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.

Master of Science / Department of Statistics / Gary L. Gadbury / Discerning the metabolic or enzymatic role of a particular gene product, in the absence of information indicating sequence homology to known gene products, is a difficult task. One approach is to compare the levels of metabolites in a wild-type organism to those in an organism with a mutation that causes loss of function of the gene. The goal of this project was to develop an approach to analyze metabolite data on wild-type and mutant organisms for the purpose of identifying the function of a mutated gene. To develop and test statistical approaches to analysis of metabolite data for identification of gene function, levels of 141 lipid metabolites were measured in leaves of wild-type Arabidopsis thaliana plants and in leaves of Arabidopsis thaliana plants with known mutations in genes involved in lipid metabolism. The mutations were primarily in fatty acid desaturases, which are enzymes that catalyze reactions in which double bonds are added to fatty acids. When these enzymes are mutated, leaf lipid composition is altered, and the altered levels of specific lipid metabolites can be detected by a mass spectrometry. A randomization P-Value and other metrics were calculated for all potential reactant product pairs, which included all lipid metabolite pairs. An algorithm was developed to combine these data and rank the results for each pair as to likelihood of being the actual reactant-product pair. This method was designed and tested on data collected on mutants in genes with known functions, fad2 (Okuley et al., 1994), fad3 (Arondel et al., 1992), fad4, fad5 (Mekhedov et al., 2000), fad6 (Falcone et al., 1994), and fad7 (Iba et al., 1993 and Gibson et al., 1994). Application of the method to three additional genes produced by random mutagenesis, sfd1, sfd2, and sfd3, indicated that the significant pairs for fad6 and sfd3 were similar. Consistent with this, genetic evidence has indicated that sfd3 is a mutation in the FAD6 gene. The methods provide a list of putative reactions for an enzyme encoded by an unknown mutant gene. The output lists for unknown genes and known genes can be compared to provide evidence for similar biochemical activities. However, the strength of the current method is that the list of candidate chemical reactions for an enzyme encoded by a mutant gene can be produced without data other than the metabolite profile of the wild-type and mutant organisms, i.e., known gene analysis is not a requirement to obtain the candidate reaction list.

Calcific aortic valve disease (CAVD) is associated with increased presence of myofibroblasts, osteoblastic cells and, occasionally, adipocytes and chondrocytes in lesions. The ectopic cell types in diseased valves may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitial cells (VICs) that populate the valve interstitium. Notably, lesions form preferentially in the fibrosa layer, the stiffer layer of the valve leaflet. It has been shown that differentiation of VICs to myofibroblasts and osteoblasts is modulated by matrix stiffness. However, the molecular mechanisms involved in mediating stiffness-dependent mechanotransduction remain obscure. The objectives of this thesis were: (1) to determine whether VICs contain a subpopulation of multipotent mesenchymal progenitor cells and to measure the frequencies of the mesenchymal progenitors and osteoprogenitors; (2) to determine the role of β-catenin and matrix stiffness in transforming growth factor-β1 (TGF-β1)-induced myofibroblast differentiation of VICs; and (3) to preliminarily investigate the involvement of four and a half LIM domains protein 2 (FHL2) in CAVD and stiffness-dependent mechanotransduction downstream of RhoA in VICs. Firstly, VICs were found to contain a subpopulation of mesenchymal progenitors that are inducible to osteogenic, myofibroblastic, adipogenic, and chondrogenic lineages. The frequencies of mesenchymal progenitors and osteoprogenitors were significantly higher than other reported sources. Secondly, it was demonstrated that β-catenin is required in TGF-β1-induced, matrix stiffness-regulated myofibroblast differentiation. Notably, TGF-β1 was only able to induce β-catenin nuclear translocation and myofibroblast differentiation on matrices with fibrosa-like stiffness, but not on matrices with ventricularis-like stiffness. Thirdly, FHL2 was found to be upregulated and colocalized with runt-related transcriptional factor 2 (Runx2) in lesions in the fibrosa layer of diseased valves, suggesting its role in osteogenic processes in CAVD. Notably, increasing matrix stiffness increased FHL2 nuclear translocation and RhoA activity in VICs. Preliminary data showed that matrix stiffness regulates FHL2 nuclear translocation via RhoA activity. These results suggest that differentiation of the rich valve progenitor subpopulation, regulated by both mechanical and biochemical cues, may contribute to the preferential occurrence of ectopic cell types in the fibrosa in CAVD. More broadly, these results highlight the critical role of mechanical environment in modulating cellular biochemical signaling.

