Neste estudo foi implementado um pipeline bioinformático para processar e analisar dados de RNA-Seq total e fita-específico gerados em nosso laboratório a partir de amostras pareadas de tumor e tecido adjacente não tumoral de 14 pacientes com o objetivo de catalogar com alta-resolução a composição e alterações no transcritoma no PDAC incluindo genes codificadores e não codificadores de proteína. / In the present work, we applied a bioinformatic pipeline to process and analyse data from total RNA-seq strand-oriented generated in our laboratory from matched samples of tumor and non-tumor adjacent pancreatic tissue from 14 patients with the goal of generate a high resolution catalog of the composition and the alterations in the transcriptome of PDAC, including protein coding and non coding genes.

This thesis describes a study undertaken between 1992 and 2001 to explore the possible health impacts of human exposure to pesticides. The study followed the health outcomes of approximately 4000 outdoor workers over a period of up to sixty-one years. These workers comprised two subcohorts of approximately even size, one composed of agricultural workers with high insecticide exposures, and the other made up of outdoor staff from local councils in the same area with little or no occupational exposure to insecticides. Mortality and morbidity were compared between the two groups, and with the general Australian community. The study identifies significantly increased mortality among both exposed and control subjects when compared to the Australian population. The major cause of this increase was mortality from smoking related diseases. The study also identifies significant increases in mortality among exposed subjects for a number of conditions that do not appear to be the result of smoking patterns, both when compared to the control group and the Australian population. These include pancreatic cancer in some DDT exposed subjects and asthma, diabetes, and leukaemia in subjects working with more modern chemicals. There was also an increase in self reported chronic illness and asthma, and lower neuropsychological functioning scores among surviving exposed subjects when compared to controls. Diabetes was also reported more commonly by subjects reporting occupational use of herbicides.

The domestic dog, Canis lupus familiaris, has emerged as a model system for the study of human hereditary diseases. Of the approximately 450 hereditary diseases described in the dog, half have clinical presentations that are quite similar to specific human diseases. Understanding the genetic bases of canine hereditary diseases will not only complement comparative genetics studies but also facilitate selective breeding practices to reduce incidences in the dog. Whole genome screens have great potential to identify the marker(s) that segregate with canine hereditary diseases for which no reasonable candidate genes exist. The Minimal Screening Set-1 (MSS-1) was the first set of microsatellite markers described for linkage analysis in the dog and was, until recently, the best tool for genome screens. The MSS-2 is the most recently described screening set and offers increased density and more polymorphic markers. The first objective of this work was to develop tools to streamline genomic analyses in the study of canine hereditary diseases. This was achieved through the development of 1) multiplexing strategies for the MSS-1, 2) a multiplex of microsatellite markers for use in canine forensics and parentage assays and 3) chromosome-specific multiplex panels for the MSS-2. Multiplexing is the simultaneous amplification and analysis of markers and significantly reduces the expense and time required to collect genotype information. Pancreatic acinar atrophy (PAA) is a disease characterized by the degeneration of acinar cells of the exocrine pancreas and is the most important cause of exocrine pancreatic insufficiency (EPI) in the German Shepherd Dog (GSD). Although the prognosis for dogs having EPI is typically good with treatment, many dogs are euthanized because the owners are unable to afford the expensive enzyme supplements. The second objective of this work was to determine the mode of transmission of EPI in the GSD and conduct a whole genome screen for linkage. Two extended families of GSDs having PAA were assembled and used to determine the pattern of transmission. The results of this indicate that PAA is an autosomal recessive disease. The multiplexed MSS-1 was used to conduct an initial whole genome screen, although no markers were suggestive of linkage.

Zinc levels in pancreatic islets are amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genome-wide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we begin to elucidate the molecular mechanisms linking ZnT8 and type 2 diabetes by characterizing global and beta cell specific ZnT8 knockout mice. Our results associate absence of ZnT8 with a reduction in zinc sequestration into insulin vesicles, abnormal insulin granule morphology, down regulation of insulin processing enzymes, abnormal insulin secretion, elevated plasma proinsulin levels and diet-induced obesity and insulin resistance. Furthermore, we observed differential zinc uptake properties by two human ZnT8 variants. We report here that the W325 variant of ZnT8 is more efficient in mediating zinc transport than the at risk variant, R325. Cumulatively, these results suggest that ZnT8 is crucially important for zinc transport and zinc-insulin crystallization in insulin granules of the pancreatic beta cell.

No description available.

