Дисертації з теми "MEDICINA MOLECOLARE"
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Rihawi, Karim <1984>. "Caratterizzazione molecolare per la medicina personalizzata nel paziente oncologico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2021. http://amsdottorato.unibo.it/9944/1/TESI%20DOTTORATO%20Karim%20Rihawi_final.pdf.
Повний текст джерелаCirculating tumor cells (CTCs) represent a subset of cells found in the blood of patients with solid tumors, which are mainly involved in the process of metastatization. CTCs preserve primary tumor heterogeneity and mimic tumor properties, and may be considered as clinical biomarker, preclinical model, and therapeutic target. As such, CTCs can play a pivotal role as being a component of liquid biopsy which has potential in analyzing the genomic landscape of patients with cancer, supervising treatment responses, monitoring minimal residual disease, and managing non-invasive therapy resistance. Compared with traditional tissue biopsy, liquid biopsy is noninvasive and real-time. Therefore, CTCs can be used to tailor treatment in oncology patients. The aim of this work is to obtain through the detection of CTCs a multi-level molecular characterization using different tumor samples from the same patient or the same sample but taken throughout different stages of the disease. In our study 190 patients were enrolled; among them, 19 had a CTC count > 4. We report the results of these patients highlighting the strengths and the pitfalls of the techniques utilized as well as the clinical implications of the genetic analyses which followed the identification and isolation of CTCs.
LIBERATOSCIOLI, LAURA. "Biologia molecolare della paraoxonasi." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2008. http://hdl.handle.net/2108/548.
Повний текст джерелаDe, Rocco Daniela, and Rocco Daniela De. "STUDIO CLINICO E MOLECOLARE DELLA SINDROME DI BERNARD-SOULIER." Doctoral thesis, Università degli studi di Trieste, 2015. http://hdl.handle.net/10077/10848.
Повний текст джерела2013/2014
La sindrome di Bernard-Soulier (BSS) è una rara piastrinopenia ereditaria causata da alterazioni a livello del complesso glicoproteico GPIb-IX-V, presente sulla membrana piastrinica e responsabile della adesione delle piastrine in seguito a danno vascolare. La BSS si trasmette come malattia autosomica recessiva (BBSA1) e i pazienti affetti presentano piastrine giganti e severi episodi di sanguinamento. Tuttavia in tempi recenti sono state descritte delle famiglie con una forma dominante nota come BSSA2. In questi pazienti la piastrinopenia è moderata e le piastrine presentano un volume leggermente aumentato. Finora sono state individuate solo 5 varianti in eterozigosi nel BSSA2:, 4 nel gene GP1BA e 1 in GP1BB. Fatta eccezione per p.Ala172Val del gene GP1BA che è relativamente frequente nella la popolazione Italiana, le altre 4 sono state descritte in singole famiglie. I pochi casi di cui disponiamo, soprattutto per la forma recessiva non ci permettono di avere informazioni sui meccanismi patogenetici e sulla sua evoluzione nel tempo. Per questo motivo è stato istituito un Consorzio Internazionale per lo studio della BSS grazie al quale è stato possibile raccogliere i dati clinici e molecolari di 132 famiglie. Tutte le informazioni sono state inserite in un database (BSS Consortium database) attualmente gestito dal nostro laboratorio e consultabile dai gruppi di studio che hanno aderito al Consorzio. Inoltre per aumentare le informazioni sulle varianti identificate nel BSSA1 abbiamo incrementato i dati molecolari delle famiglie del Consorzio con i dati di altre 79 famiglie descritte in letteratura, raggiungendo un totale di 211 famiglie. Tutte le mutazioni identificate in queste famiglie sono state poi inserite in un database pubblico disponibile in rete (LOVD: Leiden Open Variation Database). La raccolta e l’elaborazione dei dati ci ha permesso di chiarire alcuni aspetti clinici e molecolari della malattia. Tuttavia data l’eterogeneità genetica e l’elevata espressione fenotipica gli studi genotipo-fenotipo si sono rivelati difficili da eseguire. Nonostante le molte informazioni acquisite, il database risulta ancora incompleto e limitato; per questo motivo è necessario raccogliere nuovi casi e inserire assieme alle varianti anche i relativi studi funzionali che si rivelano indispensabili per poter definire l’effetto delle varianti sul complesso GPIb-IX-V. Nell’ambito invece dello studio e caratterizzazione della forma meno grave di BSS (BSSA2) sono stati selezionati 120 pazienti piastrinopenici senza diagnosi caratterizzati da piastrine grandi. In questi pazienti sono stati analizzati i geni GP1BA, GP1BB e GP9 e sono state identificate 11 diverse varianti: 1 nonsense, 2 mutazioni di framshift, 1 mutazione nel codone di inizio e 5 varianti missense. Gli studi funzionali eseguiti sulle varianti missense per stabilire il loro ruolo patogenetico sono ancora in corso. Tuttavia se gli studi dovessero confermare la loro patogenicità 11 pazienti su 120 risulterebbero BSSA2 e questa forma dovrebbe essere considerata una tra le piastrinopenie ereditarie più frequenti in Italia. In conclusione grazie a questo studio è stato possibile raccogliere la più ampia casistica di pazienti affetti da BSSA1 fin’ora descritta e ottenere numerose informazioni sia sulla clinica che sulle mutazioni coinvolte. Il BSS Consortium database permetterà ai clinici che hanno partecipato allo studio di osservare nel tempo l’andamento della malattia nei pazienti e di ottenere informazioni utili per stabilire un corretto protocollo per la presa in carico dei pazienti. Infine la caratterizzazione di nuove forme di BSSA2 rappresenta il punto di partenza per descrivere al meglio la malattia BSSA2 sia dal punto di vista clinico che molecolare. In futuro sarà quindi indispensabile estendere il BSS Consortium database anche alla forma BSSA2.
XXVII Ciclo
XXVII Ciclo
1979
LAZZARETTI, CLARA. "Azione Molecolare E Cellulare Degli Ormoni Della Riproduzione." Doctoral thesis, Università degli studi di Modena e Reggio Emilia, 2022. http://hdl.handle.net/11380/1278344.
Повний текст джерелаClassically, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone (LH) receptor (LHCGR) -driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signalling, showing at the same time the activation of steroidogenic and pro-apoptotic events. These signals can be impaired by estrogens inducing anti-apoptotic events via nuclear receptors and non-genomic action of a G protein-coupled estrogen receptor (GPER). The aim of the project is to better understand the role of estrogens/gonadotropins and their membrane receptors in regulating ovarian physiology and the selection of the dominant follicle. In this study it was demonstrated that GPER heteromerizes both with FSHR and LHCGR at the cell surface of HEK293 cells overexpressing the two receptors as well as human primary granulosa lutein cells (hGLC). FSHR/GPER heteromers reprogram cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. On the other hand, GPER/LHCGR complex does not affect the LH and hCG-induced cAMP production and do not compromise the activation of the cAMP/PKA pathway, as it is indicated by similar CREB and ERK1/2 phosphorylation and same progesterone production in hGLC treated with siRNA against GPER, and the mock-treated one. Interestingly, GPER displace the LHCGR/Gαq coupling and consequently impedes the intracellular Ca2+ release and IP1 accumulation in LHCGR-GPER co-expressing HEK293 cells upon LH and hCG treatment compared to LHCGR-expressing cells. Also, it was demonstrated that in presence of GPER the kinetic of FSHR internalization through early and late endosomes is reduced, suggesting its ability to blockade FSHR at the intracellular level and reduce FSHR recycling on cell membrane. Indeed, FSHR internalization is necessary for GPER to inhibit FSH-induced cAMP response. According to our results, estrogens are selectively involved in the regulation of pro- and anti-apoptotic signals and receptor internalization through FSHR/GPER complexes and in modulation of LHCGR-mediated signaling cascade. Our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR and LHCGR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.
Jakimovska, Frosina. "Characterization of the interaction between nucleophosmin (NPM) and highmobility group a (HMGA) proteins." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2760.
Повний текст джерелаABSTRACT HMGA proteins are members of the high mobility group (HMG) non-histone, architectural chromosomal proteins. The HMGA family consists of: A1a, A1b and A2, the first two being isoforms produced by alternative splicing. These are small proteins, about 100aa residues long, characterized by three basic stretches of AT-hooks which bind to the AT-rich sequences on the DNA minor groove. They are normally present in rapidly proliferating cells (embryo) whereas upon differentiation the levels of these proteins decrease until they are nearly absent from adult cells. Overexpression of these proteins has been correlated with various types of neoplastic transformations. The interaction network of HMGA has been an object of study for years in our laboratory and one of the most intriguing protein-protein interactions studied is the one between HMGA and nucleophosmin. Nucleophosmin (NPM, B23, numatrin or NO38) is one of the most abundant phosphoproteins, mainly localized in the nucleoli but it also shuttles between the nucleus and the cytoplasm. This versatile protein has been proven to be involved in various cellular functions and related to both proliferative and growth-suppressive roles in the cell. The first evidence of the interaction HMGA2-NPM in our lab has been obtained by in vitro assays (affinity chromatography). The step ahead was to see if this interaction occurs in vivo and if it does, to try to find ts functional significance. Thus,immunoprecipitation (IP) experiments were performed.The first one gave the proof thet interaction between HMGA2 protein and NPM (transfected and endogenous) occurs. The second IP gave the evidence that interaction occurs between HMGA1a and NPM too. This in vivo protein-protein interaction was to be given a functional significance. We tested by siRNA HMGA1a treatment if downregulation of HMGA1a expression influences the expression of the SOD2 gene (regulated by NPM). The effect of the HMGA1a silencing has proven to be very slight on the SOD2 gene (a 14% of expression reduction). EMSA experiments have been performed in order to test the effect of NPM on the DNA binding properties of the HMGA2 protein with the DNA probes E3 and HCRII. In both cases it has been observed that NPM increases the DNA binding affinity of the HMGA2 protein.
1979
Vignini, Arianna. "Sindrome metabolica e rischio cardiovascolare: un nuovo approccio allo studio degli effetti a livello molecolare." Doctoral thesis, Università Politecnica delle Marche, 2007. http://hdl.handle.net/11566/242476.
Повний текст джерелаDotti, Isabella. "Towards molecular medicine:optimization of the methods for gene expression analysis in clinical samples." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2626.
Повний текст джерелаThe advent of molecular “-omics” technologies enabled an unprecedented view into the inner molecular mechanisms of cancer and enhanced optimism towards a patient-tailored vision of medicine. The successful application of these molecular approaches in the discovery of candidate biomarker has accelerated the shift towards personalization of medicine. Indeed, biomarkers hold great promise for refining our ability to establish early diagnosis and prognosis, and to predict response to therapy. The develoment of clinically useful biomarkers would be impossible without access to human biological specimens and associated patient data, since they complete the molecular information gained from laboratory research. Furthermore, with the advances of sensitive molecular technologies, human bio-specimens can be now successfully used for wide analysis at all molecular levels (DNA, RNA and proteins), in addition to conventional cytologic and histologic investigations. However, despite the hundreds of reports on tumor markers, only a few markers have proven clinically useful. The insufficient experience in clinical application of molecular methods combined with the high complexity of clinical material represent the major obstacles for the development of clinically useful biomarkers. Thanks to the possibility to have access to the fresh and archival samples from the hospital, our laboratory can investigate the potential of technological innovations and the current technical pitfalls directly on clinical material. The work in my thesis is strictly correlated to this activity. In particular, the first part is focused on the technical optimization of molecular methods for gene expression analysis in biological fluids and especially in urine samples. In this context we validated a new experimental kit for total RNA extraction from urine samples and tested the potential of a colorimetric approach for PCR product detection. The major part of the study is focused on the technical optimization of molecular methods for gene expression analysis in archival material. This activity is in step with one of the main objectives of the European project called “Archive tissues: improving molecular medicine research and clinical practice-IMPACTS”, in which my laboratory and other 20 European centres are directly involved. In this phase the comparison of the experiences between laboratories and their active collaboration are essential for a more rapid validation of protocols dedicated to RNA (but also DNA and protein) analysis. In particular, we investigated some molecular aspects involved in the pre-analytical phase (tissue fixation procedures) and analytical phase (RNA extraction, RNA quantification and integrity assessment, qRT-PCR) of tissue processing. The final objective of this activity will be the definition of common technical guidelines for a reliable quantification of molecular biomarkers for diagnosis, prognosis and therapy directly in human archival samples. Finally, my thesis includes the clinical application of molecular methods for the quantification of candidate biomarkers in two archival case studies (a breast cancer and an adrenal gland cancer case study). In the breast cancer case study we showed that a panel of seven genes (involved in different cell pathways) is associated to patients’ survival. The adrenal gland tumor case study is part of a preliminary study about the angiogenetic process in rare human cancers.
1979
Agostini, Stefania. "Il sistema [MV(N)(PNP)]2+ (M=Tc-99m, Re-188) nella scintigrafia miocardica e in medicina nucleare molecolare." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425059.
Повний текст джерелаSinigaglia, Milena. "Dissection of the effects induced by VEGF165, VEGF121 and Semaphorin 3A in endothelial cells." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3258.
Повний текст джерелаThe development of new blood vessels is a complex and highly regulated process that requires the coordinated action of different growth factors. Vascular Endothelial Growth Factor (VEGF) is a powerful inducer of angiogenesis, acting through the metabolic activation, proliferation and migration of endothelial cells. The major angiogenic member of the VEGF family is VEGF-A, a gene which is transcribed to give rise to at least 4 major splicing variants, of 206, 189, 165 and 121 amino acids in humans. The angiogenic effect of the most abundant isoform (VEGF165) is basically mediated by its interaction with VEGFR2 (KDR) and VEGFR1 (Flt-1) as well as with the co-receptor Neuropilin-1 (NP-1). The shortest isoform (VEGF121) binds VEGFR1 and VEGFR2 only, but not NP-1. NP-1, together with PlexinA1, also acts as a receptor of Semaphorin 3A (Sema3A), a factor also involved in vascular patterning, besides its very well known role in axonal guidance. The aim of this project has been the detailed analysis of the peculiarity/specificity of the effect of VEGF165, VEGF121 and Sema3A in endothelial cells. We first exploited an Adeno Associated Virus (AAV)-based gene delivery to unravel the in vivo effect of these cytokines. In particular, we observed that the prolonged expression of the main isoform of VEGF, VEGF165, acted as a powerful inducer of angiogenesis, stimulating the development of both a larger capillary network and the formation of an impressive new set of arterioles. Most surprisingly, an unexpected effect of VEGF165 was also the recruitment to the sites of its expression, of a vast set of mononuclear cells. These cells derived from the bone marrow and expressed a broad set of monocytic markers (CD45+, CD11b+); their presence correlated with the formation of arterioles and the maturation of the VEGF-induced vasculature. Strikingly, VEGF121 (the shorter form unable to bind NP-1) was, on the contrary, unable to induce maturation of the newly formed capillaries to mature arteries and to recruit cells, an observation that suggested that these monocytes might be essential in blood vessel maturation. We found that cell recruitment both in vitro and in vivo strictly depends on NP-1 and that Sema3A, a NP-1 activator, acts as a powerful recruiter of these cells. The expression of VEGF and Sema3A receptors by CD11b+ cells together with the VEGFR2 interaction with NP-1, indicate that these cells could be target of a peculiar VEGF and/or Sema3A induced signalling pathway. Taken together, these results prompted us to develop an in vitro model to unravel the signalling features elicited by the VEGF165, VEGF121 and Sema3A on endothelial cells. In particular, we wanted to dissect these pathways through a proteomic approach involving the detection of the phosphoproteome of endothelial cells expressing specific receptor after treatment with the various ligands of interest. Due to the large amount of growth factors required for this kind of experimentation, we first established a Baculovirus-based system to generate the recombinant factors. For this purpose, we engineered suitable plasmids encoding VEGF165, VEGF121 and Sema3A endowed of a cleavable histidine tag at the N-terminus. Once the recombinant protein expression conditions were set up, we moved to optimize protein purification by affinity chromatography, together with the removal of the tag. The functionality of baculovirus-expressed VEGF165 and VEGF121 was ascertained by the analysis of VEGF receptor phosphorylation, as well as proliferation assays on endothelial cells; a cell contraction assay confirmed that the recombinant Sema3A produced was as active as protein expressed in mammalian cells. To dissect the differential signalling induced by each of the recombinant factors, we explored their transduction pathways in endothelial cell lines expressing the main receptors (KDR and NP1). We set up the lysis conditions and bi-dimensional SDS-PAGE, together with the optimal phospho-proteome staining. The findings obtained, together with phospho-enrichment approaches, strongly supported that peculiar signalling pathways exist in endothelial cells, selectively triggered by VEGF165, VEGF121 and Sema3A, as demonstrated by the differential pattern of phosphorylated proteins induced by the recombinant proteins. Furthermore, we first demonstrated a close similarity in the phospho-tyrosine proteome induced by VEGF165 and Sema3A, at least in the cellular system examined.
