Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate. The compounds melatonin and sPDZD2 have been suggested to act through the Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation. In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation. Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Type 1 diabetes (T1D) is a chronic disease, characterised by the destruction of pancreatic beta cells, which results in lack of insulin expression. Most current therapies rely on the exogenous administration of recombinant insulin. Islet transplantation has been shown to be a more effective alternative treatment, but it is also limited by the lack of available islets for transplantation. The recently awarded work of Yamanaka and colleagues has shown that fully differentiated cells can be reprogrammed towards their pluripotent, undifferentiated state, through overexpression of a simple combination of four key transcritption factors (TFs). The studies presented in this thesis sought to investigate whether a combination of a small group of key pancreatic TFs would be able to drive both mouse embryonic stem cells (mES) and fully differentiated rat and human acinar cells of the pancreas towards insulin-producing cells. When administered in a timely manner to mES cells, the pancreatic TFs Pdx1 and MafA were able to induce the formation of cells that synthesised insulin de novo. Further studies aimed at investigating if a small number of TFs would be able to reprogramme the exocrine pancreatic cells towards insulin expressing cells, since, unlike endocrine cells, exocrine cells are highly abundant in the pancreas. Studies performed in both a rat exocrine cell line (AR42J-B13) and in human exocrine cells demonstrated that the combination of the TFs Pdx1, MafA, Ngn3 and Pax4 was able to generate glucose responsive β-like cells in both models. In addition, Pax4 was found to be determinant for the functionality of the generated β-cells. The functionality of these cells was further demonstrated by their ability to prevent the onset of hyperglycemia upon transplantation into a diabetic mouse model. The work presented in this thesis has shown that cultured exocrine cells may be a promising alternative for generating a replenishable supply of β-cells for transplantation.

One of the current goals in cancer research is to discover and validate novel molecular targets that may be useful for diagnostic and therapeutic purposes in fighting this disease. The PRL phosphatases (PRL-1, PRL-2, and PRL-3) are low molecular weight protein tyrosine phosphatases with unknown biological function(s) that have gained attention from cancer researchers in the past couple of years, mainly due to reports that these phosphatases may play important roles in tumor progression and metastasis. Motivated by the particular urgent need for molecular targets in pancreatic cancer this work was undertaken to determine what role PRL proteins played in pancreatic cancer biology and to determine if targeting PRLs would be effective in treating this disease. In this dissertation, it was found that both PRL-1 and PRL-2, but not PRL-3 are upregulated in pancreatic adenocarcinomas, suggesting that some cancer cells are dependent upon their activity for continued proliferation and survival. To validate this hypothesis, siRNAs were used in cell-based assays to evaluate the biological consequences of PRL-1 and/or PRL-2 inhibition. It was found that perturbations in PRL phosphatase signaling result in reduced proliferation, migration and especially the ability to grow in soft agar. Oligonucleotide microarray analysis revealed that many Erk and/or Akt dependent stress and growth factor inducible genes were differentially regulated between pancreatic cancer cells treated with PRL-targeting siRNA and their non-targeting siRNA treated counterparts. Subsequently, PRL knockdown was found to alter serum induced as well as amino acid deprivation induced Akt and Erk phosphorylation in multiple pancreatic cancer cell lines, suggesting that PRLs function upstream of these key pathways. Interestingly, we show that PRL proteins in cell free assays exhibit higher activity on doubly phosphorylated phosphatidylinositol substrates than tyrosine-phosphorylated peptides, suggesting that the biological substrate(s) might include non-protein molecules. These data support the hypothesis that PRL-1 and PRL-2 might play important biological roles in pancreatic cancer cells and further studies should be undertaken to determine the usefulness of these phosphatases as potential molecular biomarkers and targets.

Background: Palliative gemcitabine has been shown to prevent the deterioration of well-being and to prolong survival of patients with pancreatic cancer in phase III randomized controlled trials (RCTs). It is unknown whether the efficacy reported in RCTs has translated into effectiveness in routine clinical practice. Objectives: 1) To describe the characteristics of patients with pancreatic cancer treated with palliative gemcitabine at the regional cancer centres (RCCs) of Ontario, 2) To describe: clinical benefit at two months, defined as stable or improved well-being; time to treatment discontinuation; and overall survival, 3) To identify factors associated with clinical benefit, and 4) To compare the effectiveness of gemcitabine with its reported efficacy in RCTs. Methods: This was a retrospective analysis of prospectively collected data. The study included patients with pancreatic cancer treated with palliative gemcitabine at the RCCs of Ontario between 2008 and 2011. Information about well-being was patient self-reported as captured by the Edmonton Symptom Assessment System (ESAS) at the RCCs. The proportions of patients that achieved clinical benefit were reported. Time to treatment discontinuation and overall survival were calculated using Kaplan –Meier survival analysis. Logistic regression was used to identify factors associated with clinical benefit. iii Results: The study population included 423 patients. Only 168 (39.1%) patients completed a pre-treatment ESAS. Patients completing a pre-treatment ESAS were not different than those that did not. Patients treated at RCCs were not different than those in RCTs. The median age of the study population was 65 years, 50% were male, 57% had stage IV disease and 94% had adenocarcinoma morphology. Thirty-seven percent of patients achieved clinical benefit at two months. Median time to treatment discontinuation and overall survival was 2 and 5.7 months, respectively. Stage and pre-treatment wellbeing were associated with clinical benefit at two months. Similar proportions of patients at RCCs and RCTs experienced clinical benefit. Time to treatment discontinuation and survival were similar as well. Conclusions: Efficacy of gemcitabine in RCTs has translated into effectiveness for patients treated at the RCCs of Ontario. It is unknown if this is true for patients not treated at the RCCs. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2012-08-01 17:50:00.185