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. We demonstrated that curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of survivin, VEGF and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on downregulation of Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. Curcumin decreases expression of Sp1, Sp3 and Sp4 in blader cancer cells indicating that the cancer chemotherapeutic activity of curcumin is due, in part, to decreased expression of Sp transcription factors and Sp-dependent genes. Betulinic acid (BA) and curcumin are phytochemical anticancer agents, and we hypothesized that both compounds decrease EGFR expression in bladder cancer through downregulation of specificity protein (Sp) transcription factors. BA and curcumin decreased expression of EGFR, Sp1, Sp3, Sp4 and Sp-dependent proteins in 253JB-V and KU7 cells; EGFR was also decreased in cells transfected with a cocktail (iSp) containing small inhibitory RNAs for Sp1, Sp3 and Sp4 showing that EGFR is an Sp-regulated gene. Methyl 2-cyano-3,11-dioxo-18?-olean-1,12- dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid which inhibits proliferation of KU7 and 253JB-V bladder cancer cells. CDODA-Me also decreased expression of specificity protein-1 (Sp1), Sp3 and Sp4 transcription factors. Similar results were observed for a structurally-related triterpenoid, methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), which is currently in clinical trials for treatment of leukemia. Celastrol, a naturally occurring triterpenoid acid from an ivy-like vine exhibits anticancer activity against bladder cancer cells. Celastrol decreased cell proliferation, induced apoptosis and decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-dependent genes like Fibroblast growth factor receptor 3 (FGFR3). In vivo studies using KU7 cells as xenografts showed that celastrol represents novel class of anticancer drugs that acts, in part, through targeting downregulation of Sp transcription factors.

Zinc levels in pancreatic islets are amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genome-wide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we begin to elucidate the molecular mechanisms linking ZnT8 and type 2 diabetes by characterizing global and beta cell specific ZnT8 knockout mice. Our results associate absence of ZnT8 with a reduction in zinc sequestration into insulin vesicles, abnormal insulin granule morphology, down regulation of insulin processing enzymes, abnormal insulin secretion, elevated plasma proinsulin levels and diet-induced obesity and insulin resistance. Furthermore, we observed differential zinc uptake properties by two human ZnT8 variants. We report here that the W325 variant of ZnT8 is more efficient in mediating zinc transport than the at risk variant, R325. Cumulatively, these results suggest that ZnT8 is crucially important for zinc transport and zinc-insulin crystallization in insulin granules of the pancreatic beta cell.

Pancreatic cancer (PC) is known to be highly resistant to chemotherapy. Transporters, which regulate the influx and efflux of substrates across the plasma membrane, may play a role in PC drug resistance. ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which play major roles in cancer drug resistance. Given that 90% of PC express a mutant K-ras oncogene and that PC are highly hypoxic, I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote drug resistance in PC. Using normal and PC cell lines either overexpressing mutant K-ras or subjected to hypoxic treatment, mRNA expression was profiled for 48 ABC transporters. My findings indicate that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in PC cells in part by inducing the expression of ABC transporters. Similar to ABC transporters, I investigated whether amino acid transporters would mediate drug resistance in PC. The xc" amino acid transporter (xc") mediates cellular uptake of cystine for the biosynthesis of glutathione, a major detoxifying agent. Because the xc" has been regulates the growth of various cancer cell types, and x," is expressed in the pancreas, I postulated that the xc" may be involved in growth and drug resistance in PC. The xc" transporter is differentially expressed in normal pancreatic tissues and is overexpressed in PC in vivo. UsingPC cell lines, I found that cystine uptake via the N.: was required for growth and survival in response to oxidative stress, and that expression of the xc" correlated with gemcitabine resistance. Accordingly, inhibition of xc" expression via siRNA reduced PC cell proliferation and restored sensitivity to gemcitabine. I also identified the anti-inflammatory drug sulfasalazine as a mixed inhibitor of the x,-, which acts to inhibit cell proliferation via reducing xc" activity and not by reducing NFKB activity. My findings thus indicate that the xc" plays a role in PC growth in part by contributing to glutathione synthesis to promote PC cell proliferation, survival, and drug resistance.