1980
Biondi, Roberta <1989>. "Evidenziazione sierologica e molecolare di Chlamydia pneumoniae e Simkania negevensis in pazienti affetti da patologia aterosclerotica carotidea." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2018. http://amsdottorato.unibo.it/8499/1/Tesi%20PhD%20Roberta%20Biondi.pdf.
Повний текст джерелаAtherosclerosis is a multifactorial and complex pathology. Some studies show a possible involvement of microorganisms, but the results are contradictory. The aim of this study is to evaluate the presence of anti- Chlamydia pneumoniae and anti-Simkania negevensis antibodies in sera of patients waiting for carotid endarterectomy (CEA) surgery. In addition, after the surgery it was possible to search DNA of microorganisms into the removed pathological tissue. For the serological evaluation it was used a home-made immunoenzymatic technique, for the molecular evaluation a nested PCR. For this study, 50 patients were enrolled. The obtained results pointed out a positivity of the 28% for IgG anti-Simkania negevensis and of the 10% for IgA; for C. pneumoniae the percentages were of 20% of IgG and 8% for IgA. Molecular data of the PCR obtained were: 34% of positivity for S. negevensis and 26% for C. pneumoniae. The results of this study support the hypothesis that these microorganisms can develop a quiescent or persistent status of the infection, in which it is possible to observe, through technique of PCR, DNA of them into the pathological tissue.
VALLEGA, BERNUCCI DU TREMOUL LUCA. "Impatto del COVID-19 sui decessi ospedalieri. Analisi della casistica in relazione alla diagnosi molecolare di infezione da SARS-CoV-2." Doctoral thesis, Università degli studi di Genova, 2021. http://hdl.handle.net/11567/1046367.
Повний текст джерелаCOVID-19 disease related to SARS-CoV-2 infection causes respiratory symptoms ranging from asymptomatic forms to fatal pneumonia. The lack of specific therapies capable of stopping viral replication aroused great concern, especially due to the high percentage of critically ill patients. Several factors, both geographical and socio-economic, condition mortality rates in different countries, but the most relevant is the infection rate, which reflects in the estimate of the ratio between deceased patients and cases of confirmed infection. Currently the most reliable method for diagnosis is (rt)-Real Time PCR molecular analysis of viral RNA on respiratory tract tissue samples taken with a nasopharyngeal swab, although the accuracy of the test is variable depending on the sampling site (nasal or pharyngeal). In this study we considered people who died in two major hospitals in the Liguria region during the first wave of the sars-cov 2 pandemic, comparing the certified causes of death with the diagnostic results obtained through the molecular diagnosis using nasopharyngeal swabs. The aim is to develop a valid tool useful for assessing the actual impact of the pandemic on the overall mortality rate. Data showed a good match between the diagnoses of death with COVID-19 and the finding of at least one positive swab (89.68%), although in 45 cases (10.32%) the clinical diagnosis is not associated with any molecular confirmation. The analysis revealed an increase in the mortality rate (60.3%), although infection is reported as a cause of death only in 41.3%. The study confirms limitations of the diagnostic test and the negative repercussions for the mortality rate estimation. All this increases the uncertainty about the real impact of the pandemic.
Chiacchiera, Fulvio. "Characterization of a novel p63/p73 interacting protein." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2627.
Повний текст джерелаI tumori sono tra le maggiori cause di morte nelle popolazioni occidentali. D'altra parte anomalie congenite nello sviluppo nonostante non siano ugualmente frequenti richiedono uno sforzo notevole in termini di assistenza e cure da parte delle istituzioni e delle famiglie coinvolte. La comprensione dei processi molecolari alla base di queste patologie è quindi di fondamentale importanza per la medicina. Diverse evidenze sperimentali dimostrano come geni coinvolti nello sviluppo embrionale e nel differenziamento sono spesso coinvolto nella genesi dei tumori. In particolare membri della famiglia di p53 rivestono un ruolo fondamentale nell'omeostasi della cellula e le loro funzioni risultano spesso alterate nei tumori ed in alcune malattie genetiche. a livello molecolare l'attività di queste proteine è finemente regolata tramite una serie di modificazioni post-trascrizionali ed interazioni proteiche. Ogni singolo interattore risulta quindi un possibile bersaglio per nuove strategie farmacologiche. In questo lavoro presentiamo la caratterizzazione del prodotto del gene umano c16orf35, un nuovo interattore di p63e p73 isolato da uno screening volto a cercare nuovi interattori di p53 di Drosophila melanogaster. C16orf35 è una proteina nucleare evolutivamente molto conservata ed espressa ubiquitariamente. è in grado di associare con compartimenti cellulari specifici definiti "stress granules" e "p-bodies" in cui gli RNA subiscono diversi tipi di modificazioni strutturali. L'aumento forzato dei livelli cellulari di c16orf35 induce la formazione degli stress granules ed inibisce la proliferazione di cellule tumorali in coltura. Ciò suggerisce un possibile ruolo di questa proteina nelle vie che regolano la crescita cellulare.
1979
Licastro, Danilo. "Positive selection of hearing loss candidate genes,based on multiple microarray platforms experiments and data mining." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2645.
Повний текст джерелаSecondo le stime del World Health Organization, le perdite uditive colpiscono circa 278 milioni di persone in tutto il mondo. Approssimativamente 1 bambino ogni 100, nasce con problemi d’udito. Nonostante l’identificazione negli ultimi 10 anni di più di 100 loci genetici associati a fenotipi di perdita uditiva, non tutti i corrispettivi geni causativi sono stati identificati. Normalmente utilizzando un approccio sperimentale di linkage tradizionale non è sempre possibile identificare un intervallo genomico sufficientemente corto da essere analizzato per la ricerca di mutazioni. Il lavoro presentato in questa tesi ha lo scopo di selezionare un set limitato di geni potenzialmente coinvolti nelle perdite uditive non sindromiche, utilizzando la combinazione di un approccio biologico e bioinformatico. Il punto di partenza dell’analisi è stato il gene GJB2. Il gene GJB2 codifica la Connessina 26, proteina coinvolta nella formazione delle gap junction tra le cellule, ma anche implicata in più del 50% dei casi di perdite uditive non sindromiche. Per questa ragione è stato suggerito un ruolo chiave nella biologia dell’orecchio, che va oltre la sua funzione di proteina canale. In questa tesi è stato esaminato il profilo d’espressione genica di cellule HeLa transfettate con la forma naturale e con delle forme mutate della Connessina26. Le analisi dei dati hanno identificato numerosi geni differenzialmente espressi e si è quindi deciso di passare ad un approccio informatico per ridurne il numero. Questa analisi ha permesso di identificare 19 geni in 11 loci privi di geni causativi selezionandoli in base alla loro espressione rispetto librerie di cDNA prodotte da orecchio. Sono stati quindi identificati i geni omologhi in topo per 5 dei 19 geni, con lo scopo di verificare la loro rilevanza con la perdita uditiva. Per tutti questi 5 geni è stata confermata l’espressione nell’organo di corti in topo e con Real-time RT-PCR nelle linee cellulari transfettate impiegate negli esperimenti di microarray. Il progetto proseguirà ora con lo screening di mutazioni nei geni candidati in famiglie di pazienti selezionate.
According to WHO estimates hearing impairment affects 278 million people worldwide. Approximately 1/1000 children are born with a significant hearing impairment. To date approximately 100 genetic loci involved in deafness have been described. Despite the fact that such a large number of genetic locations associated with deafness phenotypes are known, not all the genes involved have been identified yet. Using a traditional linkage approach, however, it is not always possible to map a locus to intervals short enough to be amenable for costly mutation analysis. So far no more than 40 deafness genes have been identified and these encode very heterogeneous proteins. The work presented in this thesis aims to identify a limited set of candidate genes with high potential to be involved in Non-Syndromic Hearing Loss using a combination of biological and bioinformatics approaches. The starting point of the analysis was the GJB2 gene. The GJB2 gene encodes for the gap junction protein Connexin26 and is responsible for more than half of the non-syndromic hearing loss cases. For this reason it has been proposed that this protein might play a wider role in the biology of the ear, beyond its mere channel function. I therefore performed whole genome expression profiles of HeLa cells transfected with the wild type form of the GJB2 gene and compared them to that of cells transfected with mutant forms of this gene to shed light on its function. Initially this experiment yielded a bewildering number of differentially expressed genes (4,984). Thus I devised an in silico strategy to narrow down this number, focusing on genes which were positionally linked to specific non-syndromic hereditary hearing loss conditions, as well as found within human ear cDNA libraries, thus potentially causative of the disease. This further analysis yielded 19 genes within 11 loci. In order to assess their relevance to hearing loss, the mouse homologs of these genes were identified for 5 of them and indeed they were all found to be expressed in the mouse organ of corti. These five genes were also validated by Real-time RT-PCR in the human cell line used for the microarray experiments.
1979
Farra, Rossella. "siRNAs targeted against cell cycle related genes as tools to down regulate cell-proliferation in hepatocellular carcinoma and vascular smooth muscle cells." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2651.
Повний текст джерелаABSTRACT Exuberant and non-controlled cellular proliferation underlines the ethio-pathogenesis of many human pathological conditions, including tumour and non-tumour diseases. Thus, the possibility to control this complex process can be extremely useful in terms of prevention/control of disease progression, especially in the light of the limited efficacy of current therapeutic approaches. In this project we draw our attention on the down regulation of cell proliferation in the context of two human diseases, namely hepatocellular carcinoma (HCC), as an example of tumour pathology and in stent- restenosis, example of non-tumour pathology. To explore the possibility to down regulate cell growth, we targeted the transcription factor E2F1 and the serum response factor (SRF) and cyclins E1/E2, all genes implicated in cell cycle progression. As tools to down regulate the expression of the target genes, we used small interfering RNAs, short double stranded RNA molecules able to induce the specific degradation of a homologue mRNA. The presented results indicate that SRF depletion impairs cell proliferation, in primary VSMC and in the most differentiated HCC cell line HepG2, but not in the less differentiated HuH7 and JHH6. Additionally, the depletion of CyE1, CyE2 and E2F1 is effective in preventing all HCC cell expansion, regardless of the differentiation status. However, whereas in HepG2 the major mechanism is the induction of apoptosis, in HuH7 and JHH6 it is the down modulation of cell growth. In conclusion, our project based on the inhibition of cell growth in tumour and non tumour cells by means of siRNAs, can contribute to better understand the complex mechanisms regulating cell proliferation in HCC and in vascular smooth muscle cells. Moreover, these data support the rationale to continue the studies for the development of future novel anti HCC and in-stent restenosis approaches based on the use of siRNAs.
1977
Rossi, Tatiana. "Molecular epidemiology of influenza viruses in three consecutive epidemic season,2005-2007." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2643.
Повний текст джерелаEvery flu season a global network of scientists attempts to identify several predominant flu strains and include them into the next season’s flu vaccine. The objective of this research is describing the main antigenic and molecular features of influenza viruses involved in three consecutive seasons. The prospect that a new influenza virus with a pandemic potential will emerge in humans is of particularly serious concern. We still do not have sufficient understanding of influenza infection in humans to predict whether the H5N1 viruses will became established in humans and, if they do, they will retain their higly virulent phenotype. Therefore monitoring and studying the emerging viruses is important to limit an eventual pandemic. Moreover laboratory - based influenza surveillance is important for identifying and isolating strains candidate to enter new vaccines, the main tool of primary prevention. Although viral isolation remains the basic step for the antigenic and molecular characterization of circulating strains, the introduction of molecular methods has enormously amplified the sensitivity of virological surveillance in recent years. The widespread use of sequencing allowed a better assessment of viral genetic variation.
1979
Moimas, Silvia. "A gene transfer approach, based on Adeno-Associated Viral (AAV) vectors, to study the process of vessel maturation and stabilization." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3311.
Повний текст джерелаThe main goal of angiogenic gene therapy is the formation of functional new blood vessels adequate to restore blood flow in ischemic tissues. Angiogenesis is a complex process, consisting in the sprouting of new capillaries from pre-existing vessels to form an immature vascular network, which subsequently undergoes functional maturation and remodelling. Many factors are involved in this process and, among them, the VEGF family members are universally recognized as the key players. During my PhD I exploited gene transfer by vectors based on the Adeno-Associated Virus (AAV) to express several factors involved in the angiogenic process, in an attempt to define the molecular and cellular mechanisms of vessel maturation and stabilization. Most experiments were performed by vector injection in the mouse and rat skeletal muscle, followed by detailed histological, immunohistochemical and functional analysis. First of all the angiogenic effect driven by two main VEGF isoforms, VEGF165 and VEGF121 was compared. AAV-VEGF165 and AAV-VEGF121 appeared equally able to induce endothelial cell proliferation, leading to the formation of new CD31 positive capillaries. However, only the longest VEGF165 isoform was capable to recruit -SMA positive cells around growing capillaries and therefore giving rise to small arteries. The acquisition of a smooth muscle cell layer can be considered as marker of vessel maturation. This was also confirmed by a permeability assay, which showed that VEGF121-induced vessels were more permeable compared to those induced by VEGF165. Interestingly, the presence of -SMA positive vessels was paralleled by the recruitment of CD11b positive mononuclear cells from the bone marrow, cells which were not recruited by VEGF121. The presence of these infiltrating cells in close proximity to the newly formed arterioles suggested their possible role in smooth muscle cell recruitment and vessel maturation. Real-time PCR allowed observing that the infiltrating CD11b positive cells expressed a cocktail of cytokines implicated in vessel maturation, such as TGF- and PDGF-B. As a proof of concept of the paracrine activity of these cells in vessel maturation, we developed an AAV-PDGF-B vector, which, when co-injected with AAV-VEGF121, was arteriogenic even in absence of cellular infiltration. Thus, the expression of PDGF-B partially substitutes for the cells observed in the muscles injected by AAV-VEGF165 to form arterial vessels. To verify the functionality of the vessels induced by AAV-VEGF165 we delivered this vector to different animal models of tissue ischemia: a flap ischemia model and an in vivo chamber for tissue engineering based on an artero-venous loop. In both the models, VEGF165 expression induced the formation of -SMA positive vessels, which turned out to improve flap survival in the flap models, and to promote the formation of new vascularized tissue in the chamber. Despite the presence of several arteries, other vessels formed by VEGF165 were abnormally enlarged and leaky, often forming vascular lacunae. This observation indicated that VEGF gene transfer might not be sufficient for the formation of a fully functional vascular network, and that other factors might be required in order to achieve functional competence of the neovessels. We observed that the combined expression of VEGF165 with Angiopoietin-1, which is known to stabilize endothelial and mural cell interactions, resulted in a significant reduction of vessel permeability and improved blood flow, as assessed by positron emission tomography (PET) and single photon emission tomography (SPECT). These findings reveal that a fine control of the expression of angiogenic factors is needed to achieve the formation of stable and functional vessels. The presence of -SMA positive cells might be considered as a first step in vessel maturation but further stabilization factors have to take part to the process in order to tighten the cell-cell junctions. Moreover, we showed that a detailed histological and functional analysis ex vivo might not be sufficient to characterized the new vasculature, requiring imaging techniques such as PET or SPECT.
XX Ciclo
1978
Carrer, Alessandro. "AVV-mediated delivery of Semaphorin 3A influences tumor miscroenvironment and inhibits tumorigenesis in vivo." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3131.
