGylated NPs. As for Fospeg®, the use of the delivery system led to a significant reduction of m-THPC dark toxicity.. As expected for PEGylated NPs, the efficiency of cell internalization of m-THPC entrapped in PEG PLGA was reduced by 50% with respect to that in the standard solvent, but surprisingly cytotoxicity induced in irradiated A549 cells was quite comparable. At 24 h post-injection in vivo biodistribution of bare and PEGylated PLGA NPs compared to Foscan® was assessed in mice, showing very similar drug accumulation in the major organs but reduced skin uptake for both NP formulations. Thus, even if m-THPC release in the presence of serum proteins was measured in vitro, PEGylated PLGA NPs appeared potentially useful as stealth and biodegradable PS delivery systems. The premature release of the PS from the delivery system was completely avoided with the covalent link of m-THPC to the silane matrix of highly PEGylated ORMOSIL NPs (Prof. F. Mancin, University of Padova). This type of NPs exhibited a very low extent of cell internalization in vitro due to their high degree of PEGylation, making NP targeting an essential prerequisite to enhance intracellular drug delivery. In addition to FA, the RGD peptide and the antibody Cetuximab, which bind respectively the integrin α5ß3 receptor and epidermal growth factor receptor (EGFR), were exploited as targeting agents for ORMOSIL NPs and the specific uptake and photo-toxicity of m-THPC delivered by conjugated NPs were evaluated in vitro. The study revealed how the characteristics of the targeting agents are of crucial importance in determining the performances of targeted PEGylated nanosystems. In fact, the hydrophobic FA was very likely buried in the PEG layer and was unable to drive the selective uptake of ORMOSIL NPs while RGD peptide and Cetuximab antibody displayed some selectivity toward cells over-expressing their receptors (HUVEC cells over-expressing integrin α5ß3 receptors and A431 cells over-expressing EGFR). Unfortunately, the enhanced and selective uptake of m-THPC obtained by the two latter targeted ORMOSIL NPs was not accompanied by efficient and selective photo-induced cytotoxicity; it appeared that the selectivity of NP uptake was achieved in scarce drug cell loading conditions, determining only low PDT efficacy. The assessment of the biocompatibility of NPs is of fundamental importance for their safe use in nanomedicine. Since ORMOSIL NPs are not well characterised from this point of view, a toxicological characterization of empty ORMOSIL NPs were carried out in vitro in normal (CCD-34Lu) and cancer (A549, NCIH-2347) lung cells. The study included traditional cell viability and cytotoxicity tests (MTS test, LDH release assay, ROS production, cell membrane permeabilization measurements and electron microscopy analyses) in combination with a genome-wide analysis of gene expression profiles of cells exposed to NPs. The results pointed out that different types of cells respond quite differently to NPs and PEGylation of NPs highly affected the cytotoxicity profiles. PEGylation of ORMOSIL NPs completely abolished the toxicity of the nanosystem in CCD-34Lu and NCIH-2347 cells. On the contrary PEG ORMOSIL NPs induced necrotic cell death of A549 by increasing the permeability of the plasma membrane. At sub-lethal concentrations alteration of gene expression and inflammation were measured in A549 cells exposed to. The different response to PEG NPs is very likely explained considering the peculiarity of the cell type and the particular interaction of NPs with cell and internalization mechanisms. In fact, it was shown clearly that NPs internalized in A549 cells localized in and affected the morphology and the functioning of pulmonary surfactant containing lamellar bodies, peculiar of alveolar type II cells of which A459 cells represents an in vitro models.