No description available.

Familial Pancreatic Cancer (FPC) is an autosomal dominant condition with greater then 80% of genetic causes unknown. We hypothesize that an integrative approach employing germline exome sequencing and somatic microarray analysis of FFPE DNA will identify novel tumour suppressor genes (TSGs). 55 FPC and 21 sporadic PDAC tumours were analyzed on the Affymetrix Oncoscan FFPE Express 2.0 SNP microarray and compared to data from 33 germline FPC cases analyzed on the Illumina GAIIx Analyzer. We have demonstrated that FPC is genetically heterogeneous with recurrent loss at CDKN2A/p16, TP53 and SMAD4. Analysis of 2 sisters has shown a shared loss region involving DCLK3 and SERPINF1. By an integrative approach, we have identified ATPAF1-AS1 and MAP3K6 as potential TSGs. Germline variants of these genes have been confirmed by Sanger sequencing and somatic fluorescence in-situ hybridization. Future functional studies will better characterize the importance of these regions and novel putative TSGs in FPC.

Familial Pancreatic Cancer (FPC) is an autosomal dominant condition with greater then 80% of genetic causes unknown. We hypothesize that an integrative approach employing germline exome sequencing and somatic microarray analysis of FFPE DNA will identify novel tumour suppressor genes (TSGs). 55 FPC and 21 sporadic PDAC tumours were analyzed on the Affymetrix Oncoscan FFPE Express 2.0 SNP microarray and compared to data from 33 germline FPC cases analyzed on the Illumina GAIIx Analyzer. We have demonstrated that FPC is genetically heterogeneous with recurrent loss at CDKN2A/p16, TP53 and SMAD4. Analysis of 2 sisters has shown a shared loss region involving DCLK3 and SERPINF1. By an integrative approach, we have identified ATPAF1-AS1 and MAP3K6 as potential TSGs. Germline variants of these genes have been confirmed by Sanger sequencing and somatic fluorescence in-situ hybridization. Future functional studies will better characterize the importance of these regions and novel putative TSGs in FPC.

No description available.

Type 1 diabetes is caused by immune destruction of pancreatic β cells. There is increasing evidence that genes outside the MHC region contribute to the pathogenesis of type 1 diabetes. Cytokines due to their role in immune regulation seem to play a crucial role in the pathogenesis of the disease. Three hundred and eight patients with type 1 diabetes and 150 normal controls were genotyped for polymorphism in the genes for IFN-γ, IL-4, IL-6, and TGF-β1. All assays employed in this study were PCR based. The IFN-γ CA repeats was an octa-allelic repeat and the 3 / 3 genotype showed a significant association with type 1 diabetes (p=0.0001). The IL-4 C (-590) T polymorphism did not show a significant association with type 1 diabetes. The GG genotype of G (-174) C of the IL-6 gene polymorphism showed a strong association with the susceptibility towards type 1 diabetes (p= 0.002). The TC genotype of the TGF-β1 T (+869) C polymorphism also showed a significant association with type 1 diabetes (p= 0.003). The association of the 3 I 3 genotype of the IFNG CA repeats and no association of IL-4 C (-590) T polymorphism may support the idea of dominance of the TH1 cytokine profile and type 1 diabetes suggesting a cell mediated disease. The IL-6 G (-174) C result attests an existing hypothesis of the important role of IL-6 in the onset of type 1 diabetes and its development. Immunosuppression of the TGF-β1 may have been initiated after deviation of the TH1/TH2 cytokine milieu. The GC of the IL-6 G (-174) C and the TC of the TGF-β1 T (+869) C showed strong association with diabetic nephropathy. Haplotype studies showed that cytokine function might be as a result of a cytokine network rather than individual cytokines. Further, the genetic susceptibility may be influenced not only by genetic composition but by the gender of patients as well as age at onset of type 1 diabetes. In conclusion these results suggest a contribution of the IFNG CA repeats, the TGF-β1 T (+869) C, and the IL-6 G (-174) C to the genetic susceptibility of type l diabetes and may have future therapeutical values.