Повний текст джерелаLe semaforine di classe 3 costituiscono una piccola famiglia di proteine che sono state inizialmente studiate per la loro capacità di dirigere la crescita del cono assonico durante lo sviluppo. Tuttavia, tali molecole sono state recentemente coinvolte in ulteriori processi biologici, tra cui merita particolare attenzione il processo angiogenetico, in cui diverse semaforine sembrano partecipare attivamente anche se con ruoli e funzioni variegati. Tale eterogeneità funzionale ha spesso dato origine a risultati apparentemente contraddittori, dovuti per lo più alla scarsa conoscenza del reale coinvolgimento delle diverse semaforine nell’angiogenesi. Questo studio è stato incentrato sul ruolo della semaforina-3A (Sema3A), analizzando nel dettaglio i mutamenti apportati al microambiente locale da una sua overespressione in vivo. Questa proprietà non è stata finora riportata in letteratura e si esplica principalmente attraverso il reclutamento di cellule mononucleate di derivazione midollare. Ciò è stato inizialmente osservato nel muscolo scheletrico di topi wild-type a seguito di trasferimento genico della Sema3A mediate tecnologia AAV. Sia a 15 giorni che a un mese dopo l’iniezione del vettore virale è stato possibile osservare la presenza di un imponente numero di cellule mononucleate che infiltravano le fibre muscolari. Attraverso analoghi esperimenti mediante vettori AAV, il nostro laboratorio aveva precedentemente dimostrato un simile richiamo di cellule a seguito di espressione di VEGF165, un potente fattore pro-angiogenetico (Arsic et al, Mol. Ther., 2003). Viceversa, molte indicazioni suggerivano che Sema3A fosse in realtà un fattore anti-angiogenetico, come successivamente è stato appurato (Acevedo et al, Blood, 2008 & Zacchigna et al, J.Clin.Invest., 2008). Era perciò ragionevole pensare che i due fattori (VEGF165 e Sema3A) reclutassero in situ due popolazioni cellulari differenti, in grado di accompagnare attività biologiche contrapposte. Diversi approcci sperimentali, tuttavia, ci hanno condotto a sostenere l’ipotesi che entrambe le molecole in realtà richiamino la medesima sub-popolazione mieloide agendo attraverso l’attivazione di neuropilina-1 (Nrp-1), un recettore in grado di legare sia VEGF165 che Sema3A. Da notare che VEGF121, un’isoforma del gene VEGF carente dell’esone 7, quando overespressa nel muscolo scheletrico non causa il reclutamento di cellule infiltranti, coerentemente con la sua incapacità di legare Nrp-1. Oltre a ciò, l’espressione ectopica di VEGF121, contrariamente a quanto accade per VEGF165, non determina la formazione di arteriole (vasi di media grandezza, ricoperti da cellule della parete vascolare come periciti e cellule muscolari lisce), pur causando un eguale aumento del letto vascolare attraverso iper-proliferazione capillare. Ciò ci ha indotto a pensare che la presenza di cellule richiamate attraverso Nrp-1 (chiamate da noi NEM) sia in realtà indispensabile per la maturazione dei vasi neo-formati. In effetti, la co-iniezione di AAV-VEGF121 e NEM ci ha permesso di osservare a 15 giorni un fenotipo chiaramente arteriogenico (presenza di vasi α-SMA+), contrariamente al fenotipo esclusivamente capillogenico determinato dalla sola iniezione di AAV-VEGF121. Attraverso una caratterizzazione estensiva delle cellule richiamate da VEGF165 o Sema3A, abbiamo potuto riconoscere i NEM come una popolazione mieloide precedentemente mai descritta e differente da altre popolazioni midollari coinvolte nell’angiogenesi. In aggiunta, attraverso l’analisi della loro espressione genica abbiamo potuto riconoscere alcuni geni tipicamente espressi da macrofagi polarizzati in senso M1, noti per essere pro-infiammatori e anti-tumorigenici. I NEM sono stati inoltre visti esprimere alti livelli di PDGFβ e TGFβ, due molecole coinvolte nella maturazione vascolare. Il fenotipo M1-like e la capacità di indurre la maturazione della vascolatura sono entrambe caratteristiche potenzialmente in grado di influire negativamente sulla crescita tumorale. Abbiamo quindi deciso di investigare più nel dettaglio come il reclutamento di questo particolare tipo cellulare influisse su un modello tumorale ottenuto tramite xenotrapianto di cellule singeniche direttamente nel muscolo scheletrico. Muscolo precedentemente condizionato mediante trasferimento genico di VEGF165, VEGF121 o Sema3A. In linea con l’ipotesi da dimostrare, la precedente espressione di VEGF165 o Sema3A rende meno efficace l’attecchimento del tumore, rallentandone la crescita, mentre il trasferimento del gene codificante per VEGF121 addirittura accelera tale crescita, come d’altra parte prevedibile per un fattore angiogenetico. Una dettagliata analisi morfologica della rete vascolare tumorale ci ha permesso di constatare come il richiamo di NEM mediante pre-condizionamento da Sema3A causasse una più matura struttura vascolare (in termini di mural cell coverage, di dimensioni e di tortuosità dei vasi). Per avvalorare definitivamente il ruolo dei NEM nell’inibizione tumorale osservata dopo trasferimento genico, abbiamo purificato tali cellule direttamente da muscoli scheletrici di topi precedentemente iniettati con AAV-Sema3A. Tali cellule sono poi state amministrate direttamente in masse tumorali in crescita in topi singenici. Esse sono risultate effettive nel rallentare la crescita tumorale in fasi tardive, consistentemente con una loro capacità di sostenere la maturazione della vascolatura tumorale. Un’analisi più approfondita di ha permesso di rilevare diversi segni di una “normalizzazione” vascolare a seguito della somministrazione dei NEM, essendo i vasi più maturi, meno dilatati, meno tortuosi e, dato importante, meno permeabili.
1980
Bisso, Andrea. "New microRNAS regulating the P53 signaling pathway." Doctoral thesis, Università degli studi di Trieste, 2010. http://hdl.handle.net/10077/3594.
Повний текст джерелаA vast body of evidence from clinical and basic research studies has demonstrated that the p53 pathway acts as an essential barrier in preventing cancer onset and development. ------------------------ A vast body of evidence from clinical and basic research studies has demonstrated that the p53 pathway acts as an essential barrier in preventing cancer onset and development. p53 receives and integrates a wide variety of cytotoxic and genotoxic stress signals from upstream sensors translating them into different cellular outcomes, ranging from apoptosis, cell cycle arrest, se-nescence, DNA repair or other tumor-suppressive responses. p53 exerts its role mainly at the transcriptional level and its timely activation/inactivation in response to stress depends on a complex repertoire of post-translational modifications and interactions with proteins. The crucial role of the p53 pathway in tumor suppression is highlighted by the fact that almost all tumors select for its functional inactivation, either by directly mutating the p53 gene or by altering the expression and functions of key p53 regulators and effectors. Therefore, identification of cellular factors that modulate this pathway and that could be altered in cancer cells, thus allowing to eva-de p53 control, is crucial for understanding cancer development and for designing novel effecti-ve therapeutic approaches. In this context, the aberrant expression or function of microRNAs (miRNAs) might be highly relevant. microRNAs are small non coding RNAs that finely regulate gene expression by binding the 3’UTR of their target mRNAs, thus altering their translation, stability and localization. It has been shown that several miRNAs modulate critical cellular processes deregulated in cancer, ac-ting either as oncogenes or tumor suppressors. On this basis, the aim of this thesis has been the identification and characterization of novel miRNAs able to modulate p53 functions by altering either upstream regulators (i.e. stress-activated kinases) or cofactors of p53. With this purpose we initially selected a panel of twenty-one candidate oncogenic miRNAs from the literature. First, we tested these miRNAs in a functional screening for their ability to modula-te p53-dependent functions in response to cisplatin (apoptosis) and nutlin-3 (cell cycle arrest). Second, miRNAs were also screened for their ability to impair p53 transcriptional activity through a reporter-based assay. We identified five candidate miRNAs from the first approach, and three from the second. miR-26a was identified through both approaches, suggesting that it might repersent a critical modulator of p53 functions. We demonstrated that miR-26a overe-xpression is able to dampen p53 transcriptional activity towards several p53 target promoters (Bax, Pig3 and p21). Moreover, we have shown that miR-26a strongly reduces p53-dependent apoptosis upon DNA damage in different cell lines, to a similar extent as obtained by RNAi-mediated p53 knock-down. At the molecular level, we observed that miR-26a impairs p53 activation by targeting multiple stress-activated kinases that phosphorylate p53, such as ATM, HIPK2 and PKCδ. Accordingly, miR-26a reduces p53 phosphorylation on Ser15 and Ser46 upon DNA damage. As a consequen-ce, miR-26a overexpression allows cells to proliferate in the presence of oncogenic stress, bypassing the induction of senescence (OIS) driven by RASV12 oncogene similar to what obtai-ned by knockdown of either p53 or its activationg kinase ATM. Considering our data and the reports describing miR-26a overexpression in several tumors (e.g. glioblastomas), we speculate that aberrant expression of miR-26a might represent an oncogenic process by preventing activation of the p53 pathway and thus relieving a primary barrier against transformation. For all these reasons, the perspective to block miR-26a expression/functions could represent a new important way to tackle tumors.
XXI Ciclo
1981
Mazzone, Graciela Lujan. "Role of unconjugated bilirubin in the endothelial dysfunction." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2647.
Повний текст джерелаAtherosclerosis, a progressive cardiovascular disease, is characterized by the accumulation of cholesterol in macrophage deposits (foam cells) and the formation of atherosclerotic plaques in the walls large- and medium- sized arteries. The earliest events in the development of atherosclerosis involve progressive modifications in the endothelial micro-environment. This endothelial dysfunction is a complex of multi-step mechanisms, for which reduced NO levels have been reported as a marker, is characterized by increasing expression of adhesion molecules (AMs), which mediate the diapedesis (migration) of inflammatory and immunocompetent cells through the endothelial layer into the arterial wall. NO is synthesized intracellularly by nitric oxide enzymes (eNOS and iNOS) and is regulated by a variety of stimuli. NO acts as an autocrine or paracrine hormone, as well as intracellular messenger, with a critical role in vascular endothelial growth factor-induced angiogenesis and vascular hyperpermeability in vitro. The over-expression of AMs is orchestrated by pro-inflammatory cytokines, particularly TNF-alpha. The two major subsets of AMs participating in these processes are the selectins, in particular E-selectin, and the immunoglobulin gene superfamily, in particular vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Transcriptional regulation of these inflammatory genes requires the participation of several proteins, inducible activators, as: NF-kappa B and (CRE)-binding protein (CREB). The most abundant form of NF-kappa B is an heterodimer of p50 and p65; which is sequestered in the cytoplasm in an inactive form through interaction with the I kappa B inhibitor proteins. Signals that induce NF-kappa B release dimmers to enter to the nucleus and induce gene expression. Pyrridoline dithiocarbamate (PDTC) a metal-chelating compound inhibits NF-kappa B by blocking ubiquitine ligase activity towards phosphorylated I kappa B, in turn downregulating the expression of E-selectin, VCAM-1 and ICAM-1. CREB is a widely expressed DNA-binding protein, downstream target of cAMP, activated by phosphorylation on serine 133. A regulatory site, on the gene promoters of both E-selectin and VCAM-1, binds both NF-kappa B and CREB transcription factors. Unconjugated bilirubin (UCB), long considered to be simply a waste end product of heme metabolism and a marker for hepatobiliary disorders, is now thought to function as an endogenous tissue protector by attenuating free radical-mediated damage to both lipids and proteins. There is increasing epidemiological evidence supporting an inverse association between cardiovascular disease and plasma levels of bilirubin. Recent studies indicated that bilirubin may be protective in the development of atherosclerotic diseases by inhibiting the proliferation of vascular smooth muscle cells by mechanisms yet to be established. It has been proposed that UCB can interfere with the atherosclerotic disease development by inhibiting the trans-endothelial vascular cell adhesion molecule (VCAM-1)-dependent migration of monocytes into the intima. The aim of this study is to investigate the effect of the UCB in the endothelial dysfunction. Specifically UCB effects on NO production, AMs expression and the regulatory transcription factors involve in the inflammatory response. Variable doses of free bilirubin (Bf) (the active form of UCB in plasma), simulating upper normal (15 nM) and modestly elevated levels (30 nM) for plasma, were evaluated in two models of endothelial cells. A) H5V, murine microvascular endothelial cell line, and B) HUVEC (Human Umbilical Vein Endothelial Cells), isolated from the vein of human umbilical cord. TNF-alpha (20 ng/mL) was added in order to reproduce, in vitro, the endothelial dysfunction and describe UCB contribution on its effects. UCB alone reduced the viability in H5V cells by MTT assay in a dose dependent manner after 24 hours while no effect was observed in the LDH released. In the first set of experiments NO production in H5V cells was evaluated, a time-depended increase on NO basal and a dose-dependent decrease on NO concentration after TNF-alpha (20 ng/mL) were observed. NO reduction related TNF-alpha was seen at all times studied. The effect of UCB was studied in co-treatments with TNF-alpha for 24 and 48 hours. UCB (Bf 15 and 30 nM) significantly reversed the reduction of nitrite content induced by TNF-alpha at 48 hours. The gene expression analysis was performed by Real Time PCR technology with specific primers for eNOS, iNOS, E-selectin, VCAM-1 and ICAM-1. In H5V cells, TNF-alpha increased the expression of all the genes studied (except eNOS) at 2, 6 and 24 hours. The co-treatment with UCB, at a Bf that did not themselves affect the expression of the three adhesion molecules, blunts the over-expression of E-selectin, Vcam-1 and iNOS induced by a pro-inflammatory cytokine such as TNF-alpha. The inhibitory effect of UCB was usually modest (20-30%) and detected at 2 and/or 6 hours, but had disappeared 24 hours. Furthermore, a synergistic effect between TNF-alpha and UCB was seen on the expression of iNOS at 24 hours, indicating a biphasic regulation. Moreover, no effects were seen on eNOS. Similar results were observed in the regulation of the gene expression of the AMs and viability in HUVEC cells, indicating the lack of species specific effect. However, no effect of TNF-alpha or UCB was seen in the expression of iNOS, eNOS or NO content. Western blot analysis in H5V cells confirmed that TNF-alpha induced the expression of E-selectin, VCAM-1 and ICAM-1 in a time-dependent manner. This effect was blunted after 24 hours by the presence of UCB (Bf 15 and 30 nM). The contribution of NF-kappa B pathway in UCB effects was investigated by addition of a specific inhibitor, PDTC. The co-treatment with PDTC and UCB for 2 hours produced an additive reduction of TNF-alpha effect on Eselectin, VCAM-1, and iNOS in H5V cells. In addition, UCB prevented the nuclear translocation of NF-kappa B induced by TNF-alpha. Failure of UCB to alter TNF-alpha -induced phosphorylation of CREB (at Ser 133) suggested that the CREB pathway was not involved in the UCB inhibition. The results obtained in the present study shows that unconjugated bilirubin, even at upper-normal physiological (15 nM) and mildly elevated (30 nM) Bf can modulate gene expression and endothelial cell function. Furthermore, UCB may regulate NO levels by a biphasic regulation of iNOS, and in addition influences the expression of the endothelial adhesion molecules. In summary, these data indicates that bilirubin limits the over-expression of the adhesion molecules and regulates the NO metabolism in the pro-inflammatory state induced by the cytokine TNF-alpha. Even though UCB alone does not alter these markers. UCB effects are mediated in part by a modulation of the NF-kappa B transcription factor. These results support the concept that modestly elevated concentrations of bilirubin may help prevent atherosclerotic disease as suggested by epidemiological studies.
