A series of in vitro trials have been conducted to determine what relationship exists between zinc and the insulin mechanism. There were no significant differences of the zinc contents in total pancreases excised from the rats thirty minutes after the intraperitoneal glucose dose or from the non-dosed controls. When pancreases were cut into three pieces, and treated in three ways: Incubated without glucose (IWG) followed by isolation of the islets; incubated with glucose (IWG) followed by isolation of the islets; islets were isolated, then incubated with glucose (IWG). The zinc content in the islets was significantly higher (p < 0.01) in the first group than the others. The insulin release and zinc movement were studied in zinc-deficient status. In both Experiment 3 and 5 each pancreas was divided into two, one was treated IWG, one was treated IWOG. There were significant (p < 0.01) differences of zinc content in the islets between lWG and IWOG groups in zinc-supplemented pair-fed and ad lib. controls but not in zinc-deficient rats. In Experiment 5 the zinc contents in the islets of IWOG group were significantly (p < 0.01) lower in zinc-deficient rats than in zinc-supplemented pair-fed rats. Insulin release from the islets incubated with glucose was significantly (p < 0.01) less in zinc-deficient group than in zinc-supplemented pair-fed group. Glutathione contents of the pancreases were measured in Experiment 3, no significant difference was found between zinc-deficient group and both zinc-supplemented groups. Rats were intraperitoneally dosed with glucose or with saline in Experiment 6. After 30 minutes pancreases were excised and the isolated islets were treated with IWG or IWOG. There was no significant difference of zinc content in the islets treated IWOG in glucose-dosed and saline0dosed groups, but significant (p < 0.01) difference was found in the islets treated IWG. Insulin release from the islets treated IWG was significantly (p < 0.01) less in glucose-dosed group than in saline-dosed group. It is concluded that zinc is released from the islets as a component of insulin on glucose stimulation, zinc-deficiency results in an impairment of insulin release or synthesis, and insulin secretion was reduced in the isolated islets taken from animals prestimulated with glucose in vivo.

Pancreatic cancer (PC) is the fourth leading cause of cancer death in Western societies. Despite significant progress in understanding the molecular pathology of PC and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility. Affymetrix Genechipfi oligonucleotide microarrays were used to interrogate mRNA expression of PC and normal pancreas to identify molecular pathways dysregulated in PC. Analysis of these data identified altered expression of numerous components of the S100 Calcium Binding Protein Family, Retinoic Acid signalling pathway and the HOX transcriptional network in PC compared to normal pancreas. These pathways were assessed using immunohistochemistry (IHC) and in-situ hybridisation (ISH) in a cohort of patients with PC. Increased protein expression, of S100A2, S100A6 and S100P was observed in 43%, 60% and 48% of PC respectively. Expression of S100A2 was associated with a poor outcome (p = 0.009), whilst increased expression of S100A6 (p = 0.0008) and S100P (p = 0.0005) were associated with an improved outcome. Additionally, S100A2 expression was identified as an independent marker of outcome in resected tumours. Aberrant expression of retinoic acid signalling components was demonstrated in PC cell lines using semi-quantitative RT-PCR. ISH demonstrated expression of Retinoic Acid Induced 3 (RAI3), an orphan G protein coupled receptor normally expressed in the fetal lung, in 68% of PC, and this co-segregated with an improved overall survival (p = 0.026).Ectopic protein expression of HOXB2, a transcription factor normally expressed in the developing hindbrain and modulated by retinoic acid, was observed in 15% of early PanIN lesions and 38% of PC specimens. Expression of HOXB2 was associated with non-resectable tumours and was an independent predictor of poor survival in resected tumours. Suppression of HOXB2 protein expression using small interfering RNA, resulted in epithelioid trans-differentiation in the Panc-1 PC cell line, however no alteration in proliferation rates were observed compared to controls. This thesis has shown that transcript profiling and tissue validation has identified potential markers of early diagnosis and outcome in PC. Furthermore, pathways and molecules previously thought to be associated with normal human development have been implicated to play a role in the development and progression of PC. Further analyses of these markers will determine any potential role in future diagnostic and therapeutic strategies.