Il recente progresso apportato dalla nanotecnologia nella manipolazione della materia ha portato al conseguente sviluppo di diversi tipi di nanoparticelle e nanodevices per applicazioni biomediche. In campo oncologico, oggetti dalle dimensioni nanometriche si sono dimostrati particolarmente interessanti in qualità di sistemi per la veicolazione di farmaci, poiché si presume che l’ingegnerizzazione dei nanoveicoli e la loro funzionalizzazione con specifici ligandi/anticorpi possa portare ad un miglioramento dell’efficacia e della selettività delle terapie antitumorali sfruttando meccanismi di targeting del tumore sia passivi che attivi. L’utilizzo di sistemi di veicolazione è particolarmente importante nel caso di terapie nelle quali la somministrazione dei farmaci in formulazioni acquose conduce a fenomeni di aggregazione con conseguente diminuzione di attività e di disponibilità nel circolo sanguineo, o nel caso di farmaci con scarsa selettività per il tumore. Appartengono a queste categorie la maggior parte dei fotosensibilizzanti utilizzati in terapia fotodinamica (PDT), poiché farmaci di natura idrofobica e con scarsa selettività di accumulo nei tessuti maligni. Negli ultimi decenni, la PDT si è dimostrata una promettente tecnica di trattamento del cancro in alternativa alle terapie convenzionali che invece generalmente dimostrano alta tossicità sistemica e fenomeni di farmaco-resistenza. La PDT si basa sulla somministrazione di un fotosensibilizzante (PS) che accumulatosi nel tumore, e dopo essere stato attivato con opportune lunghezze d’onda di luce, è in grado di reagire con l’ossigeno molecolare che lo circonda generando specie reattive dell’ossigeno (ROS) altamente citotossiche con conseguente danno cellulare e vascolare. In questa tesi di dottorato, tre diversi nanosistemi quali liposomi, nanoparticelle PLGA (poly-(D,L-lactide-co-glycolide)) e nanoparticelle di silice organicamente modificata (ORMOSIL), sono stati presi in considerazione per la veicolazione del fotosensibilizzante di seconda generazione meta-tetra(hydroxyphenyl)chlorin (m-THPC, Temoporfin) in cellule tumorali in vitro. In particolare, sono state valutate l’efficienza di veicolazione del farmaco, la tossicità buia e fotoindotta delle diverse formulazioni di m-THPC. Per migliorare la biodisponibilità e la selettività per il tumore della m-THPC, nella progettazione dei nanoveicoli sono state considerate quali strategie essenziali la pegilazione e il targeting delle particelle, in modo da prolungare la circolazione dei nanosistemi nel flusso sanguineo e in modo da sfruttare meccanismi attivi di targeting del tumore. Per la veicolazione della m-THPC utilizzando liposomi unilamellari sono state saggiate in vitro quattro diverse formulazioni liposomiali pegilate (Fospeg®, fornito dalla ditta Biolitec Research) con lunghezza (PEG750, PEG2000, PEG5000) e densità del PEG (2%, 8%) variabili, utilizzando come linee cellulari fibroblasti di polmone normali (CCD-34Lu) e cellule tumorali di epitelio polmonare (A549). Se paragonate al farmaco somministrato in forma libera in soluzione (Foscan®, etanolo/PEG 400/acqua (20:30:50, vol/vol)), le formulazioni liposomiali di m-THPC hanno mostrato una ridotta internalizzazione in entrambe le linee cellulari, ma nello stesso tempo la presenza del sistema di veicolazione ha portato alla significativa riduzione della tossicità buia del farmaco. La riduzione della tossicità buia del farmaco è risultata proporzionale all’aumento della densità di PEG sulla superficie del liposoma mentre la lunghezza delle catene di PEG sembra essere ininfluente nel limitare l’effetto tossico della m-THPC al buio. Comunque, la ridotta internalizzazione della m-THPC veicolata tramite Fospeg® influenza in modo solo parziale la fototossicità misurata in cellule A549, mentre l’efficienza d’induzione di mortalità in seguito a trattamento fotodinamico è risultata paragonabile tra le diverse formulazioni saggiate. Indipendentemente dalla veicolazione tramite Fospeg® o Foscan®, è stata riscontrata la medesima localizzazione intracellulare della m-THPC (apparato del Golgi e reticolo endoplasmatico) suggerendo il possibile rilascio del farmaco dalla formulazione liposomiale in presenza di proteine del siero, essendo la m-THPC solamente fisicamente intrappolata all’interno dei liposomi. Il rilascio della m-THPC è stato confermato dal fatto che liposomi nei quali viene legata covalentemente rodamina vengono effettivamente internalizzati dalle cellule e, differentemente dalla m-THPC, si accumulano nei compartimenti acidi intracellulari. Nonostante il rilascio del fotosensibilizzante dai liposomi, la formulazione Fospeg® è comunque stata utilizzata per veicolare selettivamente la m-THPC in cellule cancerose tramite la coniugazione dei liposomi con acido folico, essendo i recettori del folato sovraespressi in diversi tumori umani. Quindi sono state valutate l’internalizzazione