La malattia cardiovascolare è la causa di morte e disabilità più importante nel mondo occidentale e tra le malattie cardiovascolari, quella vascolare aterosclerotica rappresenta la percentuale maggiore. Le lesioni aterosclerotiche si creano principalmente nelle arterie di medio e grande calibro con parete muscolare elastica e possono determinare ischemia nei tessuti a valle della lesione stessa. Numerose osservazioni epidemiologiche hanno riscontrato una significativa correlazione inversa tra incidenza di malattia cardiovascolare ischemica e livelli serici di bilirubina. Sono stati pubblicati studi di prevalenza su campioni di popolazione maschile e sulla popolazione generale, studi prospettici su soggetti con iperbilirubinemia non coniugata (Sindrome di Gilbert) o nella popolazione generale. Questi studi hanno confermato l'importanza dei valori di bilirubinemia nella valutazione del rischio d'incidenza di malattia ischemica coronarica. In alcuni casi, il valore di bilirubinemia è addirittura identificabile come fattore di rischio indipendente di malattia. Analoghe osservazioni sono state documentate nella prevalenza della malattia vascolare periferica aterosclerotica. L'endotelio è un tessuto in grado di rispondere ai vari stimoli con la produzione di sostanze vasoattive, tra cui l'ossido nitrico (NO) sintetizato principalmente dagli enzimi ossido nitrico sintetasi (iNOS ed eNOS), e le molecole di adesione (AMs), E-selectina, VCAM-1 e ICAM-1. Il primo evento che si verifica nella insorgenza di una lesione aterosclerotica è la comparsa della disfunzione endoteliale. Per disfunzione endoteliale si intende una successione di condizioni patologiche che coinvolgono l'endotelio vascolare determinate da una ridotta biodisponibilità di NO e un aumento dell'espressione delle molecole di adesione. Questi eventi favorirebbero l'adesione di linfo/monociti e la successiva migrazione nell'intima. Una riduzione di NO e l'incremento dell'espressione dalle AMs nelle cellule endoteliali stesse avviene nel processo infiammatorio attraverso l'induzione di fattori di trascrizioni tra cui NF-kappa B e CREB. Questo progetto di ricerca si pone come obiettivo lo studio degli effetti della bilirubina non coniugata (UCB) a concentrazioni fisiologiche normali o lievemente incrementate sull'insorgenza della disfunzione endoteliale, prima manifestazione della malattia aterosclerotica. Specificamente, studiare l'effetto della UCB libera (Bf), che è la porzione attiva di UCB in plasma non legata all'albumine, nella regolazione della produzione di NO e l'espressione delle AMs. Lo studio è stato condotto utilizzando due modelli in vitro di cellule endoteliali. a) H5V, linea cellulare immortalizzata di topo e b) HUVEC cultura primaria di cellule umane, della vena ombelicale (Human Umbilical Vein Endothelial Cells). La citochina TNF-alpha (20 ng/mL) è stata utilizzata per indurre lo stato di disfunzione endoteliale. Lo studio ha evidenziato che livelli elevati di bilirubina non coniugata libera (Bf 100 nM) riescono a ridurre significativamente la vitalità cellulare valutata attraverso il test MTT senza provocare variazioni nella liberazione di LDH. Inoltre è stato evidenziato un incremento basale del livello extracellulare di NO in maniera tempo dipendente. Il trattamento con TNF-alpha provoca una riduzione dose dipendente del livello extracellulare di NO a tutti i tempi di trattamento. UCB riesce a modulare l'effetto di TNF-alpha dopo 48 ore di cotrattamento. Questo effetto si correla con un incremento nell'espressione genica di iNOS dopo 24 ore. A per tempi brevi di trattamento (2 e 6 ore) UCB riduce l'espressione genica di E-selectina, VCAM-1 in entrambi tipi cellulari, dimostrando l'assenza di un effetto specie-specifico. Inoltre è stata anche osservata, nelle cellule H5V una riduzione dell'espressione di iNOS dopo 2 ore. Alla riduzione dell'espressione genica delle AMs segue la riduzione della espressione proteica dopo 24 ore di trattamento nelle cellule H5V. Un effetto inibitorio additivo all'azione dell'UCB si è ottenuto inibendo in modo specifico il fattore di trascrizione NF-kappa B con PDTC. In seguito a trattamento con UCB si è riscontrata una riduzione della localizzazione di NF-kappa B nel nucleo e nessun effetto sulla fosforilazione del fattore di trascrizione CREB. Questi risultati confermano il coinvolgimento di UCB a concentrazioni fisiologiche normali (Bf 15 nM) o lievemente elevate (Bf 30 nM) nella regolazione dal metabolismo del NO e nella modulazione dell'espressione delle AMs indotte da TNF-alpha, anche se UCB non influisce di per sé su codesti marcatori. Questo lavoro conferma che l'effetto della bilirubina, in moderate concentrazione fisiologiche, è coinvolta nella prevenzione delle malattie aterosclerotiche, secondo quanto suggerito dagli studi epidemiologici.
1977
Tocco, Francesca. "Modulation of p53 activities by the prolyl-isomerase PIN1 and the bromodomain protein BRD7." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2622.
Повний текст джерелаABSTRACT: MODULATION OF p53 ACTIVITIES BY THE PROLYL-ISOMERASE PIN1 AND THE BROMODOMAIN PROTEIN BRD7 The tumour suppressor p53 belongs to a family of transcription factors that play key roles in maintaining genomic stability and cellular homeostasis. The orchestration of the appropriate cellular responses depends on the fine regulation of p53’s functions through post-translational modifications and interaction with other proteins. In many years of intense study a considerable knowledge on p53 activity has been achieved, yet a greater insight is needed on the specificity of its response. In the first part of this thesis a novel mechanism in the regulation of p53-mediated apoptotic response has been disclosed. It has been demonstrated that upon severe stress signalling p53 dissociates from iASPP, an anti-apoptotic co-factor that inhibits p53 apoptotic functions, and that key roles in this process are played by the prolyl isomerase Pin1. Moreover, it emerged that phosphorylation at p53 Ser46 is required for Pin1-mediated dissociation of the p53-iASPP complex thus providing a mechanistic explanation for the relevance of this site in p53 mediated apoptosis. Notably, the role of Pin1 in assisting the dissociation of p53 from iASPP appears to be independent from Pin1-induced acetylation of p53 and dissociation from Mdm2, further confirming that Pin1 may modulate p53 activity at different levels. A different approach to gain insight on the mechanisms governing p53 regulation is the analysis of p53 protein interaction profiles. The bromodomain containing protein Brd7 was identified as a common interactor of the p53 family proteins in a yeast two hybrid screening conducted in our lab. The presence of the bromodomain and evidences from literature made Brd7 a promising candidate for modulating the p53 pathway at the transcriptional level. This protein and its functional interaction with p53 have been therefore characterized in the second part of this thesis. Upon depletion of Brd7 expression in cells it has been demonstrated that Brd7 is required for efficient cell-cycle arrest in U2OS cells upon challenging with genotoxic stimuli. This effect appeared to be due to a reduction in p21 expression that occurred upon Brd7 depletion and under stress condition. The down-regulation of p21 as a consequence of Brd7 silencing occurred at the transcriptional level and proved to be p53-depedent. Taken together the data reported in the second part of this thesis suggest a role for Brd7 as a positive regulator of p53 transcriptional activity during cell-cycle arrest response and that this function might be exerted by regulating p53-mediated transcription on a chromatin context. Further analysis is needed to dissct the role of this functional interaction. Yet, preliminary investigation suggest that Brd7 might be an important modulator of p53 response and that it can be an important means for p53 to crosstalk with other signalling pathway. Together, the data presented in this thesis contribute to achieve greater knowledge on the mechanism that govern p53 response. As the p53 pathway is compromised to some degree in almost all human cancers, this would be also of great relevance in designing new targeted strategies for cancer treatment.
Riassunto: Modulazione delle attività di p53 da parte della prolyl isomerasi Pin1 e della proteina contenente dominio Bromo Brd7 L’oncosoppressore p53 appartiene a una famiglia di fattori di trascrzione che svolge un ruolo fondamentale nel mantenimento della stabilità genomica e dell’omeostasi cellulare. L’attuazione di un’appropriata risposta cellulare dipende molto dalla regolazione fine delle funzioni di p53. Ciò avviene attraverso modificazioni post-traduzionali e interazioni con altre proteine cellulari. In molti anni di intenso studio si è raggiunta una notevole conoscenza sull’ attività di p53 ma ancora non è stato definito cosa regoli la specificità della risposta. Nella prima parte di questa tesi è stato portato alla luce un nuovo meccanismo nella regolazione della risposta apoptotica mediata da p53. E’ stato dimostrato che, in seguito a stress intensi, p53 si dissocia da iASPP, un co-fattore anti-apoptotico che inibisce le funzioni apoptotiche di p53 e che la prolyl-isomerasi in1 gioca un ruolo fondamentale in questo processo. In aggiunta è emerso che la fosforilazione di p53 al residuo Ser46 è necessaria al distacco di p53 da iASPP mediato da Pin1, fornendo una spiegazione meccanicistica alla nota rilevanza di questo siro per l’apoptosi mediata da p53. Interessantemente, il ruolo di Pin1 nell’assistere la dissociazione di p53 da iASPP sembra essere indipendente dalla sua capacità di favorirne l’acetilazione e il distacco da Mdm2, così confermando che Pin1 può modulare l’attività di p53 a diversi livelli. Un differente approccio per avere delucidazioni sui meccanismi che governano la regolazione di p53 è l’analisi del profilo di interazione proteica. La proteina Brd7 (contenente dominio Bromo) è stata identificata come comune interattore dei membri della famiglia di p53 in uno screening di doppio ibrido in lievito, condotto nel nostro laboratorio. La presenza del dominio Bromo e alcune evidenze di letteratura hanno reso Brd7 un candidato promettente come modulatore trascrizionale della via di segnalazione di p53. Brd7 e la sua interazione funzionale con p53 sono stati caratterizzati nella seconda parte di questa tesi. Dopo aver bloccato l’espressione di Brd7 in cellule è stato dimostrato che Brd7 è necessario per un efficiente arresto del ciclo cellulare in seguito a danni genotossici. Questo effetto sembra essere dovuto ad una riduzione nell’espressione di p21 che avviene I seguito alla deplezione di Brd7 e in condizioni di stress.Tale riduzione avviene a livello trascrizionale ed è p53-dipendente. Insieme I dati riportati nella seconda parte della tesi indicano un ruolo per Brd7 come regolatore positivo dell’attività trascrizionale di p53 durante l’arresto del ciclo cellulare e ciò può avvenire attraverso una regolazione a livello della cromatina. Insieme, I dati presentati in questa tesi contribuiscono ad una maggiore conoscenza sui meccanismi che governano la risposta di p53. Dato che la via di segnalazione di p53 è compromessa a qaulche livello in quasi tutti i tumori, questo potrebbe essere rilevante per disegnare nuove terapie mirate per la cura del cancro.
1978
Morgese, Fabio. "Identification of putative interactors of Fanconi anaemia proteins by yeast to hybrid system: characterization of two novel genes highly expressed during spermatogenesis." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3134.
Повний текст джерелаFanconi Anaemia (FA) is a rare human genetic disease characterized by bone marrow failure, malformations, chromosomal instability and cancer susceptibility. Thirteen genes belonging to a common pathway have been identified, but their function is still unclear even if evidence indicates a role in DNA-repair. In the attempt to gain new insights on FA-BRCA pathway, this work aimed at finding and characterizing novel putative interactors of the FA proteins. Using the yeast two-hybrid system, we screened a cDNA library of human testis and rescued two clones. Clone 54, which encoded for a putative ubiquitin-conjugating enzyme E2 (UBE2U), was first found to interact with FANCD2 and then with FANCL (E3 ubiquitin-ligase of the FA pathway), FANCC, FANCE and FANCF by direct interaction mating in yeast. Different assays indicated that the expression of this gene is limited to mouse and human testis (specifically in spermatocytes and spermatides). Interestingly, even mouse Fancd2 showed a high expression level in these two cell types, supporting the hypothesis of an interaction between the two proteins and a role of the FA-BRCA pathway during spermatogenesis. In order to confirm the binding between UBE2U and FANCD2, we transiently transfected cell lines with a tagged UBE2U. However, since we failed to detect the protein at any level, we tried to validate the interaction using mouse testis extract. Using the specific antibody we generated, we were however not able to confirm the binding, but, before excluding definitively the interaction, we should further investigate using more suitable antibodies. Clone 4, encoding for a novel putative exonuclease (ISG20L2), was instead found to interact with the C-terminus of FANCG. Though it was ubiquitously present at low levels in all the cells tested, it showed a stronger expression in mouse testis. In transiently transfected cells, ISG20L2 was detected primarily in nucleoli by immunofluorescence, but it was revealed also in the cytoplasmic fraction by western blot. Both nuclear and cytoplasmic distributions of the protein were confirmed at endogenous levels, after production of a specific antibody. Coimmunoprecipitation studies between ISG20L2 and FANCG did not confirm their interaction, but this might be in agreement with a recent report for ISG20L2 as a nucleolar exoribonuclease, not directly involved with DNArepair.
1976
Saracino, Ilaria Maria <1980>. "Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3499/1/saracino_ilariamaria_tesi.pdf.
Повний текст джерелаSaracino, Ilaria Maria <1980>. "Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3499/.
Повний текст джерелаTiberi, Luca. "Human notch1 and pin1 unveil a molecular circuitry involved in tumorigenesis." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2623.
Повний текст джерелаCancers arise as somatic cells mutate and escape the control of cell cycle, survival or death. These processes are normally regulated by several signalling pathways and recently several studies highlight the potential importance of the Notch1 signaling pathway in human cancer. Notch activation has been involved in several mouse models of mammary carcinogenesis and recently also in human breast cancer. Importantly loss of Numb, a negative regulator of Notch1, has been observed in about 50% of breast carcinomas (Pece et al., 2004). Expression of Notch1 and Ras correlates in breast carcinomas and expression of the Notch1 pathway ligand Jagged1 is a potential markers for breast cancer progression (Reedijk et al., 2005). Furthermore, the Notch1 interplay with pathways such as Wnt, c-Myc, is subverted in breast cancer, thus suggesting that Notch could be a target for drug-based therapy of breast cancer . Intriguingly also the prolyl-isomerase Pin1 is crucial in breast cancer and in several human malignancies through modulation of these pathways. Alterations of Pin1 have been implicated in the amplification of oncogenic signals, as demostarted by its cooperation with the Neu/Ras pathway in mammary tumorigenesis (Wulf et al., 2001) . The aim of this work was to unveil a possible role of Pin1 in the regulation of Notch1 pathway in breast cancer. Pin1 directly interacts with phosphorylated Notch1, and increases Notch1 cleavage by gamma-secretase. Accordingly, Pin1 contributes to Notch1 transforming properties in human breast cells. Notch1 in turn up-regulates Pin1, thus establishing a feed-forward loop that amplifies Notch1 signalling. Importantly human breast cancers bearing elevated levels of Pin1 have also deregulated expression of activated Notch1 and Hes-1 and these data underscore the relevance of our observations for human carcinogenesis.
La pathway di Notch1 è molto importante in molti processi cellulari come, il mantenimento delle cellule staminali, il differenziamento e nel controllo del cell-fate decision. Infatti la deregolazione della pathway di Notch1 è stata associata a molti disordini durante lo sviluppo embrionale e a molti tipi di tumori umani. Notch1 è un recettore di membrana e la sua attivazione passa attraverso il legame con un ligando che provoca due successivi tagli del recettore, liberando un frammento citoplasmatico chiamato dominio intracellulare di Notch1 (N1ICD). N1ICD trasloca nel nucleo e modula l'espressione di molti geni bersaglio. Recentemente è stato trovato un coinvolgimento della pathway di Notch1in molti tumori umani compreso quello della mammella. Inoltre Notch1 interagisce con molte pathway coinvolte in questo tipo di tumore, come la pathway di Ras, Wnt e c-myc. Interessantemente le varie proteine coinvolte in queste pathway sono regolate direttamente da segnali di fosforilazione in Serine o Treonine che precedono una Prolina. Recentemente la prolil-isomerasi Pin1, un enzima che lega e isomerizza questo tipo di legami fosfo Ser/Tre-Pro, è emersa come un importante regolatore di molto substrati fosforilati che a loro volta sono coinvolti nel ciclo cellulare, nella replicazione del DNA e nell'apoptosi. Pin1 è over-espressa in molti tumori umani e recentemente è stato trovato un suo coinvolgimento nel tumore della mammella e nella regolazione della pathway di Ras ed ErbB2. Durante il mio dottorato abbiamo trovato che Pin1 lega il recettore Notch1 e ne regola la sua attivazione. Pin1 potenzia il taglio di Notch1 da parte della gamma secretasi, portando ad un incremento dei livelli proteici di N1ICD e aumentando la capacità trasformante di Notch1. Inoltre abbiamo trovato che N1ICD è in grado di indurre la trascrizione di Pin1 così da generare un circuito positivo tra le due proteine. Analizzando 150 tumori umani della mammella abbiamo osservato una forte correlazione tra l'over-espressione di Pin1 e l'attivazione della pathway di Notch1. Interessantemente questo circuito molecolare tra Notch1 e Pin1 potrebbe svolgere un ruolo molto importante nel tumore della mammella.