The characteristics of an inhibitor of infectious pancreatic necrosis virus (IPNV) found in normal rainbow trout serum (RTS) were studied. The serum inhibitor had a molecular weight of approximately 150 kDa and was dependent on divalent cations, either Ca����� or Mg�����. It was stable at temperatures up to 50��C and at a pH range between 4-10. The inhibitor directly inactivated the virus and the inhibition level was dependent on cell densities and on the time at which virus was exposed to RTS. The level of virus inhibition by RTS was altered by the cell line in which virus was produced. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines. Most of the salmonid sera tested showed inhibition, while non-salmonid sera did not inhibit IPNV replication. Rainbow trout continuously showed a significant level of inhibition in their serum after 23 weeks post hatch. Three isolates of IPNV were passaged five times in RTG-2 cells with either MEM-10 or MEM-10 with 1% rainbow trout serum and virus from each passage were tested for RTS sensitivity in vitro and virulence in vivo. The mortality level in brook trout fry was highly variable during viral passages, ranging between 30-89%. The RTS sensitivity and virulence were changed during viral passages, and these changes were dependent on cell culture conditions and IPNV isolate used. It was found that an IPNV crayfish isolate passaged in RTG-2 cells with MEM-10 showed significantly increased RTS sensitivity. This was, however, not correlated with decreased virulence. All three isolates showed identical antigenicity patterns with a panel of 11 monoclonal antibodies, irrespective of viral passage conditions. Clones prepared from an IPNV-Jasper (Ja) population which had been twice passed through brook trout were heterogeneous with respect to RTS sensitivity, serotype, and cDNA sequences. Eight percent of clones (4/50) were very sensitive to RTS (Ja-S), as was the parent strain, and eighty four percent of clones (42/50) showed a mid-range of RTS sensitivity. The final eight percent of clones (4/50) were RTS resistant (Ja-R). Enzyme immunodot assay revealed that Ja-S clones and Ja-R clones differed by several epitopes. Ja-S and Ja-R had significant differences in their cDNA sequences for the capsid protein VP2. These two strains shared 80.7% and 86% identity in nucleic acid and in amino acid sequences, respectively. / Graduation date: 2001

The pancreas is an endodermally derived organ consisting of three major cell lineages. The endocrine cells, organised into the Islets of Langerhans, regulate blood glucose homeostasis by producing and secreting hormones such as glucagon and insulin into the bloodstream. The major part of the pancreas consists however of acinar cells that produce digestive enzymes that are transported via a highly branched ductal system to the duodenum where they function in breakdown of food. Early in pancreas development a dorsal and ventral evagination of the foregut epithelium appear, resulting in the formation of the dorsal and ventral pancreatic bud. These pancreatic buds subsequently grow, branch and differentiate to form the mature pancreas via a process controlled by intrinsic factors, such as transcription factors, and extracellular signals. Insulin promoter factor 1 (Ipf1), also known as Pdx1 (for Pancreatic duodenal homeobox gene 1), is required for pancreas development. Although the evagination of pancreatic buds still occurs in Ipf1/Pdx1 mutant mice, the subsequent proliferation, branching and differentiation is impaired, resulting in complete pancreatic agenesis. Gene array profiling identified several candidate Ipf1/Pdx1 target genes, including FgfR2IIIb, ErbB3, Ptf1a/p48, Pax6 and Nkx6.1, in pancreatic progenitor cells. Together these genes provide a mechanistic explanation for the pancreatic growth arrest observed in Ipf1/Pdx1 deficient mice. In addition, Spondin1, which has not previously been described in the pancreas, was identified to be regulated by Ipf1/Pdx1. The spatial and temporal expression pattern of Spondin1 defines Spondin1 as a marker for early pancreatic progenitor cells. The Notch signalling pathway controls cell type specification and differentiation during pancreas development. The Fringe family of proteins have previously been shown to regulate Notch signalling by altering the interaction between Notch receptors and their ligands, hence affecting the cellular response. Manic Fringe (MFng) is transiently expressed in pancreatic pro-endocrine cells between E9.5 and E14.5. The expression of MFng is regulated by Ngn3, which may suggest a role for MFng in pro-endocrine cell maturation. The lack of a pancreatic phenotype in transgenic mice overexpressing MFng in the pancreatic epithelium and in MFng null mutant mice, however, provide evidence that MFng is dispensable for the specification, differentiation and function of the adult pancreas. Inhibitors of DNA binding (Id) proteins are generally known as inhibitors of differentiation, a feature they mainly perform by forming non-functional heterodimers with bHLH proteins, thereby inhibiting downstream targets of the bHLH proteins. Id proteins also promote cell proliferation by interacting with the cell cycle machinery. In the developing pancreas Id2 and Id3 are co-expressed in an overlapping manner during the period of massive proliferation and expansion of the pancreatic epithelium, suggestive of a role for the Id proteins during these processes. In addition, Id4 expression is also detected in the embryonic pancreas, albeit at lower levels. Gain- and loss- of- function analyses suggest however that specification, differentiation and function of the adult pancreas are largely independent of Id function.