<p>Abstract</p><p>The trefoil factor family of secreted proteins contains three members; trefoil factor 1 or TFF1, trefoil factor 2 or TFF2, and trefoil factor 3 or TFF3. These three proteins share a conserved 42-43 amino acid domain containing 6 cysteine residues resulting in three disulfide bonds that holds the protein in a characteristic three-loop or "trefoil structure" known as the P domain. TFF1 is primarily localized to the stomach and secreted by the gastric mucosa while TFF2 and TFF3 are primarily localized to the colon and duodenum and secreted by the goblet cells. All three of these proteins play a protective role in the gastrointestinal tract where they are normally localized and have been identified as possible tumor suppressors, however, these proteins are also upregulated in cancer within tissues where they are not normally expressed including the breast, pancreas, prostate, and liver. The mechanisms by which two of these factors, TFF1 and TFF3, promote tumorigenesis remain largely undefined. In this dissertation we will attempt to elucidate these mechanisms as well as the regulation of these two proteins in both pancreatic and prostate cancer. Many of the underlying genetic and molecular mechanisms involved in the development of both pancreatic and prostate cancer remain largely unknown and as a result, therapeutic and diagnostic tools for treating these diseases are not as effective as they could be. By deciphering the role of TFF1 and TFF3 in these cancers, they could potentially serve as new therapeutic targets or biomarkers for treating both diseases.</p><p>Chapter 2 of this dissertation will examine the functional role of TFF1 promoting tumorigenesis in pancreatic and prostate cancer. We will show that TFF1 expression is critical for the viability of both pancreatic and prostate cancer cells and that reduction of TFF1 expression in these cells results in decreased tumorigenicity when implanted in immunocompromised mice. It will also be demonstrated that TFF1's function in promoting tumorigenicity is its ability to assist tumor cells overcome the tumor suppressive barrier of senescence. Thirdly, we show that the form of senescence that TFF1 assists in allowing the cells overcome is oncogene-induced senescence (OIS). Lastly, a cell cycle array identifies the potential downstream target p21CIP, a cyclin-dependent kinase inhibitor and OIS marker, whose expression is induced by loss of TFF1 expression.</p><p>In Chapter 3 of this work, we examine the role of another trefoil factor family member, TFF3, and its role in promoting prostate tumorigenesis. Just as with TFF1, it appears that TFF3 3 expression is critical for prostate cancer cell viability and tumorigenicity using the same experimental techniques used in Chapter 2. Using a genetically defined model of prostate cancer, a PI3-kinase-dependent regulatory mechanism of TFF3 emerges in this prostate cancer context. Using this system we begin to see a divergence in both regulation and function of TFF1 and TFF3 in prostate cancer. Finally, a mouse model expressing TFF3 was developed to monitor the histopthological changes associated with expression of this protein. Initial characterization of this model suggests a hyperplastic phenotype coinciding with TFF3 expression in the prostate.</p><p>The two studies in this dissertation establish a role of TFF1 and TFF3 in both prostate and pancreatic tumorigenesis and demonstrate that ablation of expression of both proteins is a potent inhibitor of tumorigenesis. With this knowledge, it is possible that TFF1 and TFF3 may become a potential therapeutic target or diagnostic marker for better treatment of prostate and pancreatic cancer.</p> / Dissertation

Genetically engineered cells have the potential to solve the cell availability problem in developing a pancreatic tissue substitute for the treatment of insulin-dependent diabetes (IDD). These cells can be beta-cells genetically engineered so that they can be grown in culture, such as the betaTC3 and betaTC tet mouse insulinomas developed by Efrat et al; or, they can be non-beta cells genetically engineered to secrete insulin constitutively or under transcriptional regulation. The aim of this work was to thoroughly characterize and improve the secretion dynamics of pancreatic substitutes based on genetically engineered cells. One issue involved with the continuous beta-cell lines is the remodeling of the cells inside an encapsulated cell system, which may affect the insulin secretion dynamics exhibited by the construct. To evaluate the effect of remodeling on the secretion properties of the construct, we used a single-pass perfusion system to characterize the insulin secretion dynamics of different alginate beads in response to step-ups and downs in glucose concentration. Results indicated that the secretion dynamics of beads indeed changed after long-term culture. On the other hand, data with a growth-regulated cell line, betaTC tet cells, showed that the secretion profile of beads can be retained if the cell growth is suppressed. A major concern associated with genetically engineered cells of non-beta origin is that they generally exhibit sub-optimal insulin secretion characteristics relative to normal pancreatic islets. Instead of relying on molecular tools such as manipulating gene elements, our approach was to introduce a glucose-responsive material acting as a control barrier for insulin release from a device containing constitutively secreting cells. Proof-of-concept experiments were performed with a disk-shaped prototype based on recombinant HepG2 hepatomas or C2C12 myoblasts, which constitutively secreted insulin, and concanavalin A (con A)-based glucose-responsive material as the control barrier. Results demonstrated that the a hybrid pancreatic substitute consisting of constitutively secreting cells and glucose-responsive material has the potential to provide a more physiologic regulation of insulin release than the cells by themselves or in an inert material.