1979
Gazzin, Silvia. "Effect of bilirubin on expression and localization of PGP and Mrp1 in the central nervous system." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2625.
Повний текст джерелаINTRODUZIONE A basse concentrazioni la bilirubina non coniugata (unconjugated bilirubin, UCB) prodotta dalla degradazione dell’emoglobina, sembra essere un potente anti-ossidante, mentre è estremamente dannosa ad alte concentrazioni, causando encefalopatia nei neonati con severo ittero. Il 70% dei bambini che presentano kernittero muoiono entro sette giorni di vita, mentre il 30% dei sopravvissuti manifesta irreversibili conseguenze come sordità, ritardo mentale e danni cerebrali permanenti. L’encefalopatia dovuta ad alti livelli di bilirubina rappresenta oggi la maggior causa di riammissione ospedaliera nei neonati entro il primo mese di vita. Storicamente gli studi riguardanti le modalità di ingresso della bilirubina nel sistema nervoso centrale si sono concentrati sulla barriera emato-encefalica (blood brain barrier, BBB), costituita dai microvasi e dai capillari del cervello (micro vessels, MV). Tali studi hanno dimostrato come solamente la bilirubina non coniugata e non legata all’albumina del sangue, definita “bilirubina libera” (free bilirubin, Bf) sia capace di attraversare le membrane cellulari e diffondere nel tessuto. Tuttavia i microvasi non sono l’unica interfaccia sangue-tessuto presente nel cervello. Una seconda barriera è costituita dai plessi coroidei (CP). Questi, collocati nei ventricoli del cervello, mediano il passaggio delle molecole dal sangue al liquido cefalorachidiano e viceversa, posseggono una ampia superficie di scambio, il più alto flusso sanguigno del sistema nervoso centrale ed un fenotipo barriera meno restrittivo rispetto ai microvasi del parenchima. L’ingresso della bilirubina nel cervello sembra essere attivamente controllato da due trasportatori appartenenti alla famiglia delle “ATP dependent transporters”, Mrp1 e Pgp. Tali trasportatori potrebbero mantenere bassa la concentrazione della bilirubina limitandone l’ ingresso a livello di barriere o agendo direttamente a livello delle cellule del parenchima. Nonostante l’ impatto di questi trasportatori sulla disponibilità nel sistema nervoso centrale non solo della bilirubina ma egualmente di atre molecole potenzialmente tossiche, così come dei principi attivi, la loro espressione e localizzazione nelle interfacce sangue-cervello non sono del tutto chiare. Per tali motivi il lavoro di questi tre anni di tersi è stato incentrato a: Ia) chiarire il livello di espressione proteica relativa di Mrp1 e Pgp nelle due principali barriere cerebrali, la BBB (blood brain barrier, barriera emano encefalica) e la BCSFB (blood CerebroSpinal Fluid barrier, barriera emato liquorale); Ib) Definire l’andamento della loro espressione nel corso dello sviluppo post-natale in situazione fisiologica. II) Valutare l’effetto di elevati livelli serici di bilirubina sull’espressione di Mrp1 e Pgp nelle barriere emato encefaliche, come prima linea di difesa verso la bilirubina nel kernittero. Per raggiungere questo secondo obiettivo abbiamo utilizzato il ratto Gunn, considerato il modello in vivo per la sindrome di Crigler-Najjar e il kernittero. I ratti Gunn presentano elevati livelli di bilirubina serica ed un quadro clinico simile a quanto si riscontra nell’uomo. L’iperbilirubinemia, nel ratto, è dovuta ad una mutazione nell’enzima responsabile della coniugazione del pigmento, passaggio fondamentale per la sua successiva eliminazione. Nell’omozigote (jj) la bilirubina totale nel sangue (TBS) è molte volte più alta che nell’eterozigote (Jj) in cui l’allele non mutato codifica per l’enzima nella sua forma attiva, sufficiente a mantenere livelli di bilirubina normali. RISULTATI Ia) Attraverso una quantificazione relativa dell’ espressione proteica, ottenuta tramite Western blot, abbiamo dimostrato una espressione speculare dei due trasportatori nelle interacce sangue cervello. Mentre i microvasi sono caratterizzati dalla forte espressione di Pgp, ed i livelli di Mrp1 sono 15-20 volte inferiori rispetto ai plessi, questi ultimi presentano una elevata espressione di Mrp1 ed una quasi completa assenza di Pgp. Per quanto riguarda l’espressione di Mrp1 nei plessi coroidei (CP), abbiamo potuto evidenziare una differenza, con la massima espressione nel plesso del 4° ventricolo rispetto ai ventricoli laterali. Tramite immunofluorescenza abbiamo poi evidenziato per entrambe i trasportatori una localizzazione lato sangue, con Pgp luminale nei vasi e Mrp1 baso-laterale nel plessi coroidei. Ib) Anche l’andamento dell’espressione durane lo sviluppo post-natale differisce. Mentre Mrp1 è sin dalla nascita (2 giorni di vita) altamente espresso in entrambe le barriere, Pgp è inizialmente espresso a livelli più bassi (4,6 volte meno) rispetto all’ adulto (60 giorni). Contemporaneamente anche la densità dei vasi nel parenchima aumenta. II) Nel modello iperbilirubinemico rappresentato dal ratto Gunn, la TBS (jj) e molte volte più alta che nell’ eterozigote (Jj) e tale differenza permane per tutto l’arco di tempo esaminato (0-60 giorni dalla nascita). Al contrario la bilirubina libera (calcolata) è elevata solo nelle prime due settimane di vita, quando il rapporto bilirubina-albumina nel sangue è superiore all’unità. Poi, il rapido aumento della concentrazione ematica di albumina determina un significativo calo della Bf. Mentre l’analisi degli effetti (macroscopici) dell’iperbilirubinemia sullo sviluppo degli emisferi cerebrali non evidenzia differenze tra Jj e jj; in questi ultimi la crescita del cervelletto è severamente inibita. Già a 17 giorni di vita l’ipoplasia del cervelletto si manifesta con una differenza nel peso del 50% nei jj rispetto agli animali normo bilirubinemici di pari età. Durante tale periodo anche l’espressione dei due trasportatori nelle barriere è modificata. L’espressione proteica di Pgp nella BBB degli animali iperbilirubinemici è aumentata ad ogni età presa in esame. Tuttavia tale incremento non modifica in maniera importante la quantità del trasportatore nei MV durante lo sviluppo post natale, rimanendo quindi poco espresso (5 volte meno rispetto all’adulto) almeno fino ai 17 giorni di vita. Contemporaneamente la presenza Mrp1 nella BCSFB è inibita. Già a 9 giorni nel plesso del 4° ventricolo Mrp1 è il 50% rispetto al controllo (pari età, Jj). Anche se nei plessi dei ventricoli laterali l’inibizione dell’espressione è inferiore, nell’insieme la quantità di Mrp1 è fortemente ridotta negli animali iperbilirubinemici. Contrariamente, nei ratti Jj, Mrp1 ha un andamento simile a quello descritto nella sezione (Ib). CONCLUSIONI I risultati da noi ottenuti sottolineano importanti differenze tra le due barriere. La barriera emato-encefalica si sviluppa durante il primo periodo post-natale, in un ambiente caratterizzato dalla forte presenza di membrane cellulari. Similarmente l’espressione di Pgp è inizialmente bassa ed incrementa molto durante lo sviluppo post-natale. Al contrario I plessi coroidei appaiono precocemente in età embrionale, contribuiscono allo sviluppo del cervello e posseggono il più alta espressione di enzimi di fase II, coinvolti nel metabolismo di potenziali sostanze tossiche, del cervello. Un alto livello di Mrp1 sin dalla nascita suggerisce un suo coinvolgimento nel trasporto di qualche sostanza importante nello sviluppo del cervello o in un suo precoce coinvolgimento nel mantenimento dello stato ossido riduttivo, o nell’eliminazione di metabolici dal sistema nervoso centrale. Elevati livelli di bilirubina, come nel modello Gunn, modulano sia l’espressione di Pgp nella BBB, che di Mrp1 nella BCSFB. Tuttavia l’incremento nell’espressione di Pgp nei microvasi non sembra essere sufficiente a contrastare efficacemente l’ingresso della bilirubina libera, molto elevata fino al 17 giorno di vita. La simultanea riduzione di Mrp1 nei plessi coroidei, può facilitare l’ingresso o ridurre l’efflusso della bilirubina nel liquido cefalo rachidiano, consentendo l’accumulo e conseguente danno dei tessuti esposti.
................................................................ ....................... .. .BACKGROUND The unconjugated bilirubin (UCB), a heme degradation product, has been suggested to be a potent antioxidant at low concentration while it seems to be extremely dangerous at higher concentrations, causing encephalopathy in severely jaundiced neonates. Around 70% of children with kernicterus die within seven days, while the 30% survivors usually suffer irreversible sequels, including hearing loss, paralysis of upward gaze, mental retardation, and cerebral palsy with athetosis. Bilirubin encephalopathy is actually the leading cause of hospital readmission of newborns within the first month after birth. Historically the studies concerning the bilirubin entry the central nervous system have focused on the blood brain barrier (BBB), located at the level of the endothelial cells forming the brain micro vessels (MV), leading to the “free bilirubin theory”. It consists in the idea that only the free unconjugated bilirubin, the part of bilirubin exceeding the binding ability of the serum albumin, is able to cross the cell membranes and diffuse in tissue. In brain a second blood brain barrier is present. It is located at the level of the epithelial cells forming the choroids plexuses, between the blood and the cerebrospinal fluid (blood-cerebrospinal fluid barrier, BCSFB). Despite the largest surface area available for the exchanges, the high blood flux, the strategically position between two circulating fluids and the more leaky phenotype, limited studies have been made concerning its role in limiting the bilirubin entry the brain. Two ATP dependent transporters, the Multidrug Resistance-associated Protein 1 (Mrp1) and the MultiDrug resistance Protein (Pgpor MDR1), appear to be actively involved in UCB trafficking. The transporters play an important role in keeping extra cellular bilirubin concentration, such as potentially toxic compounds, below toxic levels by limiting the entry of UCB from blood to brain, or else in controlling intracellular bilirubin levels in parenchyma cells. Despite the importance of Mrp1 and Pgp on BBI their pattern of expression and cellular localization remains still unsettled. Based on these considerations - The first aim of the thesis was clarify the relative protein expression of these transporters at the two major BBI protecting the brain from toxic insults (Ia), and to identify their post-natal developmental profile of expression and cellular localisation (Ib). Similarly, no data about the Mrp1 and Pgp expression on BBI during the bilirubin encephalopathy are available. - The second aim of the thesis was investigate a relation between the high level of blood bilirubin and Mrp1 and Pgp expression in brain barriers in vivo using the Gunn rat (II), in witch the symptoms closely correlate to the human kernicterus and Crigler-Najjar syndrome type I. In this animal model, a mutation in the enzyme responsible for the conjugation and subsequent elimination of bilirubin, leads to the total absence of the enzymatic activity in the homozygous animals (jj), causing a severe life long hyperbilirubinemia. In the heterozygous Gunn rats (Jj), the enzymatic activity, until if reduced, is present and result in normal serum bilirubin levels. RESULTS Ia) By quantitative Western blot, we have demonstrated a mirroring expression of the two transporters at the blood brain interfaces in the adult rat. On the BBB the Pgp is strongly expressed and the Mrp1 amount is 15-20 times lower than in CPs. At the contrary, the CPs are characterized by the high expression of Mrp1, with a difference between the lateral ventricle (LV) and the 4th ventricle (4thV) CP, the former being a lower Mrp1 expression than in the last. In both LV and 4thV CPs, Pgp is virtually absent. By immunofluorescence we revealed that both ABC transporters are located at the blood side, the Pgp luminal on MV, and Mrp1 basal on CPs. Ib) With respect to the post-natal development, the Mrp1 expression is high since the early post-natal age and do not change significantly from birth to adult life in both barriers. By contrast, Pgp expression is weak a P9 and increase 4.6 fold with maturation on MV. Synchronously the density of Pgp stained MV in parenchyma seems increasing. II) In the homozygous Gunn rat (jj) the total bilirubin in serum is several time higher than in the heterozygous (Jj) animals all life long. By contrast the (calculated) free bilirubin is extremely elevated until the first week of life, when the bilirubin-albumin ratio exceed the unit, then drop due to the developmental increasing albumin concentration in blood. While no differences in Cx weight have been found between Jj and jj rat at every postnatal age, the cerebellum development is strongly impaired by the bilirubin toxic effect, displaying a Summarymarked hypoplasia, with about the 50% of weight loss respect the Jj control at 17 days after the birth. Concerning the ABC transporters, the differential pattern of expression between blood brain interfaces is maintained. But, in jj Gunn rats, the Pgp expression at the BBB is up-regulated at every post natal age analysed, also if this increase do not seems to be sufficient to confer protection at list until P17, when the amount of the transporter in the MV is about 5 times lower than in adult (P60). At the same time the Mrp1 expression on the BCSFB is down regulated. Since P9 the amount of Mrp1 in the 4thV CP of jj rats drops around to the 50% respect the amount in the littermates. In the LV CP the decrease is less marked, but in any case the Mrp1expression in both CPs is strongly impaired. This down regulation seems to be post-transcriptional. In the Jj animals, the Mrp1 relative expression is already high in both plexuses at early postnatal stages. A significant difference was noted only between LV CP (76%) and 4thV CP at P60 (100%). CONCLUSIONS All together these results indicate that the two barriers differ: The BBB develops after the birth and is surrounded by the lipid rich parenchyma environment, in agreement with the transporter preference for the lipid compounds and the strong post-natal developmental increase of the Pgp amount on MV. The CPs develops early in the foetal life, are involved in the guidance of the brain development and posses the highest phase II metabolising enzymes in the brain. The Mrp1 amount in CPs is similar to the adult level since the birth and may be involved in the transport of some compounds important in the brain development, in the detoxification or in the maintenance of the redox state (GS- sulfo- conjugates, LC4, etc.). In Gunn rats, as model for Kernicterus and Crigler-Najjar syndrome type I, the Pgp offered protection is not sufficiently modulate until P17, when the amount of the free bilirubin is elevated and could cross the brain barriers. The simultaneously down regulation of Mrp1 at the BCSFB may facilitate the entry of the bilirubin or strongly impair their clearance in the central nervous system, leading to the accumulation in brain and subsequent damage of tissue.
1974
Paroni, Federico. "Molecular mechanisms of pancreas development and insulin regulation in beta cells." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2763.
Повний текст джерелаType 1 or “juvenile” Diabetes is an autoimmune disease in which the insulin secreting beta-cells, essential for glucose homeostasis, are destroyed by a target immune attack. To date, patients with type 1 Diabetes can rely only on exogenous insulin injection to control the glucose levels, but, unfortunately, the related chronic and devastating complications cannot be prevented. Identification of beta-cell-specific genes playing critical roles during the development will help to create new sources of beta-cells from stem/progenitor cells. Interestingly, endocrine cell fate appears to be governed by the “peaked” expression of the transcription factor Ngn3. However, Ngn3’s targets are still not well identified. As for pancreas development also insulin gene regulation lacks important information. Therefore the aims of this Thesis work have been to test an set-up an efficient system for the transient expression of Ngn3, mimicking its physiological behavior; identify the major factors that lie directly downstream the Ngn3 expression and characterize a novel beta-cell specific insulin gene regulator. As result from this work, an innovative system mimicking pancreatic differentiation and Ngn3 pulsed expression has been developed. Furthermore, a novel Ngn3’s downstream target, the zinc-finger protein “OVO_like 1, has been identified opening a new, and interesting, scenario of Ngn3 gene’s regulation. Last, but not least, the beta-cell-insulin-gene regulator A2.2 has been characterized and a reproducible purification system has been developed.