In this thesis, I describe studies identifying and characterizing two putative stem cell populations of the neural and pancreatic lineages. The mechanisms governing the emergence of the earliest mammalian neural cells during development and the ontogeny of neural stem cells remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition, however an instructive process has also been proposed. I utilized mouse ES cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals, and what cellular fate will result. Individual ES cells were found to rapidly transition directly into neural cells by a default mechanism, a process shown to be independent of suggested instructive factors. Further, I provide evidence that the default neural identity is that of a primitive neural stem cell, the earliest identified stem cell of the neural lineage. The exiguous conditions used to reveal the default state were found to present primitive neural stem cells with a survival challenge, which could be mitigated by survival factors or genetic interference with apoptosis. I also report the clonal identification of multipotent precursor cells, PMPs, from the adult mouse and human pancreas. These cells proliferate in vitro to form clonal colonies and display both pancreatic and neural cell multipotentiality. Importantly, the newly generated beta cells demonstrate glucose-dependent calcium responsiveness and regulated insulin release. PMP colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. Moreover, genetic lineage-labeling experiments excluded the neural crest, and established the embryonic pancreatic lineage, as the developmental source of PMPs. The PMP cell was further found to express insulin in vivo, and insulin+ stem cells were shown to contribute to multiple pancreatic and neural cell populations in vivo. These findings demonstrate that the adult mammalian pancreas contains a population of insulin+ multipotent stem cells, capable of contributing to the pancreatic and neural lineages. In the final section of this thesis, I consider the relationships between neural and pancreatic tissues, as well as discussing the relevance of these two novel stem cell populations.

No description available.

No description available.

The aggressive nature of pancreatic cancer, characterized by invasiveness, resistance to treatment, rapid progression, and its high prevalence in the population urges the need for developing more effective treatments. Many studies have attributed resistance to therapeutics of pancreatic cancer to activity of the transcription factor nuclear factor kappa B (NF-kB). NF-kB is regulated by the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is a key mediator of pathways such as insulin, wnt, and PI3K/Akt and has roles in proliferation, glucose metabolism, apoptosis, motility and neuroprotection. Depending on the cellular context, GSK-3 activity can promote or inhibit cell survival. GSK-3 inhibition was recently reported to have anti-cancer effects against pancreatic cancer cells. This effect was in part attributed to suppression of NF-kB. In this thesis, I showed that while blocking GSK-3 disrupts NF-kB, and has anti-survival effects on pancreatic cancer cells, it does not sensitize to the chemotherapeutic drug gemcitabine. NF-kB inhibition by curcumin also resulted in similar effects. These results questions previous reports that NF-kB activation plays a major role in chemo-resistance of pancreatic cancer. The inhibition of NF-kB by genetic disruption of GSK-3 was previously reported to sensitize mouse embryonic fibroblasts and hepatocytes to TNF-alpha cytotoxicity. I therefore tested whether GSK-3 inhibition could sensitize pancreatic cancer cells to apoptosis induced by the clinically applicable member of the TNF-alpha family, TNF-alpha related apoptosis inducing ligand (TRAIL). In contrast to the results obtained with gemcitabine, the combination of genetic or pharmacological inhibition of GSK-3 and TRAIL was found to be highly synergistic in apoptosis induction. Analysis of the apoptotic mechanisms, point towards effects of GSK-3 inhibition on caspase-8 activation, consistent with inhibition of the death receptor signalling pathway. It was found that not only caspase-8 but also mitochondrial anti-apoptotic proteins such as Bcl-XL and Mcl-1 were mediating the TRAIL sensitization. Furthermore, for the first time the in vivo effects of GSK-3 inhibition in combination with TRAIL treatment was investigated. The results indicate a significant enhancement of apoptosis in pancreatic cancer xenografts with minimal toxic effects. Together, these studies provide a rationale for developing combination treatments based on GSK3 inhibition and TRAIL death receptor activation to treat pancreatic cancer.