specifica e la fototossicità di liposomi coniugati con folato (liposomi folato) rispetto a liposomi della stessa composizione ma privi di acido folico (liposomi non coniugati) in cellule KB e A549, rispettivamente positive e negative per l’espressione di recettori del folato. In cellule KB, l’internalizzazione della m-THPC si è rivelata doppia in caso di veicolazione con liposomi folato, malgrado solo una modesta parte (~15%) dei nanosistemi coniugati con folato siano effettivamente internalizzati tramite meccanismi di endocitosi mediata da recettore, essendo invece un’internalizzazione di tipo aspecifico il meccanismo prevalente per l’internalizzazione dei liposomi in entrambe le linee cellulari saggiate. In ogni caso, all’aumentato accumulo di m-THPC ottenuto tramite la veicolazione con Fospeg coniugato con folato in cellule che sovra esprimono il recettore, ne è conseguita una tossicità dopo irradiamento aumentata di circa 1.5 volte. Riguardo invece la veicolazione di m-THPC tramite particelle PLGA, formulazioni nude o pegilate sono state sintetizzate (Prof. J. Kos, Università di Lubiana) e saggiate sia in vitro che in vivo per la loro potenziale applicazione in fototerapia o in diagnosi dei tumori, sfruttando la fluorescenza del fotosensibilizzante fisicamente intrappolato all’interno delle particelle. Studi in vitro condotti su cellule A549, MCF10A neo T (derivate da tumore del seno) e U937 (cellule pro-monocitiche derivate da linfoma), hanno mostrato una ridotta internalizzazione della formulazione di m-THPC pegilata rispetto a quella nuda. Anche con particelle PLGA e come già visto per il Fospeg®, l’utilizzo di un sistema di veicolazione porta alla significativa riduzione della citotossicità buia della m-THPC. L’efficienza d’internalizzazione del fotosensibilizzante veicolato tramite particelle PLGA pegilate viene ridotta circa del 50% rispetto alla sua veicolazione nella formulazione standard ma sorprendentemente l’effetto citotossico indotto in cellule A549 irradiate è quasi paragonabile. La biodistribuzione della m-THPC (veicolata tramite nanoparticelle PLGA nude o pegilate o nella formulazione standard) è stata valutata 24 ore dopo la sua iniezione in topi, mostrando una simile distribuzione nei vari organi ma una significativa riduzione dell’accumulo a livello epidermico per entrambe le formulazioni nanoparticellari. Quindi, nonostante anche per le particelle PLGA pegilate sia stato misurato il rilascio di m-THPC in presenza di proteine sieriche, esse appaiono un buon sistema di veicolazione di fotosensibilizzanti soprattutto per le loro caratteristiche ‘stealth’ e per la loro biodegradabilità. Il rilascio prematuro del fotosensibilizzante è stato invece completamente limitato con il legame covalente della m-THPC alla matrice silanica di particelle ORMOSIL altamente pegilate (Prof. F. Mancin, Università di Padova). Tuttavia questo tipo di particelle ha mostrato un’internalizzazione intracellulare estremamente bassa derivata dall’elevato grado di pegilazione, ponendo come requisito essenziale il targeting delle particelle. In qualità di agenti di targeting per le particelle ORMOSIL pegilate sono stati valutati, oltre al folato, anche il peptide ciclico RGD e l’anticorpo Cetuximab, essendo questi ultimi in grado di legarsi rispettivamente ad integrine α5ß3 e al recettore del fattore di crescite dell’epidermide (EGFR). L’internalizzazione selettiva e la fototossicità della m-THPC veicolata tramite le tre diverse nanoparticelle funzionalizzate sono state valutate in vitro in opportuni sistemi cellulari. Tale studio ha mostrato come le caratteristiche dell’agente di targeting influenzino in modo sostanziale la selettività di tali nanosistemi pegilati. Infatti, mentre il folato altamente idrofobico si ripiega verosimilmente verso la corona di PEG rendendosi inefficace nel guidare selettivamente le particelle ORMOSIL, il peptide RGD e l’anticorpo Cetuximab hanno mostrato una certa selettività nei confronti di cellule sovraesprimenti i rispettivi recettori (cellule HUVEC sovraesprimenti recettori per le integrine α5ß3 e cellule A431 sovraesperimenti EGFR). Tuttavia, l’aumentato accumulo selettivo della m-THPC ottenuto tramite la coniugazione delle nanoparticelle con RGD e Cetuximab non ha portato ad una conseguente aumentata efficienza e selettività nell’induzione di citotossicità in seguito ad irradiamento. Tale risultato è verosimilmente imputabile al fatto che la selettività di accumulo delle nanoparticelle viene raggiunta in condizioni nelle quali la disponibilità del farmaco nelle cellule è troppo bassa, con conseguente scarsa efficacia dopo trattamento fotodinamico. La valutazione della biocompatibilità delle nanoparticelle risulta di fondamentale importanza per un’applicazione sicura della nanotecnologia in campo medico. Quindi, poiché le nanoparticelle ORMOSIL non sono ancora state ben caratterizzate da tale punto di vista, un loro