1974
Di, Girolamo Filippo Giorgio. "BIOMARKERS TO DEFINE OPTIMAL PROTEIN REQUIREMENT." Doctoral thesis, Università degli studi di Trieste, 2015. http://hdl.handle.net/10077/11139.
Повний текст джерелаDietary proteins are the source of the amino acids required by the body for tissue growth and maintenance. The Population Reference Intake (PRI) for proteins, as defined by the European Food Safety Authority (EFSA) for healthy adults, including the elderly, is 0.83 g/kg body weight/day. This amount is defined on the net balance of body protein (or “nitrogen balance”, given by the difference between dietary nitrogen intake and losses) equivalent to 0.66 g/kg/day plus a safety factor for interpersonal variability and differences in proteins quality of mixed diets. The PRI, however, is the minimum daily amount of protein needed to maintain the nitrogen balance and avoid a progressive loss of lean body mass in healthy people with moderate physical activity. Therefore nitrogen balance may not be adequate to define protein requirement in adults and especially in ageing characterized by loss of muscle mass and function (sarcopenia). Furthermore until recently the prevalent idea was that a protein intake above PRI had no further benefits and on the contrary could impair health. These believes are now under discussion, diets with higher protein intake have been shown beneficial in the prevention and treatment of conditions such as sarcopenia, COPD and type 2 diabetes mellitus. There is a need of more precise methods to define protein requirement. AIM. The aim of the present thesis is to investigate in human healthy volunteers new biomarkers adequate to define optimal protein intake. Recent studies have determined protein needs by measuring whole-body protein metabolism using stable labeled isotope-amino acids. METHODS. Our research group has applied two different metabolic methods based on the most widely used tracer, i.e. D5-Phe stable isotope, in two experimental bed rest campaigns (FP7 PLANHAB and INTERREG PANGaA) in healthy volunteers. BR is a suitable model to investigate physiologic adaptation to inactivity. MAIN RESUTLTS. FP7 PLANHAB. We applied the stable isotope infusion technique, to assess the effect of physical inactivity and/or hypoxic condition on whole body protein turnover as previously described in Biolo et al 2008. Chronic hypoxia has been associated with an overall reduction in protein synthesis and in total plasma and skeletal muscle protein content. During the PLANHAB study we investigated, through a crossover randomization, the net effects of 10 days normobaric hypoxia (4000 mt.), associated with either ambulatory conditions or BR, in 11 young (age 24±4 yr), healthy and normal weight male subjects maintained on eucaloric diets. Main results. Hypoxia in ambulatory conditions significantly decreased whole body protein turnover by reducing both protein synthesis (-8±2%) and protein degradation (-8±3%). Hypoxia during bed rest did not caused significant changes in protein metabolism. INTERREG PANGaA. The skeletal muscle loss in aging is caused mainly by the “anabolic resistance” i.e. the inadequate increase in the rate of protein synthesis in response to nutritional-metabolic stimuli, including exercise, protein and amino acid intake as well as insulin and insulin-like growth factor stimulation. As a consequence, the net protein balance becomes negative leading to sarcopenia. The effects of ageing on the anabolic resistance induced by inactivity are poorly investigated. During the PANGeA study we had the opportunity to perform the second documented experimental BR in in healthy elderly volunteers and the first comparing aged with young subjects. To evaluate the anabolic resistance associated with ageing and inactivity, we enrolled 7 young (23±1yr) and 8 elderly (59±1yr) normal weight individuals, in a 14-d experimental BR protocol. We replaced our previous infusion method with a new, simpler, safer and quicker technique, by which tracers are given orally instead of parenterally, the all procedure is completed in two hours, instead of 6, and only two blood draws versus 7 are sufficient. Main results. At baseline parameters of anabolic sensitivity were comparable between young and elderly individuals. The anabolic resistance significantly increased after BR in both groups (bed-rest effect p<0.01), with a statistically significant bed-rest×group interaction (p=0.01). Anabolic resistance increased significantly in elderly (18.5%±7.3%) more than in young (5.2%±9.4%) subjects. DISCUSSION. In the PLANHAB study, hypoxia in ambulatory conditions reduced by the same level both protein synthesis and catabolism, as measured by isotope infusions, suggesting an adaptive mechanism: the lower energy production and availability induced by hypoxia associated with ambulatory condition. These modifications could not have been revealed by the use of nitrogen balance method, showing the relevance of more sophisticated analysis. The direct evaluation of the muscle protein metabolism through an infusion of stable-labeled isotope tracer, considered the golden standard methodology, gave us, in the PLANHAB study, reliable results in the early protein metabolism changes during hypoxia and/or BR. This method however has the limit of being complex, onerous and invasive, therefore being unsuitable for clinical evaluation. In the PANGeA study we could confirm the presence of a reduced sensitivity to anabolic stimuli in the elderly population compared to the young men. The elderly subjects are therefore, more at risk to develop changes of protein metabolism induced by inactivity. The simpler, timesaving and less invasive method we have developed for the PANGeA study, on the other hand, could be applied to a wider ranges of experimental conditions and clinical settings.
XXVII Ciclo
1983
BUGLIONE, ENRICO. "Nanomeccanica per la Ricerca sul Cancro." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/304787.
Повний текст джерелаWith the term cancer are intended many species of diseases having quite different properties from each other. Despite such vast differences, the mechanisms beyond the onset of any kind of cancer are very similar and can be classified in two main groups depending on their stage. The first is related to the dysregulation of particular genes (oncogenes), that results in an impairment of the cell cycle. The second concerns the ability of cancer cells to continuously divide and migrate through tissues, that results in a highly invasive potential. From a mechanical point of view, the investigation of such features can be crucial for a deeper understanding of cancer onset and progression as well as for the study of novel pharmacological treatments. The outbreak of cancer is caused by a deficiency in the regulation of the cell cycle which, in turn, often depends on an abnormal expression of oncogenes. It is the case of the proto-oncogene c-KIT, that encodes for a mast/stem cell growth factor receptor. Its regulation relies mainly on its promoter, which is constituted by 3 distinct three-dimensional DNA structures called G-quadruplexes (G4s). Those structures can be studied by means of nanomechanical tools such as Magnetic Tweezers, which can recognize folded G4s at single-molecule level, thus enabling to study their role in the regulation of the oncogene. After the onset of cancer, a generic cell undergoes mechanical changes: it divides quickly, and it starts migrating. Both phenomena require a modification in the cell structural phenotype, eventually modifying its rigidity. Chronic lymphocytic leukemia is a case in point: malignant B lymphocytes continuously traffic between peripheral blood and lymphoid tissues. Such frequent migrations require a change in the rigidity of cells. In this case, Atomic Force Microscopy can provide a nanomechanical approach allowing to measure the stiffness of single cells from patients with leukemia, which is slightly decreased if compared to rigidity of cells from healthy donors. This feature can also allow to observe the effect of targeted therapies on the cells, evaluating their effect from a mechanical point of view.
Gola, Elisabetta. "Ruolo dell'infiammazione nella patogenesi dell'acute on chronic liver failure." Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3424533.
Повний текст джерелаBackground. L'acute on Chronic Liver Failure (ACLF) è una condizione clinica che colpisce i pazienti con cirrosi ed è caratterizzata da un peggioramento acuto della funzionalità epatica, danno ad uno o più organi ed elevata mortalità a breve termine. Recentemente è stata proposta una nuova classificazione dell'aCLF sulla base del Chronic LIver Failure-Sequential Organ Failure Assessment (CLIF-SOFA) score. Nel quadro complesso dell'aCLF, l'infiammazione sistemica sembra avere un ruolo cruciale essendo coinvolta apparentemente nello sviluppo di ACLF e nel danno d'organo; i dati in letteratura riguardanti la risposta infiammatoria in questa condizione clinica sono scarsi e lacunosi. In questo scenario, la nostra ricerca è stata indirizzata nell'identificare e descrivere il profilo citochinico pro e anti infiammatorio nei pazienti con ACLF e senza ACLF, al fine di caratterizzare il legame tra la risposta infiammatoria e ACLF. E' stata inoltre studiata l'espressione genica dei principali mediatori dell'infiammazione (NF-kB, TNF-α, IL-1ß, CASP-1) e dell'apoptosi (CASP-3) nei Peripheral Blood Mononucleated Cells (PBMCs) dei pazienti con o senza ACLF. Scopo. Lo scopo di questo studio è stato quello di valutare il profilo citochinico pro e anti infiammatorio all'ammissione e durante la prima settimana di ricovero in pazienti con e senza ACLF in modo da caratterizzare il link tra risposta infiammatoria e ACLF e valutare il possibile ruolo dell'inflammasoma e della sua iper-attivazione nella patogenesi dell'aCLF. Materiali e metodi. Sono stati raccolti i dati (parametri vitali quali: pressione arteriosa sistemica, frequenza cardiaca e saturazione O2, dati anamnestici, dati di laboratorio: emocromo, bilirubina totale e diretta, AST, ALT, GGT, ALP, creatinina, urea, sodio, potassio, PT, INR, albumina, ammoniemia, emocolture, urocolture, di microbiologia e scores clinici ed epatici, Child-Pugh, MELD score, all'ammissione in ospedale e al momento dell'inclusione-ricovero in unità specializzata, giorno 1) di 72 pazienti consecutivi con cirrosi e ospedalizzati per complicazioni della patologia epatica in corso (età = 59.1 ± 12.4 anni, MELD= 17.6 ± 7.8), 21 di questi con ACLF (secondo il CLIF-SOFA score). I livelli di citochine circolanti (TNF-α, IL-6, IL-10) sono stati misurati tramite test ELISA (Enzyme-Linked ImmunoSorbent Assay) nei giorni 2 e 7 dopo il ricovero. Il rapporto TNF-α/IL-10 è stato usato per valutare l'equilibrio pro/anti infiammatorio. I livelli di espressione genica di NF-kB, caspasi-3, caspasi-1, TNF-α e IL-1ß sono stati misurati nei Peripheral Blood Mononucleated Cells (PBMCs) di pazienti con e senza ACLF. Risultati. L'infezione batterica è stata riscontrata nel 52.4% e 37.3% di pazienti con e senza ACLF rispettivamente (p= NS [0.35]). I livelli di TNF-α e IL-6 sono risultati significativamente più alti in pazienti con ACLF che in pazienti no-ACLF (TNF-α: p=0.015; IL-6: p=0.011). Il rapporto TNF-α/IL-10 è risultato maggiore nel gruppo con ACLF vs no-ACLF (p=0.019). Al giorno 7 dal ricovero, il gruppo ACLF ha mostrato una riduzione significativa dei livelli di IL-10 rispetto al gruppo no-ACLF (ΔIL-10= -1.58 pg/ml-IQT: -6.15 to 0.42 pg/ml vs 0.05 pg/ml-IQT: 0.00 to 4.61 pg/ml, p=0.026). L'analisi dell'espressione genica mostra un aumento significativo dei livelli di espressione di NF-kB (p=0.03), caspasi-3 (p=0.03), caspasi-1 (p=0.05), TNF-α (p=0.05) e IL-1ß (p=0.05) nei PBMCs di pazienti con ACLF vs no-ACLF. L'analisi statistica è stata condotta mediante test di significatività non parametrico di Mann-Whitney. Conclusioni. I risultati hanno evidenziato un significativo e progressivo squilibrio pro infiammatorio nei pazienti con ACLF vs no-ACLF; il pathway effettore (espressione genica di NF-kB, caspase-1 and IL-1ß) è risultato significativamente over-espresso in PBMCs di pazienti con ACLF vs no-ACLF
BASON, RAMONA. "EPIGENETIC CHARACTERIZATION OF TUMOR INFILTRATING CD4+ T REGULATORY CELLS." Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/917092.
Повний текст джерелаCorradini, Fabio. "Caratterizzazione molecolare delle mutazioni nei geni BRCA." Doctoral thesis, Università Politecnica delle Marche, 2009. http://hdl.handle.net/11566/242012.
Повний текст джерелаCalzolari, Claudia <1976>. "Diagnosi e prognosi molecolare nel linfoma canino." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/92/1/definitiva_per_PDF_claudia.pdf.
Повний текст джерелаCalzolari, Claudia <1976>. "Diagnosi e prognosi molecolare nel linfoma canino." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/92/.
Повний текст джерелаCaroppo, V. "EFFECTS OF CYSTEINYL LEUKOTRIENES ON PLATELET ACTIVATION." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/363389.
Повний текст джерелаZambon, Elisa <1985>. "La diagnostica molecolare nel laboratorio di patologia clinica veterinaria." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/7007/1/Zambon_Elisa_Tesi.pdf.
Повний текст джерелаThe first part of the present study concerns the LAMP technique (Loop-Mediated Isothermal Amplification), an isothermal amplification technique recently developed (Notomi et al., 2000). LAMP has many advantages over traditional PCR: it doesn’t require sophisticated instruments like thermal cyclers, it can be performed by unskilled staff, it is a highly sensitive and specific technique and it is very tolerant to inhibitors. All these characteristics make it suitable to be used outside diagnostic laboratories, as POCT (Point-of-care testing), with the advantage of not having to send the sample and obtaining results as accurate as PCR tests and in very short times. We designed and optimized assays to detect bacteria that require a very long time for cultivation or that are not even cultivable. We drew assays for the diagnosis of viral diseases that require to be diagnosed as soon as possible. We developed a test to assess two genetic diseases of the dog and two food contaminating bacteria. All tests were carried out using real-time technique to decrease the risk of cross-contamination. Finally, we developed a colorimetric method for showing results which can be applied to all of the assays we optimized. The second section presents the molecular study of a subject who had myeloperoxidase deficiency at the automated cytochemistry analysis (ADVIA ® 2120 Hematology System). The study was conducted through amplification and comparison of the PCR products obtained from the pathological subject and on two subjects with wild-type phenotype. The products were sequenced using an automated sequencer in order to find the responsible mutation for the MPO deficiency in the indicated subject.
Zambon, Elisa <1985>. "La diagnostica molecolare nel laboratorio di patologia clinica veterinaria." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/7007/.
Повний текст джерелаThe first part of the present study concerns the LAMP technique (Loop-Mediated Isothermal Amplification), an isothermal amplification technique recently developed (Notomi et al., 2000). LAMP has many advantages over traditional PCR: it doesn’t require sophisticated instruments like thermal cyclers, it can be performed by unskilled staff, it is a highly sensitive and specific technique and it is very tolerant to inhibitors. All these characteristics make it suitable to be used outside diagnostic laboratories, as POCT (Point-of-care testing), with the advantage of not having to send the sample and obtaining results as accurate as PCR tests and in very short times. We designed and optimized assays to detect bacteria that require a very long time for cultivation or that are not even cultivable. We drew assays for the diagnosis of viral diseases that require to be diagnosed as soon as possible. We developed a test to assess two genetic diseases of the dog and two food contaminating bacteria. All tests were carried out using real-time technique to decrease the risk of cross-contamination. Finally, we developed a colorimetric method for showing results which can be applied to all of the assays we optimized. The second section presents the molecular study of a subject who had myeloperoxidase deficiency at the automated cytochemistry analysis (ADVIA ® 2120 Hematology System). The study was conducted through amplification and comparison of the PCR products obtained from the pathological subject and on two subjects with wild-type phenotype. The products were sequenced using an automated sequencer in order to find the responsible mutation for the MPO deficiency in the indicated subject.
FAZZARI, MARIA. "NOVEL APPROACHES OF ¿PERSONALISED MEDICINE¿ AS PROOF-OF-PRINCIPLE FOR CDKL5-RELATED PATHOLOGIES." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/548108.
