Copies of author's previously published articles inserted. / Bibliography: leaves 152-176. / xiv, 177 leaves, [4] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Explores aspects of the relationship between metabolism and proliferation of colonic epithelial cells from rats and humans. Emphasis is placed on developing and integrating in vivo and in vitro models for both metabolic and proliferative studies. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1990

The activity of dihydrofolate reductase, a key enzyme in the de novo biosynthesis of thymidylate, purines, and some amino acids, had previously been found to be increased in a methotrexate-resistant mouse leukemia (L5178Y) cell line as compared to the activity in the parental methotrexate-sensitive cell line. The increased activity was composed of two forms of the enzyme, one of which, form 2, was highly insensitive to inhibition by methotrexate. Both forms were purified to near homogeneity and using the antibodies prepared against them, it could be demonstrated that the two forms are antigenically distinct. The increased dihydrofolate reductase activity present in the methotrexate resistant cells resulted from an overproduction of both forms of the enzyme due to the presence of abundant mRNA coding for these enzymes. An increase in the dihydrofolate reductase gene copy number could be demonstrated in the resistant cells. mRNA coding for form 1 and form 2 enzymes was greatly enriched by polysome immunoprecipitation and complimentary DNA (cDNA) was synthesized in vitro from these enriched mRNA molecules. Evidence was found for the presence of methotrexate-insensitive forms of dihydroflate reductase in the blast cells of three out of eight acute myelogenous leukemia patients, and in two (distinct from the above) of the eight patients the activity was significantly increased. In contrast to the overproduction of dihydrofolate reductase protein in the methotrexate-resistant mouse cell line, increased enzyme activity in a methotrexate-resistant human promyelocytic leukemia (HL-60) cell line could not be correlated with an increase in the enzyme protein. Furthermore, the amounts of dihydrofolate reductase mRNA and gene-dosages were similar in the parental metho-trexate-sensitive and methotrexate-resistant cells. The enzyme from the resistant cells differed significantly in some of its physical and kinetic properties from that present in the parental cells. An increase in dihydrofolate reductase activity resulting from a modification of the enzyme rather than gene amplification has not to date been reported in the literature and may present a novel mechanism of resistance to methotrexate. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Il est maintenant admis que l’activité anti-tumorale du TNF implique une destruction de la vascularisation de la tumeur par induction d’apoptose des cellules endothéliales. Si le traitement au TNF en thérapie humaine permet des rémissions complètes, il n’est applicable qu’aux membres isolés de la circulation systémique car les doses requises de TNF sont létales pour le patient. Mettre en évidence les mécanismes qui contrôlent l’apoptose de la cellule endothéliale devrait permettre de développer de nouvelles stratégies thérapeutiques favorisant l’effet thérapeutique du TNF tout en évitant le choc septique qu’il engendre.<p><p>Nous avons abordé cette question en utilisant le modèle de la cellule endothéliale d’aorte bovine (BAEC) stimulée au TNF afin d’étudier les mécanismes qui contrôlent le processus apoptotique et ce au cours des phases précoces de celui-ci.<p><p>D’une part, nous avons identifié par une approche pharmacologique au moins trois voies de contrôle négatif d’induction de mort cellulaire par le TNF dans les cellules endothéliales :la première faisant intervenir une des protéines kinases C, une seconde la PI3-kinase et une troisième la protéine kinase p38. Cette dernière semble spécifiquement activée par le TNF et pourrait donc constituer une nouvelle cible pharmacologique visant à sensibiliser les cellules endothéliales de la vascularisation de la tumeur à l’action apoptotique du TNF. <p>D’autre part nous avons mis en évidence et identifié par une approche protéomique au moins deux protéines montrant une rapide diminution de phosphorylation lorsque les cellules endothéliales sont stimulées par le TNF en présence de cycloheximide. Ces deux événements semblent en aval de l’activation d’une protéase de la famille des caspases. La première protéine est la sous-unité régulatrice de type II alpha (RIIα) de la protéine kinase A. Cependant, la relation entre cette déphosphorylation et le processus d’apoptose n’a pas pu être mise en évidence, une stimulation de l’activité PKA n’affectant pas l’induction d’apoptose des BAEC. La seconde est la protéine HDGF, connue pour être sur-exprimée dans certains cancers, mais dont la régulation par phosphorylation ainsi que son implication éventuelle lors du processus apoptotique n’avaient encore jamais été envisagées.<p><p>Enfin, nous avons voulu mettre en évidence le rôle potentiel de la protéine hnRNP K qui subit une modification post-traductionnelle précoce lors de l’apoptose des BAEC. Notre étude, menée après surexpression de la protéine étiquetée, suggère une modification autre qu’une dégradation. Cependant, il ne nous a pas été permis de lui attribuer un rôle puisque la surexpression de cette protéine n’affecte pas l’apparition de différents marqueurs associés à l’apoptose. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Recent work has shown that specific ALDH isoenzymes can contribute to the underlying pathology of different diseases. Many ALDH isozymes are important in oxidizing reactive aldehydes resulting from lipid peroxidation, and, thus, help maintain cellular homeostasis. Increased expression and activity of ALDH isozymes are found in many human cancers and are often associated with poor prognosis. Therefore, the development of inhibitors of the different ALDH enzymes is of interest as means to treat some of these disease states. Here I describe the results of assays designed to characterize the site of interaction and the mode of inhibition for the unique compounds that function as inhibitors of aldehyde dehydrogenase 2 and determine their respective IC50 values with intent to develop structure-activity relationships for future development.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.