profilo tossicologico è stato tracciato in vitro in cellule polmonari normali (CCD-34Lu) e tumorali (A549, NCIH-2347). Nello studio sono stati combinati esperimenti tradizionali di valutazione della vitalità cellulare e della citotossicità (test MTS, saggio del rilascio di LDH, valutazione della produzione di ROS, misure di permeabilizzazione di membrana, analisi di microscopia elettronica) con un’analisi dei profili di espressione genica estesa all’intero genoma di cellule esposte alle nanoparticelle. I risultati hanno mostrato come diversi tipi di cellule rispondono in modo abbastanza differente all’esposizione alle nanoparticelle e come la pegilazione influisce fortemente sui profili di citotossicità. Infatti, la pegilazione delle particelle ORMOSIL è in grado di abolire completamente la tossicità dei nanosistemi in cellule CCD-34Lu e NCIH-2347 mentre le stesse particelle pegilate inducono morte per necrosi in cellule A549, aumentandone la permeabilità di membrana. Inoltre nelle medesime cellule, concentrazioni sub-letali di nanoparticelle inducono infiammazione e alterazione dell’espressione genica. La differente risposta all’esposizione alle nanoparticelle pegilate delle cellule A549 è spiegabile considerando la peculiarità di questo tipo cellulare, e in particolare l’interazione delle particelle stesse con le cellule e il loro meccanismo d’internalizzazione. Infatti, è stato mostrato in modo chiaro che le nanoparticelle vengono internalizzate in corpi lamellari contenenti il surfattante polmonare, peculiari di cellule alveolari di tipo II, delle quali le cellule A549 rappresentano un modello in vitro. Tale accumulo delle nanoparticelle nei corpi lamellari porta alla modifica della morfologia degli stessi e una pesante alterazione della loro funzionalità.

LLa photophysique du calcium green a été étudiée grâce au montage de fluorescence résolue en temps sous microscope. Cette sonde est caractérisée par des durées de vie différentes selon l'état de complexation au calcium. Elle n'est pas sensible a la viscosité et peu sensible a la polarité, mais sensible a l'hydrolyse, a l'adsorption et au ph. La molécule sonde le milieu dans son état fondamental. Il existe deux conformations stables de la sonde, caractérisées par leurs durées de vie. La durée de vie courte est due au transfert de charges intramoléculaire. Des études ex vivo sur des cellules ont été faites : étalonnage, détermination de la concentration en calcium, mesure d'adsorption de la sonde par anisotropie de fluorescence.

L’ostéosarcome est un cancer primitif rare de l’os dont les approches thérapeutiques actuelles sont encore malheureusement insuffisantes pour espérer une totale guérison, et pour lequel la recherche de nouvelles molécules efficaces est constante. De plus, après exérèse de la tumeur, le défaut généré peut nécessiter de recourir à des matériaux aidant la reconstruction osseuse. Or le potentiel inflammatoire de ces matériaux est bien documenté. Les inhibiteurs de phosphodiestérase de type 4, parmi lesquels des molécules à motif pyridazinone, ont d’ores et déjà fait leurs preuves en tant qu’agents anti-cancéreux et anti-inflammatoires dans un certain nombre de modèles cellulaires. L’objectif de ce travail a été d’évaluer les effets anti-cancéreux et anti-inflammatoires de nouvelles molécules à motif pyridazinone dérivant de précurseurs agrosourcées. Les effets anti-inflammatoires ont été validés sur la production cytokinique, le potentiel migratoire et le maintien des capacités phagocytaires des neutrophiles primaires humains in vitro et dans un modèle murin d’inflammation in vivo. Ces molécules ont également montré leur potentiel thérapeutique dans des modèles d’ostéosarcomes in vitro où elles sont en capacité de bloquer la prolifération et la migration cellulaire tout en induisant l’apoptose des cellules cancéreuses. In vivo ces molécules sont capables de ralentir la progression tumorale et de maintenir la qualité minérale osseuse. Les résultats obtenus indiquent que ces nouvelles molécules à motif pyridazinone pourraient s’intégrer dans des polythérapies pour leurs potentiels thérapeutiques à la fois anti-cancéreux et anti-inflammatoire
Osteosarcoma is a rare primary bone cancer for which current therapeutic approaches are unfortunately insufficient to provide a total cure. So the need for new molecules development is constant. In addition, after tumor excision, bone defect may require materials to fill and support bone rebuilding. The inflammatory potential of such materials is well documented. Phosphodiesterase type 4 inhibitors, including pyridazinone-scaffold based molecules, have already proven themselves as anti-cancer and anti-inflammatory agents in a number of in vitro models. The goal of this work was to assess the anti-cancer and anti-inflammatory effects of new pyridazinone-scaffold based molecules derived from agrosourced precursors. Anti-inflammatory effects have been assessed on cytokine production, migratory potential and the phagocytic abilities of human primary neutrophils in vitro and in an in vivo model of inflammation in mice. These molecules have also shown their therapeutic potential in in vitro osteosarcoma models where they decreased cell proliferation and migration while inducing apoptosis in cancer cells. In vivo, these molecules were able to decrease tumor growth and maintain mineral bone quality. These results indicated that new pyridazinone scaffold-based molecules might be integrated into a multi-therapy, both for their anti-cancer and anti-inflammatory therapeutic potential