Повний текст джерелаDi, Girolamo Nicola <1987>. "Method-Comparison and Reference Interval Determination in Animal Medicine." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7650/2/Tesi_dottorato_Definitiva.pdf.
Повний текст джерелаDi, Girolamo Nicola <1987>. "Method-Comparison and Reference Interval Determination in Animal Medicine." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7650/.
Повний текст джерелаCARLETTI, GIORGIA. "Studio molecolare della produzione di antocianine in pianta." Doctoral thesis, Università Cattolica del Sacro Cuore, 2010. http://hdl.handle.net/10280/785.
Повний текст джерелаAnthocyanins are secondary metabolites in plants. They belong to a class of flavonoids synthesized via the phenylpropanoid pathway and they are antioxidant molecules important for plant, animal and human health. Flavonoids are widely studied in legume because they are implicated in several biological and physiological functions. In this thesis seven genotypes (two wild-types and five indipendent mutants) of Medicago truncatula (the model legume plant) affected in flavonoid metabolism have been characterized at phenotypic, physiological and molecular level. The main difference between wild-types and mutants, is the reduction in anthocyanin content in leaves. The anthocyanin accumulation during the leaf life, the flowering time and the fruit formation have been registered and compared. The two wild-types contain 6 μg cyanidin.mg-1 of fresh weight, while in the mutants the anthocyanin amount ranged from 0.12 μg cyanidin mg-1 to 2 μg cyanidin mg-1 of fresh weight. After HPLC-DAD-MS/MS analysis, the main anthocyanin present in the two wild-types is the cyanidin-3-O feruloyl. The Expression profile of genes codifying enzymes and transcriptional factor involved in flavonoid biosynthesis has been investigated. RT-PCR and qPCR results show different possible pathways of reduction of anthocyanins in the five mutants. These mutants, have been exposed to UV-B radiations and their response has been investigated measuring the chlorophyll fluorescence parameters (Fv/Fm, qP and NPQ) in untreated plants, during treatment (after 4hrs and 14hrs of treatment) and in the recovering phase. All genotypes, regardless of the anthocyanins amount, showed a decrease of the photosyntetic efficiency after 14hrs of treatment. This indicates a marginal role of these pigments in the oxidative damages protection. Mutants do not response in the same manner to the UV—B exposure and the anthocyanin amount does not increase equally in all genotypes. The anthocyanin metabolism is studied also at the gene regulation level. A novel Myb gene (MtMYBA) involved in anthocyanin pathway has been isolated and characterized. This gene is more expressed in tissues which accumulate anthocyanins in M. truncatula plants. The functional analysis has been investigated overexpressing this Myb gene under the control of 35S promoter. This construct has been used to transform Arabisopsis thaliana and Medicago truncatula plants. In addition, the MtMYBA promoter has been cloned in a plasmids containing GUS and GFP reporter genes.
CARLETTI, GIORGIA. "Studio molecolare della produzione di antocianine in pianta." Doctoral thesis, Università Cattolica del Sacro Cuore, 2010. http://hdl.handle.net/10280/785.
Повний текст джерелаAnthocyanins are secondary metabolites in plants. They belong to a class of flavonoids synthesized via the phenylpropanoid pathway and they are antioxidant molecules important for plant, animal and human health. Flavonoids are widely studied in legume because they are implicated in several biological and physiological functions. In this thesis seven genotypes (two wild-types and five indipendent mutants) of Medicago truncatula (the model legume plant) affected in flavonoid metabolism have been characterized at phenotypic, physiological and molecular level. The main difference between wild-types and mutants, is the reduction in anthocyanin content in leaves. The anthocyanin accumulation during the leaf life, the flowering time and the fruit formation have been registered and compared. The two wild-types contain 6 μg cyanidin.mg-1 of fresh weight, while in the mutants the anthocyanin amount ranged from 0.12 μg cyanidin mg-1 to 2 μg cyanidin mg-1 of fresh weight. After HPLC-DAD-MS/MS analysis, the main anthocyanin present in the two wild-types is the cyanidin-3-O feruloyl. The Expression profile of genes codifying enzymes and transcriptional factor involved in flavonoid biosynthesis has been investigated. RT-PCR and qPCR results show different possible pathways of reduction of anthocyanins in the five mutants. These mutants, have been exposed to UV-B radiations and their response has been investigated measuring the chlorophyll fluorescence parameters (Fv/Fm, qP and NPQ) in untreated plants, during treatment (after 4hrs and 14hrs of treatment) and in the recovering phase. All genotypes, regardless of the anthocyanins amount, showed a decrease of the photosyntetic efficiency after 14hrs of treatment. This indicates a marginal role of these pigments in the oxidative damages protection. Mutants do not response in the same manner to the UV—B exposure and the anthocyanin amount does not increase equally in all genotypes. The anthocyanin metabolism is studied also at the gene regulation level. A novel Myb gene (MtMYBA) involved in anthocyanin pathway has been isolated and characterized. This gene is more expressed in tissues which accumulate anthocyanins in M. truncatula plants. The functional analysis has been investigated overexpressing this Myb gene under the control of 35S promoter. This construct has been used to transform Arabisopsis thaliana and Medicago truncatula plants. In addition, the MtMYBA promoter has been cloned in a plasmids containing GUS and GFP reporter genes.
MORONI, Cinzia. "Diabete mellito e metabolismo del monossido di azoto: possibili implicazioni molecolari e prospettive future." Doctoral thesis, Università Politecnica delle Marche, 2007. http://hdl.handle.net/11566/242487.
Повний текст джерелаCONGIU, RITA. "Analisi molecolare in pazienti italiani con sindrome di Lowe." Doctoral thesis, Università degli Studi di Cagliari, 2006. http://hdl.handle.net/11584/265881.
Повний текст джерелаToppazzini, Mila. "Micrometer-scale systems for regenerative medicine applications." Doctoral thesis, Università degli studi di Trieste, 2010. http://hdl.handle.net/10077/3616.
Повний текст джерелаLa medicina rigenerativa applicata al campo ortopedico è considerata una possibile opzione terapeutica per la riparazione del tessuto osseo danneggiato. Si stanno studiano e sviluppando una gran varietà di sostituti ossei sintetici come valida alternativa agli innesti di tipo tissutale. Lo scopo di questo lavoro di tesi è la progettazione e lo sviluppo di un materiale iniettabile per il riempimento dei difetti ossei. In particolare abbiamo sviluppato un riempitivo composito iniettabile usando materiali biodegradabili di tipo polisaccaridico funzionalizzati con elementi bioattivi come mediatori dell’adesione cellulare, fattori di crescita osteoinduttivi e peptidi di tipo antimicrobico. Il costrutto finale è un composito a due fasi, le cui parti, inizialmente, sono state sviluppate separatamente. La struttura è stata progettata come composta da una parte bioattiva, costituita da microsfere disidratate di alginato e idrossiapatite recanti peptidi, immersa in una matrice veicolante rappresentata da una soluzione concentrata di acido ialuronico. Con lo scopo di ottenere le microsfere bioattive, sono state esplorate diverse tecniche per la coniugazione peptide-polisaccaride e sono stati presi in considerazione svariati sistemi di rilascio di sequenze peptidiche. In particolare sono stati considerati tre peptidi noti per la loro bioattività: peptidi di tipo RGD, sequenza favorente l’adesione cellulare, un frammento della proteina BMP-2 (bone morphogenetic protein-2) in grado di promuovere il differenziamento di cellule mesenchimali ad osteoblasti e LL-37 peptide antimicrobico umano. Per ottenere un costrutto con proprietà bioadesive, gli sforzi sono stati volti ad un miglioramento dell’interfaccia fra le sfere di alginato/idrossiapatite e il tessuto osseo. Questo è stato possibile immobilizzando sulla superficie delle sfere dei peptidi contenti la sequenza RGD e assicurandone il gusto orientamento ed un’alta resa di immobilizzazione. Dopo aver testato diverse strategie chimiche, l’immobilizzazione effettuata sfruttando la formazione di un ponte disolfuro fra ChitLac preventivamente modificato con gruppi tiolici e un peptide contenente un residuo di cisteina, è stata valutata come la miglior strategia in termini di resa di reazione; allo stesso tempo le microsfere funzionalizzate in questo modo hanno dimostrato un’alta capacità di promuovere l’adesione e la crescita di osteoblasti in esperimenti effettuati in vitro. Un altro importante tema ha riguardato l’incorporazione di un frammento della proteina BMP-2 nel costrutto al fine di promuovere il differenziamento e quindi la proliferazione cellulare. La sequenza temporale degli eventi fisiologici, ha suggerito lo sviluppo di un sistema che risulta essere la somma di due diverse strategie di rilascio: la prima caratterizzata dal semplice intrappolamento del peptide in un sistema di sfere di alginato capace di un rilascio veloce, ed il secondo caratterizzato da un rilascio lento e costante ottenuto grazie all’azione idrolitica di esterasi. Questo è stato possibile inserendo un legame enzimaticamente idrolizzabile nella struttura contenente il frammento di BMP. Questa seconda strategia ha sfruttato una chimica altamente selettiva quale la “click chemistry”. La sintesi della molecola spaziatrice recante un legame enzimaticamente idrolizzabile è stata effettuata partendo da un γ-valerolattone. Questo spaziatore è stato inoltre progettato per recare un gruppo terminale funzionale adeguato per le reazioni di click chemistry ed è stato legato al ChitLac. La reazione di cicloaddizone è stata poi effettuata fra il ChitLac funzionalizzato con lo spaziatore e il frammento di BMP anch’esso opportunamente funzionalizzato. L’idrolisi enzimatica è stata verificata, inoltre è stata eseguita un’esaustiva caratterizzazione tramite NMR ed elettroforesi capillare. E’ stata poi presa in considerazione l’incorporazione di peptidi antimicrobici nel costrutto. Partendo da dati di dicroismo circolare riguardanti l’influenza che alcuni polisaccaridi hanno sulla conformazione del peptide LL-37 in soluzione, è stata riconosciuta alla miscela LL- 37/alginato la capacità di modulare la citotossicità del peptide. La miscela ha inoltre dimostrato la capacità di mantenere l’attività antimicrobica su batteri Gram negativi e questo ci ha spinto a considerarne l’applicazione su costrutti solidi con finalità ortopediche. In quest’ambito è stato escluso, da esperimenti in vitro, un effetto causato dal semplice contatto fra cellule o batteri e una superficie di alginato caricato col peptide. L’unico possibile meccanismo di rilascio riscontrato in un costrutto di alginato/LL-37 è stato tramite la degradazione di questo. Infine, il costrutto è stato assemblato incorporando le microsfere disidratate in una soluzione concentrata di acido ialuronico, ottenendo un costrutto dall’aspetto simile ad una pasta che è in grado di facilitare il processo di iniezione del riempitivo. Misure di tipo reologico hanno indicato in incremento della componente viscoelastica aggiungendo il particolato nella soluzione di acido ialuronico, questo generalmente è associato ad una buona capacità di recuperare l’elasticità dopo l’iniezione il che è un requisito richiesto ai biomateriali usati come riempitivi ossei. I risultati ottenuti hanno dimostrato l’applicabilità del composito come sostituto osseo dotato di proprietà bioattivite (osteoconduzione, osteoinduzione, bioadesività ed effetto antimicrobico su ceppi di tipo Gram negativo).
Bone regenerative medicine is considered a potential therapeutic option for the healing of damaged bone tissue. A variety of synthetic bone graft substitutes have been investigated as alternative to current tissue based bone graft materials. In this study efforts have been made to achieve an injectable material to fill bone defects. We have designed composite injectable filler by using polysaccharides as biodegradable materials, and by functionalizing them with bioactive elements such as adhesion mediators, osteoinductive growth factors and anti-microbial peptides. The final construct is represented by a two-phase composite, whose parts have been initially built separately. The structure was thought as composed by bioactive alginate/hydroxyapatite dried microbeads functionalized with peptides, suspended in a concentrate hyaluronic acid solution as the matrix vehicle. With the aim to obtain bioactive alginate/HAp beads, different techniques of peptides-polysaccharides conjugation were considered and different peptide delivery systems were explored. In detail the peptides considered were three, the RGD active sequence with bioadhesive properties, a fragment of BMP-2 (bone morphogenetic protein-2) that promotes the differentiation of MSCs into osteoblasts, and LL-37 a human antimicrobial peptide. In order to obtain a construct with bioadhesive properties, efforts have been made to improve the tissue-alginate/HAp beads interface by immobilizing RGD peptide sequence in a manner that assure the right motif orientation and high yields. After having tested various chemical strategies, the immobilization by disulfide bridge formation between a ChitLac previously modified with a thiol group and a peptide containing a cysteine residue was evaluated to be the best in terms of yield; at the same time μbeads functionalized in that way exhibit a very high osteoblast adhesion and growth promotion in in vitro experiments. Another important topic regarded the incorporation of BMP-2 epitope fragment into the construct, to enhance differentiation and proliferation. Time-tuned physiological functionality suggested the development of a system being the sum of two delivery strategies: the first characterized by simple entrapment in alginate beads for a fast burst release and the second one giving a slow and constant release, obtained by the action of bone esterases on an enzymatically cleavable linker. This second strategy exploited a high selective chemistry such as that of click reactions. Starting by a γ-valerolactone the synthesis of the enzymatically cleavable spacer was performed. The linker was designed to contain a final functional group for click chemistry. It was bound to ChitLac and then to a modified BMP fragment by a click reaction. Enzymatic cleavage was verified and an exhaustively characterization by means of NMR and capillary electrophoresis was performed. The incorporation of antimicrobial peptides was also taken into account. Starting from the information gained by circular dichroism data on the influence on LL-37 conformation given by several polysaccharides, an optimal modulation activity of the cytotoxic effect of LL-37 was found using the peptide/alginate mixture. The demonstrated maintenance of antimicrobial activity on Gram negative strains drove to an application on solid constructs for orthopaedic applications. An effect caused by the simple contact between cells or bacteria and alginate surface loaded with the peptide, was excluded by in vitro experiments. The only possible release mechanism that an LL-37/alginate construct was able to show was related to the degradation of this one. Finally, the whole construct was assembled by incorporating the dried microbeads in a concentrate hyaluronic acid solution, obtaining a paste-like construct that can facilitate the injectability of the particulate. Rheological measurements indicated an increment of the viscoelastic component by adding particulate into hyaluronate solution; this, generally, is associated to a good capacity to regain the elasticity after the injection, as expected for a biomaterial for bone filler applications. The results collected demonstrate the applicability of the obtained composite as bone graft substitute with bioactive properties (osteoinduction, osteoconduction, bioadhesivity and antibiotic effect on Gram negative strains).
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Bortoluzzi, Alessia. "Effetto dell'infusione di albumina in un modello sperimentale di cirrosi con ascite." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422459.