The immune system recognizes and eliminates an enormous array of pathogens due to the diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSB) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations and oncogenic transformation. Thus, mechanisms responsible for monitoring global genomic integrity, such as those coordinated by the ATM (ataxia-telangiectasia mutated) kinase, must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. I show that ATM deficiency compromises TCRα recombination and the post-mitotic survival of T-cell receptor αβ (TCRαβ+) CD4+CD8+ (DP) thymocytes, providing a molecular and developmental basis for the immunodeficiency characteristic of ATM loss. Moreover, I show that in early thymocyte progenitors undergoing TCRβ recombination, ATM loss leads to cell cycle defects and developmental arrest, likely facilitating the acquisition of mutations that contribute to leukemogenesis. Using ATM deficiency as a murine model of T cell precursor acute lymphoblastic leukemia (T-ALL), I demonstrate that IL-7 signaling, a critical survival and proliferation signal during early stages of normal thymocyte development, is also required for leukemic maintenance. Moreover, we show for the first time that in normal and leukemic thymocyte precursors, interleukin 7 receptor (IL-7R) expression and function are controlled by Notch signaling, a key determinant of T cell fate. Collectively, these findings provide insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation, while establishing the groundwork for assessing the molecular events that lead to the initiation and stepwise progression of T cell leukemogenesis.

The blood system is organized as a developmental hierarchy in which rare hematopoietic stem cells (HSCs) generate large numbers of immature progenitors and differentiated mature blood cells. In this process, at least ten distict lineages are specified from multipotent stem cells, however the cellular and molecular organization of the hematopoietic hierarchy is a topic of intense investigation. While much has been learned from mouse models, there is also an appreciation for species-specific differences and the need for human studies. Blood lineages have been traditionally grouped into myeloid and lymphoid branches, and the long-standing dogma has been that the separation between these branches is the earliest event in fate specification. However, recent murine studies indicate that the progeny of initial specification retain the more ancestral myeloid potential. By contrast, much less is known about the progenitor hierarchy in human hematopoiesis. To dissect human hematopoiesis, we developed a novel sorting scheme to isolate human stem and progenitor cells from neonatal cord blood and adult bone marrow. As few as one in five single sorted HSCs efficiently repopulated immunodeficient mice enabling interrogation of homogeneous human stem cells. By analyzing the developmental potential of sorted progenitors at a single-cell level we showed that earliest human lymphoid progenitors (termed LMPs) possess myelo-monocytic potential. In addition to B-, T-, and natural killer cells, LMPs gave rise to dendritic cells and macrophages indicating that these closely related myeloid lineages also remain entangled in lymphoid development. These studies provide systematic insight into the organization of the human hematopoietic hierarchy, which provides the basis for detailed genetic analysis of molecular regulation in defined cell populations. In a pilot study, we investigated the role of a zinc finger transcription factor (ZNF145), PLZF, in myeloid development. We found that PLZF restrained proliferation and differentiation of myeloid progenitors and maintained the progenitor pool. Induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells. Taken together, these studies advance our understanding of the cellular and molecular architecture of human hematopoiesis.

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.

Gastrulation is the process by which the three germ layers are generated during vertebrate development. Nodal ligands, which form a subgroup of the Transforming Growth Factor β (TGFβ) superfamily, regulate the expression several transcription factors implicated in gastrulation. Among these are the paired-like homeodomain transcription factors Goosecoid (Gsc) and Mixl1. At the molecular level, Gsc has been described to function as a transcriptional repressor by directly binding to paired homedomain binding sites on target promoters. Here, I describe a novel mechanism of transcriptional repression by Gsc. Using a molecular and embryological approach, I demonstrate that the forkhead transcription factor Foxh1, a major transducer of Nodal signaling, associates with Gsc which in turn recruits histone deacetylases to negatively regulate Mixl1 expression during early mouse development. Post-translational modification of transcription factors by SUMO proteins represents an important mechanism through which their activity is controlled. Here, I also demonstrate that Gsc is sumoylated in mammalian cells by members of the PIAS family of proteins and this modification potentiates the repressive activity of Gsc on direct targets such as the Xbra and Gsc promoters, but not on indirect targets such as Mixl1. Taken together, work presented in this thesis describes two novel mechanisms of transcriptional repression by Gsc.