Although electronic cigarettes (ECs) have been widely promoted as safer alternatives to tobacco cigarettes, limited scientific data is currently available on their possible health effects. The current study aims to investigate the potential effects of ECs using a novel in-house designed cigarette/EC aerosol delivery system and physiologically relevant 2D and 3D in-vitro human airway models. Submerged cultures of BEAS 2B and CALU 3 bronchial epithelial cells were used to investigate the effects of nicotine and its oxidative metabolite cotinine. Results demonstrated that neither nicotine nor cotinine had any significant impact on the bronchial epithelial cell viability or IL-6/IL-8 pro-inflammatory mediators’ production. Further, treatment of submerged cultures of a number of airways related cell types to extracts of commercially available ECs of different nicotine strength, flavourings and brands showed that while the differing nicotine strengths had no impact on the cell viability, flavourings significantly influenced cell viability, with strawberry and cherry flavoured ECs demonstrating the highest cytotoxicity. Moreover, same flavours from different brands produced different effects on cells. Finally, a co-culture human airways model consisting of CALU 3 bronchial epithelial cells and MRC-5 pulmonary fibroblasts cultured at air-liquid-interface were treated to whole cigarette smoke (WCS) and EC vapour (ECV) at different exposure times (7 m, 1 h, 2 h, 3 h, 4.5 h and 6 h) as per the ISO:3308 standard smoking regime using an in-house designed bespoke, automated aerosol delivery system. Results demonstrated that while WCS caused a significant reduction in cell viability post 7 min exposure, ECV produced cytotoxic effects only at exposure times ≥ 3 h. A significant increase in oxidative stress and IL-6/IL-8 production was observed post 3 h ECV treatment, both of which are hallmark characteristics of airway inflammatory conditions like chronic obstructive pulmonary disorder. Overall, results from the current study suggest that ECs have the potential to cause substantial airways damage, and as yet, cannot be regarded as safe alternatives to tobacco cigarettes. Importantly, a standardised testing method is urgently required in order to elucidate on the long-term health effects of ECs.

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Taenia crassiceps cysticerci possess antigenic similarities to T. solium cysticerci and because of this and added to the relative easiness in its maintainance in laboratory they are used as an experimental model to T. solium studies. The objective of this study was to analyze the in vitro influence of glucose and insulin on the energetic and respiratory metabolism of T. crassiceps cysticerci exposed to low dosages of anti-helminthic drugs, albendazol and praziquantel, and also to different concentrations of glucose and insulin. Glucose is the main energy source to these parasites in their larval and adult stages and glycogen is their main energetic reserve. Glycogen may be converted into glucose in case of low glucose uptake and is stored in the cysticerci tegument. The cysticerci were collected from the peritoneal cavity of BALB/c female mice experimentally infected and maintained in the animal facilities of IPTSP/UFG. After 24hours of culture in RPMI culture medium supplemented and with glucose, glargine insulin (LANTUS®, Sanofi Aventis), albendazol and praziquantel, the cysticerci were removed and the medium was frozen in liquid nitrogen as to allow the metabolix stasis. Afterwards this culture medium was analyzes through HPLC as to permit the quantification of the following organic acids related to the carbohydrates metabolism: lactate and pyruvate, intermediary metabolism: citrate, α-ketoglutarate, succinate, fumarate, malate and oxaloacetate, fatty acids metabolism: β-hydroxibutyrate and propionate. It was possible to detect propionate and β-hydroxibutyrate secreted by T. crassiceps cysticerci which indicates the fatty acids oxidation as an alternative energy xv source used by the initial stage cysticerci which are in rapid growth. Also the detection of organic acids from the citric acid cycle indicates the in vitro aerobiosis performed by the initial stage cysticerci.