Повний текст джерелаBackground: La cirrosi epatica è associata ad alterazioni cardiovascolari che comprendono una circolazione iperdinamica, caratterizzata da una ridotta resistenza periferica e splancnica, bassa pressione media arteriosa e un'aumentata portata cardiaca. Studi sperimentali e clinici hanno dimostrato la presenza di un'insufficienza cardiaca latente chiamata "cardiomiopatia cirrotica", che è caratterizzata da una disfunzione diastolica, anomalie elettrofisiologiche, una compromessa contrattilità cardiaca in risposta a stress farmacologici o fisiologici, malgrado l'aumentata portata cardiaca di base. L'infusione di albumina viene usata nella gestione dei pazienti con cirrosi epatica avanzata allo scopo di migliorare il volume circolante efficace attraverso un incremento della pressione oncotica. Tuttavia studi recenti hanno dimostrato che l'azione dell'albumina è più complessa ed è legata alla sua azione antiossidante e alla sua capacità di legare citochine e ossido nitrico. Scopo: Lo scopo di questo studio è stato quello di valutare in un modello animale di cirrosi avanzata il ruolo del sistema beta adrenergico, dello stress ossidativo e del TNF-α sulla patogenesi della miocardiopatia cirrotica. Materiali e metodi: Sono stati utilizzati ratti maschi Wistar Kyoto 30 di controllo e 30 trattati con CCl4, per indurre la cirrosi. In ogni gruppo 15 ratti sono stati trattati con albumina (1.5 g/Kg tre giorni prima del sacrifico e 0.5g/Kg un giorno prima) e 15 con lo stesso volume di soluzione fisiologica (come intervento sham). Il cuore è stato prelevato e la contrattilità è stata misurata stimolando il cuore con concentrazioni crescenti di isoproterenolo (da 10-10 a 10-6 M). Sul tessuto cardiaco è stata analizzata l'espressione genica e proteica, mediante Real Time PCR e Western Blot rispettivamente, dei recettori beta adrenergici (beta 1 e beta2), della Adenilato ciclasi (Adcy3), della proteina G inibitoria α 2(Gαi2), dell'isoforma inducibile della ossido nitrico sintetasi (iNOS) e delle NAD(P)H ossidasi. Abbiamo poi analizzato la concentrazione plasmatica e nel liquido ascitico del Tumor necrosis factor-α (TNF-α) e la traslocazione nel nucleo del fattore nucleare NF-kB, attraverso kit ELISA. Risultati: La contrattilità è cardiaca nei ratti ascitici era significativamente diminuita rispetto ai controlli (p<0.01). Questa scoperta è stata associata con: a) un'aumentata espressione genica e proteica di beta2-AR (p<0.05) e di Gαi2 (p<0.05), b) la diminuzione dell'espressione genica di Adcy3, c) un'aumentata attività delle NAD(P)H ossidasi, d) un'aumentata concentrazione plasmatica e nel liquido ascitico di TNF-α (p<0.05), e) un'aumentata traslocazione nucleare di NF-kB e f) un'aumentata espressione proteica di iNOS. Dopo il trattamento con albumina la contrattilità cardiaca dei ratti ascitici è ritornata ai valori dei controlli (p<0.01). L'espressione proteica nel tessuto cardiaco degli animali ascitici di Gαi2 (p<0.05), iNOS (p<0.05) e l'attività delle NAD(P)H-oxidase (p<0.05) è simile a quella dei ratti di controllo. Una significativa diminuzione dei livelli di TNF- nel plasma (p<0.05) e nell'ascite (p<0.05) e della traslocazione nucleare di NF-kB negli animali cirrotici è stata notata dopo il trattamento con l'albumina. Conclusioni: I nostri risultati per la prima volta dimostrano che l'albumina esercita un effetto inotropo positivo sul cuore dei ratti cirrotici con ascite. Questo effetto coinvolge la sua capacità di contrastare gli effetti negativi sulla contrattilità cardiaca dei ROS e della pathway TNF-α-NF-kB- iNOS
CASAL, IDE ERIC. "LE CARATTERISTICHE MOLECOLARI DEL CARCINOMA PAPILLARE DELLA TIROIDE NON IODOFISSANTI." Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3427548.
Повний текст джерелаPresupposti dello studio: i carcinomi papillari tiroidei (PTCs) costituiscono la neoplasia endocrina più frequente e si caratterizzano per un decorso indolente e una bassa mortalità, dovuta principalmente alla capacità di questi tumori di concentrare lo iodio e alla possibilità di essere efficacemente trattati con iodio radioattivo (131-I). Tuttavia in circa un terzo dei pazienti, in particolare in corso di malattia persistente o ricorrente, viene persa la capacità di captare lo 131-I. Una mutazione somatica interessante l’esone 15 della serina-treonina-chinasi BRAF, realizzante la sostituzione di una valina con glutammato (V600E), rappresenta l’evento genico più frequente alla base dello sviluppo del carcinoma papillare tiroideo, documentabile nel 36-48% dei casi. Diversi studi hanno messo in evidenza che i carcinomi papillari con mutazione di BRAF hanno un atteggiamento più aggressivo, presentandosi in stadio più avanzato alla diagnosi e potendo evolvere verso istotipi meno differenziati o anaplastici nel follow-up. BRAF potrebbe pertanto rappresentare un nuovo fattore prognostico indipendente, in grado di identificare i carcinomi papillari a maggior rischio di recidiva e di perdita della capacità di concentrare lo 131-I. La mutazione di BRAF è inoltre associata ad un’aumentata espressione del trasportatore del glucosio GLUT1, suggerendo l’utilità di tecniche di imaging, quali la 18-FDG-PET-TC, nel follow-up dei pazienti con recidiva di tumore tiroideo differenziato. Scopo dello studio: 1) valutare la frequenza di mutazioni BRAF nei carcinomi papillari primitivi, nelle metastasi linfonodali al momento della diagnosi e nelle recidive e definire se tale frequenza sia maggiore nei tumori che recidivano e in particolare in quelli con recidive non iodofissanti; 2) valutare se la presenza di mutazioni BRAF si correli ai fenotipi con accelerato metabolismo del glucosio FDG-PET positivi. Pazienti e metodi: abbiamo selezionato 42 pazienti consecutivi operati per recidiva di carcinoma tiroideo differenziato; alla diagnosi tutti i pazienti erano stati sottoposti a tiroidectomia totale e a terapia radioablativa con 131-I. In questi pazienti la scintigrafia totale corporea è stata eseguita 3 giorni dopo una dose terapeutica di 131-I (2,7-5,4 GBq) dopo aver sospeso la terapia con ormone tiroideo. 23/42 pazienti sono stati sottoposti a 18FDG PET-TC. La presenza di mutazioni a carico dell’esone 15 di BRAF è stata analizzata tramite sequenziaggio e PCR allele-specifica (MASA). Risultati: tra le recidive di carcinoma papillare, in base allo staging tumorale, 10/42 (24%) erano T1, 8/42 (19%) T2, 4/42 (10%) T3 e 18/42 (43%) T4. (Di 2/42 pazienti non era disponibile la classificazione TNM). L'età media alla diagnosi era di 40, la mediana 35 anni (min 16, max 79). L'età media al momento della recidiva era di 45, la mediana 43 anni (min 16, max 82). La media dei valori di Tg durante terapia soppressiva era 181, mentre la mediana era 7 ng/ml (min.<0.5 max 3344). 11/42 pazienti presentavano positività/persistenza degli anticorpi anti-Tg durante il follow-up. BRAF risultava mutato in 19/37 (51%) recidive non iodofissanti e in 1/5 (20%) delle iodofissanti. Abbiamo quindi correlato la presenza di mutazioni BRAF con la capacità di captare il radioiodio e il 18-FDG (15 casi): di tali 15 casi, 14 erano recidive non iodiofissanti, mentre 1 era iodofissante. Nell’insieme delle recidive BRAF-mutate, tra le non iodofissanti 73% captavano il 18-FDG e 20% non lo captavano, mentre l’unica recidiva iodofissante BRAF-mutata era PET-positiva. Inoltre la presenza della mutazione correlava con gli indici di uptake più elevati. In 10 pazienti l’analisi delle mutazioni di BRAF è stata condotta simultaneamente sul tumore primitivo (K0), sulla metastasi linfonodale al momento della diagnosi (N0) e sulla prima recidiva non iodofissante (R1) asportata chirurgicamente e sulle eventuali successive (R2): BRAF risultava mutato in 2/10 (20%) tumori primitivi, in 3/7 (42%) metastasi linfonodali al momento della diagnosi e in 7/10 (70%) prime recidive non iodofissanti e 2/2 (100%) seconde recidive. Tramite sequenziaggio abbiamo inoltre dimostrato una nuova mutazione che interessa il codone 602 dell’esone 15 di BRAF e porta alla sostituzione dell’amminoacido serina con la fenilalanina (S602F). Conclusioni: nel nostro studio abbiamo dimostrato che 1) le mutazioni BRAF sono più frequenti nelle recidive non iodofissanti rispetto a quelle iodofissanti (51 % versus 20 %); 2) esse possono comparire de novo in corso di recidiva di tumore tiroideo primitivo (20% versus 42% e 70% rispettivamente nel tumore primitivo, nella metastasi linfonodale alla diagnosi e nella recidiva non iodofissante); 3) esiste una correlazione inversa tra la presenza della mutazione e la capacità di fissare lo iodio, ed un’associazione diretta tra la presenza della mutazione e la capacità di captare il 18-FDG e le recidive che mostrano la mutazione V600E hanno un indice di captazione elevato, suggerendo quindi l’esistenza di una correlazione tra il grado di captazione di 18-FDG e la presenza della mutazione; 4) una nuova mutazione di BRAF non riportata in Letteratura è presente a livello del codone 602 dell’esone 15 di BRAF e determina la sostituzione di un amminoacido polare - serina - con uno apolare - fenilalanina. In base ai dati del nostro studio sembrerebbe che BRAF si confermi come probabile marcatore molecolare di prognosi povera, in grado di promuovere lo sviluppo di recidive più aggressive, meno differenziate e meno iodocaptanti durante il follow-up.
Pirini, Francesca <1980>. "Composti perfluorurati: Valutazione degli effetti biologici e molecolari in modelli cellulari." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6259/1/PIRINI_FRANCESCA_TESI_.pdf.
Повний текст джерелаPerfluorooctanoic acid (PFOA) and perfluoronanoico acid (PFNA) are perfluorinated compounds (PFCs) largely employed during the last 60 years for several applications. Because of their resistance to degradation, they accumulate in the environment and organisms, for which the principal route of intake is the diet. The available information concerning their adverse effects on health in animals has recently increased the interest on the possible health risks in humans. Studies in humans indicate that PFCs are in serum at very high levels, especially in workers chronically exposed. Positive association with breast cancer and prostate cancer have been reported, but also estrogen -like property and variations in levels of methylation on genes promoters. In utero exposure was positively associated with global DNA hypomethylation in cord blood serum. The aim of this study was to investigate the effects of exposure to these perfluorinated on tumor and primary human cell lines ( MOLM -13, RPMI, HEPG2, MCF7, WBC, HMEC, MCF12A, belonging to different target tissues, using a wide range of concentrations (3.12 nM - 500 µM). In particular, it was assessed: the viability, cell cycle, gene expression, the global DNA methylation and gene specific methylation. The results showed that PFOA has a cytostatic effect, PFNA is mainly cytotoxic. The prevalent effect is on mammary epithelium primary cell(HMEC, MCF12A), even at concentrations found in chronically exposed workers (≥ 31.25 µM). The results don’t show significant changes in global DNA methylation at concentrations of 15.6 and 31.25 µM on the same cells, but we found variations of the methylation level on breast cancer markers genes and cell cycle, apoptosis, PPAR-α and estrogen pathways genes, after exposure to a concentration of 31.25 µM of PFCs.
Pirini, Francesca <1980>. "Composti perfluorurati: Valutazione degli effetti biologici e molecolari in modelli cellulari." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6259/.
Повний текст джерелаPerfluorooctanoic acid (PFOA) and perfluoronanoico acid (PFNA) are perfluorinated compounds (PFCs) largely employed during the last 60 years for several applications. Because of their resistance to degradation, they accumulate in the environment and organisms, for which the principal route of intake is the diet. The available information concerning their adverse effects on health in animals has recently increased the interest on the possible health risks in humans. Studies in humans indicate that PFCs are in serum at very high levels, especially in workers chronically exposed. Positive association with breast cancer and prostate cancer have been reported, but also estrogen -like property and variations in levels of methylation on genes promoters. In utero exposure was positively associated with global DNA hypomethylation in cord blood serum. The aim of this study was to investigate the effects of exposure to these perfluorinated on tumor and primary human cell lines ( MOLM -13, RPMI, HEPG2, MCF7, WBC, HMEC, MCF12A, belonging to different target tissues, using a wide range of concentrations (3.12 nM - 500 µM). In particular, it was assessed: the viability, cell cycle, gene expression, the global DNA methylation and gene specific methylation. The results showed that PFOA has a cytostatic effect, PFNA is mainly cytotoxic. The prevalent effect is on mammary epithelium primary cell(HMEC, MCF12A), even at concentrations found in chronically exposed workers (≥ 31.25 µM). The results don’t show significant changes in global DNA methylation at concentrations of 15.6 and 31.25 µM on the same cells, but we found variations of the methylation level on breast cancer markers genes and cell cycle, apoptosis, PPAR-α and estrogen pathways genes, after exposure to a concentration of 31.25 µM of PFCs.
MACCARONI, ELENA. "Caratterizzazione istologica e molecolare nelle pazienti con carcinoma ovarico sporadico ed ereditario." Doctoral thesis, Università Politecnica delle Marche, 2018. http://hdl.handle.net/11566/252973.
Повний текст джерелаIntroduction: Ovarian cancer represents the leading cause of cancer deaths among gynecological malignancies. Germline mutations in the BRCA1 and BRCA2 genes are associated with hereditary predisposition to breast and ovarian cancer. Together, germline mutations in BRCA1 and BRCA2 account for around 15% of OC cases. The present study aimed at evaluating pathological and molecular characteristics of BRCA-wild type (sporadic) and BRCA-mutant (hereditary) ovarian cancer (OC) patients. Patients and Methods: Between June 1996 and April 2017, 227 OC patients underwent genetic counselling and testing for BRCA1 and BRCA2 genes at Centro Regionale di Genetica Oncologica, Ancona. Detected BRCA mutations were divided based on their pathogenicity and type of genetic alteration (frameshift, nonsense, splice-site, missense, silent pathogenic mutations and large rearrangements). Results: Globally, 68 pathogenic mutations and 35 Variants of Uncertain Significance (VUS) were identified. Pathogenic mutations were significantly more frequent in the BRCA1 gene (83%) versus BRCA2 gene (51%) (p=0.00012). High grade serous OC was the most frequently reported histotype (56.7%), followed by endometrioid OC (17%). Median age at diagnosis was 52.03 years (range 16-83 years). No significant differences in terms of age at diagnosis were observed between BRCA-mutant vs BRCA-wild-type patients, neither between BRCA1-mutant and BRCA2-mutant patients. Among 227 patients, 160 (70.5%) had a positive family history for BRCA-related cancers, while 67 (29.5%) had a negative family history. In the first group, 94 patients (28.2%) had a pathogenic BRCA mutation, while in the second group only 4 (1.8%) were carriers of a pathogenic mutation. Finally, the Detection Rate (DR), defined as the probability to detect a pathogenic BRCA mutation, resulted significantly higher (40%) in OC patients with a positive family history compared with patients with a negative family history (6%) (p=0.0009). Conclusions: This work suggests the importance to analyze BRCA genes in OC patients in the Marche Region, as to perform comparisons among mutational and clinic-pathological features in our Region and in other geographical areas.
Marino, Flora <1977>. "Identificazione di un profilo molecolare di rischio nei pazienti pediatrici affetti da Linfoma di Hodgkin." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5369/1/marino_flora_tesi.pdf.pdf.
Повний текст джерелаPurpose: despite improvement in the treatment of advanced Hodgkin lymphoma (HL), approximately 30% of pediatric patients relapse or die as result of the disease. Current methods to predict prognosis determined by clinical and biological parameters, fail to identify these patients accurately. The aim of this study was to define a molecular profile of risk correlates with outcome in these patients. Methods: retrospective study of pediatric patients with LH homogeneously treated from 2004 onwards. Of these patients was undertaken a validation study of molecular markers already identified in exploratory studies previously. 27 best predictor genes in HL was evaluated in RT PCR in formalin-fixed paraffin embedded diagnostic lymph-node samples obtained from 37 pediatric patients with HL, including 25 responders and 12 non responders to standard treatment and compared the expression profiles of patients with favorable and unfavourable clinical outcome. Results: univariate regression analysis revealed that only the expression of CASP3 and CYCS genes, involved in the apoptotic pathway, is able to significantly predict failure to treatment in our cohort of patients. The study of the possible combinations of these genes has shown the existence of 3 risk groups that correlate with EFS: high risk (down regulation of both genes), intermediate risk (down regulation of only one of the 2 genes), low risk (up regulation of both genes). Multivariate analysis showed that CASP3 is the only variable that maintains its independence in influencing the prognosis with a risk of events more than double in patients with low expression of this gene Conclusions: The results of our cohort of pediatric patients with HL confirm the impact on prognosis of two molecular markers CASP3 and CYCS involved in the apoptotic pathway. The evaluation of the expression profile of these genes, may therefore be used in the course of staging, as a criterion of predictivity.