Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / Cell isolation method may have effects on the characteristics of the cells isolated from porcine umbilical cords. As stem cells age or approach senescence, it is hypothesized that their properties change. We expect that isolation method and age of cells will have effects on the phenotype of porcine umbilical cord (PUC) cells during in vitro expansion. We investigated the effects of three isolation methods on PUC population doublings, ability to produce colony forming units (CFU), and amount of ganglioside GD2 (GD2) expression over eleven passages. Isolation methods were explant (Exp) in which the Wharton's Jelly was removed from cords, minced and plated, enzyme digest (Dig), and stomacher assisted enzyme digestion (Stom). Cell isolates were analyzed for GD2 expression, CFU, and population doublings at early (3), middle (7), and late (11) passage. The Exp method produced greater (P<0.05) population doublings and more (P<0.05) CFU at passage 7. Explant isolates also were numerically more likely to survive to passage 11 (9/9 isolates vs 5/9 for Dig and 7/9 for Stom). In contrast, the percent cells expressing GD2 was greater (P<0.05) for Stom isolates than Exp isolates at passage 11. There were no trends for increased passage number to decreased population doubling, CFU formation, or percent GD2 positive cells. In summary, our results indicate that the Exp isolation method produced the greatest number of population doublings over 11 passages and there were minimal effects of isolation method on CFU and GD2 expression. Although Exp may be more difficult to scale up to isolate all of the PUCs in a cord, it provided greater in vitro expansion than the enzyme methods in our experiment and may provide the most cells for biotechnological and biomedical applications.