Os cisticercos de Taenia crassiceps possuem similaridades antigênicas com cisticercos de Taenia solium e por este motivo, associado ao fato de uma relativa facilidade de sua manutenção em animais de laboratório, são utilizados como modelo experimental para estudos da cisticercose por T. solium. O objetivo deste trabalho foi analisar in vitro a influência da glicose e da insulina no metabolismo energético e respiratório de cisticercos de T. crassiceps expostos aos fármacos anti-helmínticos albendazol e praziquantel em baixas doses, e diferentes concentrações de glicose e insulina. A glicose é a principal fonte de energia para estes parasitos tanto na fase larval quanto adultos e o glicogênio, que pode ser novamente convertido em glicose, é fonte de reserva energética, sendo encontrado armazenado em suas membranas. Os cisticercos foram provenientes da cavidade peritoneal de camundongos fêmeas Balb/c infectados experimentalmente e mantidos no biotério do IPTSP/UFG. Após 24 horas de cultivo em meio RPMI® suplementado e adicionado de glicose, insulina glargina (LANTUS®, Sanofi Aventis), albendazol e praziquantel, foram retirados da placa e o meio foi congelado em nitrogênio líquido para ocorrer estase metabólica. Posteriormente o meio de cultura passou por um processo de extração sendo analisado em Cromatografia Líquida de Alta Performance com detector UV (CLAE) dos seguintes ácidos orgânicos oriundos do metabolismo de carboidratos (lactato, piruvato), metabolismo intermediário (citrato, α-cetoglutarato succinato, fumarato, malato, oxaloacetato) e metabolismo de ácidos graxos (β-hidroxibutirato e propionato). Foram detectados propionato e β-hidroxibutirato secretados por cisticercos de T. crassiceps indicando a oxidação de ácidos graxos como fonte de energia alternativa, utilizada em especial nos estádios de crescimento e também em situações de baixas concentrações de substancias energéticas provenientes do hospedeiro. Em adição, a detecção de xiv ácidos orgânicos do ciclo do ácido cítrico nos cisticercos analisados confirma a aerobiose in vitro do estádio evolutivos inicial dos cisticercos.

La réparation endovasculaire (EVAR) d'un anévrisme de l'aorte abdominale (AAA) consiste en la mise en place d'une endoprothèse (EDP) par chirurgie mini-invasive au sein de l'anévrisme. Cet acte permet de prévenir la rupture des tissus endommagés impliqués dans un AAA, défini comme la dilatation localisée du diamètre de l'aorte. Lorsque l'amont de l'anévrisme englobe les artères périphériques rénales et/ou viscérales, l'AAA est qualifié de complexe. Dans ce cas, l'EDP déployée est dite « fenêtrée », en d'autres termes, perforée à l'emplacement des jonctions vers les artères périphériques. La prise en charge dans le cadre d'un AAA complexe devient alors plus limitante car l'EDP fenêtrée sera conçue sur mesure afin de correspondre à l'anatomie de l'anévrisme et à la position des artères périphériques du patient. Cela implique un délai de fabrication de plusieurs semaines, limite la prise en charge aux anévrismes stables et exclut les situations d'urgence. Dans ce contexte, l'impression 3D présente un intérêt considérable pour la fabrication d'EDP sur mesure et dans des délais très courts. Ainsi, l'objectif de ce travail de thèse est de concevoir un prototype d'endoprothèse par impression 3D d'un polyuréthane thermoplastique (TPU) de grade médical (élastomère thermoplastique). Le présent travail permettra de valider le procédé de conception et la fonctionnalité de notre 3D-EDP pour son application finale en tant que dispositif médical implantable.Dans un premier temps, l'impact du procédé de fabrication sur les propriétés chimiques, physiques et physico-chimiques du TPU a été étudié à chaque étape, des granulés à la stérilisation par rayons gamma d'une prothèse fabriquée par dépôt de filament fondu (FDM). L'évaluation préliminaire in vitro de la cytotoxicité et de l'hémocompatibilité du TPU a été réalisée après l'étape d'impression 3D et de stérilisation. Un vieillissement préliminaire du TPU en conditions oxydantes extrêmes a été réalisé afin de prédire l'évolution de ses propriétés sur le long terme. Par la suite, une stratégie de conception d'un prototype implantable par voie endovasculaire a été développée. Les propriétés de ce prototype stérilisé ont été caractérisées par différentes techniques (CES, ATG, DSC, FTIR, MEB, goniométrie, traction uniaxiale, …). Ses propriétés biologiques ont été évaluées in vitro par des tests de cytocompatibilité, hémocompatibilité et contact avec les macrophages pendant 24 heures (inflammation aigüe). L'évolution de ses propriétés physico-chimiques et mécaniques a été suivie par des études de vieillissement in vitro.La caractérisation des propriétés chimiques, physiques et physico-chimiques du TPU a montré que l'impression 3D FDM et la méthode de stérilisation par rayons gamma constituent une voie de fabrication viable d'un prototype comprimable dans un cathéter d'introduction endovasculaire. L'évaluation biologique in vitro a montré la cytotocompatibilité du prototype par la méthode de l'extrait. De plus, le prototype s'est révélé faiblement hémolytique et les plaquettes adhérant à sa surface n'étaient pas activées. La faible sécrétion de cytokines (IL-6 et TNF-a) au contact des macrophages inactivés a montré que le prototype d'EDP ne présente pas de caractère pro-inflammatoire. Enfin, les études de vieillissement ont montré un impact sur les propriétés mécaniques et de surface de notre prototype d'EDP sans toutefois compromettre sa fonctionnalité. Par la suite, la stratégie de conception pourrait évoluer vers une fonctionnalisation de l'EDP afin de prévenir les infections et les thromboses responsables respectivement de 2% et 6% des complications post-opératoires