In this thesis, I describe studies identifying and characterizing two putative stem cell populations of the neural and pancreatic lineages. The mechanisms governing the emergence of the earliest mammalian neural cells during development and the ontogeny of neural stem cells remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition, however an instructive process has also been proposed. I utilized mouse ES cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals, and what cellular fate will result. Individual ES cells were found to rapidly transition directly into neural cells by a default mechanism, a process shown to be independent of suggested instructive factors. Further, I provide evidence that the default neural identity is that of a primitive neural stem cell, the earliest identified stem cell of the neural lineage. The exiguous conditions used to reveal the default state were found to present primitive neural stem cells with a survival challenge, which could be mitigated by survival factors or genetic interference with apoptosis. I also report the clonal identification of multipotent precursor cells, PMPs, from the adult mouse and human pancreas. These cells proliferate in vitro to form clonal colonies and display both pancreatic and neural cell multipotentiality. Importantly, the newly generated beta cells demonstrate glucose-dependent calcium responsiveness and regulated insulin release. PMP colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. Moreover, genetic lineage-labeling experiments excluded the neural crest, and established the embryonic pancreatic lineage, as the developmental source of PMPs. The PMP cell was further found to express insulin in vivo, and insulin+ stem cells were shown to contribute to multiple pancreatic and neural cell populations in vivo. These findings demonstrate that the adult mammalian pancreas contains a population of insulin+ multipotent stem cells, capable of contributing to the pancreatic and neural lineages. In the final section of this thesis, I consider the relationships between neural and pancreatic tissues, as well as discussing the relevance of these two novel stem cell populations.

The aggressive nature of pancreatic cancer, characterized by invasiveness, resistance to treatment, rapid progression, and its high prevalence in the population urges the need for developing more effective treatments. Many studies have attributed resistance to therapeutics of pancreatic cancer to activity of the transcription factor nuclear factor kappa B (NF-kB). NF-kB is regulated by the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is a key mediator of pathways such as insulin, wnt, and PI3K/Akt and has roles in proliferation, glucose metabolism, apoptosis, motility and neuroprotection. Depending on the cellular context, GSK-3 activity can promote or inhibit cell survival. GSK-3 inhibition was recently reported to have anti-cancer effects against pancreatic cancer cells. This effect was in part attributed to suppression of NF-kB. In this thesis, I showed that while blocking GSK-3 disrupts NF-kB, and has anti-survival effects on pancreatic cancer cells, it does not sensitize to the chemotherapeutic drug gemcitabine. NF-kB inhibition by curcumin also resulted in similar effects. These results questions previous reports that NF-kB activation plays a major role in chemo-resistance of pancreatic cancer. The inhibition of NF-kB by genetic disruption of GSK-3 was previously reported to sensitize mouse embryonic fibroblasts and hepatocytes to TNF-alpha cytotoxicity. I therefore tested whether GSK-3 inhibition could sensitize pancreatic cancer cells to apoptosis induced by the clinically applicable member of the TNF-alpha family, TNF-alpha related apoptosis inducing ligand (TRAIL). In contrast to the results obtained with gemcitabine, the combination of genetic or pharmacological inhibition of GSK-3 and TRAIL was found to be highly synergistic in apoptosis induction. Analysis of the apoptotic mechanisms, point towards effects of GSK-3 inhibition on caspase-8 activation, consistent with inhibition of the death receptor signalling pathway. It was found that not only caspase-8 but also mitochondrial anti-apoptotic proteins such as Bcl-XL and Mcl-1 were mediating the TRAIL sensitization. Furthermore, for the first time the in vivo effects of GSK-3 inhibition in combination with TRAIL treatment was investigated. The results indicate a significant enhancement of apoptosis in pancreatic cancer xenografts with minimal toxic effects. Together, these studies provide a rationale for developing combination treatments based on GSK3 inhibition and TRAIL death receptor activation to treat pancreatic cancer.