Endovascular repair (EVAR) of an abdominal aortic aneurysm (AAA) involves the placement into the aneurysm of a stent graft (SG) by minimally invasive surgery. This procedure prevents rupture of the damaged tissue involved in an AAA, defined as a localized diameter dilation of the aorta. When the upstream portion of the aneurysm includes the peripheral renal and/or visceral arteries, the AAA is qualified as complex. In this case, the deployed SG is said “fenestrated”, in other words, perforated at the site of junctions to the peripheral arteries. Management of a complex AAA becomes more limiting as the fenestrated SG will be custom designed to match the anatomy of the aneurysm and the position of the peripheral arteries of the patient. This implies a manufacturing delay of several weeks, limits the management to stable aneurysms and excludes emergency situations. In this context, 3D printing (3DP) is of considerable interest for the fabrication of custom-made SGs in a very short time frame. Thus, the objective of this thesis work is to design a SG prototype by 3D printing of a medical grade thermoplastic polyurethane (TPU) (thermoplastic elastomer). The present work will validate the manufacturing process and the functionality of our 3DP-SG for its final application as an implantable medical device.First, the impact of the manufacturing process on the chemical, physical and physicochemical properties of TPU was studied at each step, from the pellets to the gamma-ray sterilization of a graft manufactured by fused filament deposition (FDM). In vitro preliminary evaluation of the cytotoxicity and hemocompatibility of TPU was carried out after the 3D printing and sterilization step. Aging of TPU under extreme oxidizing conditions was performed to predict the evolution of its properties in the long term. Subsequently, a design strategy for an endovascular implantable prototype was developed. The properties of said prototype were characterized by different techniques (SEC, TGA, DSC, FTIR, SEM, goniometry, uniaxial traction, ...). Its biological properties were evaluated in vitro by tests of cytocompatibility, hemocompatibility and contact with macrophages for 24 hours (acute inflammation). Moreover, the evolution of its physicochemical and mechanical properties was evaluated by in vitro aging studies.The characterization of the chemical, physical and physicochemical properties of TPU enabled the validation of a FDM printing manufacturing route and gamma ray sterilization of a crimpable SG prototype. The in vitro biological evaluation showed the non-cytotoxicity of the SG prototype by the extraction method. Moreover, the prototype was found to be weakly hemolytic and the platelets adhered on its surface were not activated. The low secretion of cytokines (IL-6 and TNF-α) upon contact with inactivated macrophages showed that the SG prototype does not exhibit a pro-inflammatory characteristic. Finally, aging studies showed an impact on the mechanical and surface properties of our SG prototype without compromising its functionality. Subsequently, the design strategy could evolve towards a functionalization of the SG prototype in order to prevent infections and thrombosis responsible for 2% and 6% of postoperative complications respectively

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O carcinoma mucoepidermóide de pulmão, um tipo histológico que deriva das glândulas mucosas traqueobrônquicas, manifesta-se com sintomas obstrutivos e tende a comprometer a traqueia. Com finalidade curativa ou paliativa da doença, atualmente há uma forte tendência na oncologia em desenvolver estratégias terapêuticas que visam à administração de compostos com elevado potencial de otimizar o efeito do tratamento com a radiação ionizante, de modo a aumentar a morte de células tumorais e preservar íntegras as células dos tecidos sadios adjacentes. A intensa busca por tais estratégias evidenciou resultados promissores apresentados pelo composto denominado Resveratrol (3,4,5-trihidroxiestilbeno), tornando-o amplamente divulgado e alvo de intensas pesquisas. O principal objetivo do presente estudo foi determinar o efeito do resveratrol em cultura celular de carcinoma mucoepidermóide de pulmão exposta a diferentes doses de radiação ionizante. Para tal, os estudos de citotoxicidade utilizando o método de incorporação do vermelho neutro, e da determinação da dose letal 50 % (DL50) da radiação ionizante, foram realizados em cultura de células da linhagem NCI-H292 [H292] (ATCC® CRL-1848TM), CCIAL069. Com base nos resultados do IC50% (401,5 μM) e da DL50 (693 Gy) foram realizados o teste in vitro do micronúcleo e os ensaios para avaliar o efeito do resveratrol no ciclo celular, reparo da lesão no DNA e processo de lesão radioinduzida, necrose e apoptose celular. Os resultados evidenciaram que o resveratrol na concentração de 30 μM apresenta uma importante capacidade em promover danos às células NCI-H292 após 24 h da irradiação.
Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Organometallic complexes containing transition metals, such as Ru(II), Os(II), Ir(III), have been moderately and recently used in medicinal chemistry as anticancer, antimalarial, antimicrobial or diagnostic agents. Current trends have led researchers to explore and define new synthetic methods in the quest for the design of new drugs and reduce the inherit associated toxic side-effects by using metal based compounds. Ferrocene based derivatives have been subjected to study for their biological and medicinal applications. Examples include ferrocenophane polyphenol, ferrocenyl quinone methides, ferrocenyl-aminoquinoline-carboxamide and a ferrocene-substituted hydroxytamoxifen, which has been proved as a potential new breast cancer therapeutic. Ferroquine displays antimalarial activity and ferrocifen is a tamoxifen-ferrocene anticancer agent. During the course of our research, we have focused on synthesizing and studying the biological activity of novel organometallic compounds containing the corresponding ferrocene moiety. We aimed to make, at least, 3 different families of compounds, which were cannabinoid receptor (CB1/CB2 receptor agonists), histone deacetylase (HDAC) selective inhibitors and general kinase inhibitors. Firstly, a review covering the state-of-the-art in bioorganometallic chemistry will be presented. Secondly, we started with the synthesis of a small library of compounds containing the following groups: ferrocenylamine, 4-oxo-1, 4-dihydropyridine and dihydroquinoline. We used aminoferrocene as a bioisostere of the adamantylamine group where previous studies of compounds containing the latter group had showed it to effectively interact with the cannabinoid receptors, CB1 and CB2. Some of our compounds displayed good to excellent potency in the nM range against the CB1 and CB2 receptors. Thirdly, we embarked to synthesise HDAC inhibitors knowing their potential as anti-cancer drugs and trying to obtain, wherever possible, enzyme isoform selectivity. One of the well-known HDAC inhibitors is suberoylanilide hydroxamic acid (SAHA). Vorinastat, as it is also known, has received Food and Drug Administration approval for treating patients with cutaneous T-cell lymphoma. The compound developed in our group by replacing ferrocene for the phenyl ring in SAHA, called JAHA, is another example of a highly potent HDAC inhibitor. Our research led to compounds where the hydroxamic acid moiety in JAHA has been replaced by a benzamide group. This transformation has produced a significant effect in delivering a potent HDAC3 selective HDAC inhibitor. This is supported by biological studies and a molecular modelling rationalisation. Next, a series of oxindole based analogues have been synthesized by the Knoevenagel condensation reaction of: 5-(pentafluorosulfanyl)-1,3-dihydro-indol-2-one and 6-(pentafluorosulfanyl)-1,3-dihydro-indol-2-one compound with: ferrocenecarboxaldehyde and pyrrole-2-carboxaldehyde. The compounds thereby synthesised have been studied against a panel of kinases, and kinase inhibitory data will be discussed and presented. In a Future directions section, we will describe the synthesis of FAAH (fatty acid amide hydrolase) inhibitors based on an aminoferrocene backbone.

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaCO-2 cell model, there was no significant change in the transport of [3H] l-proJine by the cell after co-incubation with either dapsone or cyclophosphamide (50!!M) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50!!M) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (1 OO!!M ) . With the test involving the NG11 S-401l-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after co-incubation with either phenytoin or cyclophosphamide (50!!M) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (SO!!M) (0.44 + 0.04 mM), sulphamethoxazole (SOIlM) (0.43 + 0.08mM) and carbamazepine (SO!!M) (0.47 + 0.034 mM), when coincubated with the rat microsomal system, compared to the control (0.52 + 0.07mM). There was no significant depletion in glutathione when the erythrocytes were coincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50~lM) (0.953 + 011 OmM) when co-incubated the rat microsomal system, compared to the control (1.124 + 0.032mM). Differences were considered statistically significant for p