Graduation date: 1993

Oxidative assimilation of glucose-U-C¹⁴ by several aerobic bacteria was found to involve the assimilation of radioactivity into nitrogenous cell components, principally proteinaceous, in conjunction with the reincorporation of endogenously produced ammonia. In one of these bacteria, Pseudomonas aeruginosa, if the cells were starved or treated with chloramphenicol/ prior to glucose-C¹⁴ the amount of assimilation, especially into protein, was decreased. The incorporation into nucleic acids and lipids was increased by the antibiotic, but was only slightly affected by starvation. A determination of the cytological sites of the assimilated material showed that, in control cell extracts, the soluble proteins of the cytoplasm contained most of the C¹⁴. Starved or antibiotic treated cell fractions had substantially less of the label in these proteins, whereas the radioactivity incorporated into the ribosomal ribonucleic acid and the "membrane" lipids was greater. A study of the aminoacyl-soluble ribonucleic acid synthetases in P. aeruginosa revealed that these enzymes were present only in the cytoplasm. Starving the cells resulted in decreased activity of the synthetases, but they were rapidly reactivated during oxidative assimilation. The large amount of heterologous reactions between bacterial soluble ribonucleic acids and synthetases indicated that little species specificity existed. However, cross reactions between the systems in bakers' yeast and the bacteria were poor, showing that some degree of species specificity was present in these instances. Preliminary experiments on the route of assimilation of ammonia in P. aeruginosa and in P. fluorescens gave no evidence for the direct amination of pyruvate by alanine dehydrogenase, but did demonstrate a requirement for concurrent substrate oxidation while ammonia was being incorporated. In contrast, several lines of evidence indicated that ammonia was assimilated via ∝-ketoglutarate in P. aeruginosa. / Land and Food Systems, Faculty of / Graduate

Twenty-four male university students were fed vegetarian diets containing 100 grams of protein. Seventy-five grams of protein came either from soy, non-fat dairy products or egg white. Diets were adjusted so that differences in total caloric intake, protein, carbohydrate, fat and fatty acid composition were minimal between the dietary treatments. Plasma total triglyceride and very low density lipoprotein-triglycerides were measured at the beginning, weekly throughout the experimental period, and two weeks after completion of the study. No significant differences existed in serum lipid values between treatment diets nor was any interaction between diet and week observed. A significant week effect was observed indicating that subjects fed soy, non-fat dairy products or egg whites responded in the same fashion to the diet from week to week. This relationship was true for both variables: serum triglycerides and VLDL-triglycerides. Serum triglyceride concentrations for all treatment groups combined at baseline were 79 mg/ 100 ml, increasing to 82 mg/100 ml at week 1 and decreasing to 64 mg/100 ml at week two. An increase of 84 mg/100 ml was noted at week three. Decreases were observed at week four, with serum concentrations of 65 mg/100 ml. From week four to follow-up serum triglyceride concentration rose to 83 mg/100 ml. Similar trends were noted in serum VLDL-triglyceride levels when mean concentration were combined for all treatment groups. Serum VLDL-triglyceride concentrations at baseline were 48 mg/100 ml. At week one serum VLDL-triglyceride concentrations remained unchanged with values of 40 mg/100 ml in both instances. Decreases were observed at week 4 with serum VLDL-triglyceride concentrations increased to 38 mg/100 ml. The results indicate that plasma triglycerides and VLDL-triglycerides are influenced by other dietary factors rather than by the protein source. / Master of Science

Simon, Ming-yuen Lee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 239-257). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

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Nuclear receptors play crucial roles in the transcriptional regulation of many biological processes such as development and cellular differentiation. ERRalpha is known, along with coactivator PGC-1alpha, to playa central role in the control of energy metabolism in cardiac and skeletal muscle. They activate the expression of many genes involved in mitochondrial oxidative metabolism. Here we identified PROX1, a factor that was previously shown to broadly influence metabolism, as a regulator of this pathway. Indeed, PROX1 interacts in vitro and in vivo with both ERRalpha and PGC-1alpha. To provide more insight on the hepatic functions of ERRalpha and PROX1, we performed ChIP-on-chip using mouse liver, identifying a large number of ERRalpha and PROX1 genomic targets and reinforcing their role in energy metabolism. Over 40% of the target genes were found to be common to both factors and we observed that PROX1 could be recruited to ERRalpha binding sites and act as a negative regulator o fthe ERRalpha/POC-1alpha pathway.

The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.

Self-renewal is central to the expansion of normal and cancerous stem cells. Its understanding is therefore critical for future advances in transplantation-based therapies and cancer treatment. Although the molecular machinery controlling stem cell self-renewal remains poorly defined, a number of genes important to this process have recently been identified. Two prominent genes in this group are Hoxb4 and Bmi1. Members of our group led the way to demonstrate important regulatory functions of these genes in hematopoietic stem cell (HSC) self-renewal and expansion. / The major goal of my thesis project is to dissect mechanisms that regulate self-renewal of HSCs. Our starting hypothesis was that HSC activity is regulated by complementary and independent self-renewal mechanisms: self-renewal of expansion and self-renewal of maintenance (Chapters 1-2). In order to further verify this theory, we have analyzed the genetic interaction between Hoxb4 and Bmi1. While Hoxb4 overexpression triggers HSC expansion, Bmi1 proper expression is essential to sustain long-term stem cell activity. We have also demonstrated that Hoxb4 and Bmi1 regulate distinct gene targets, likely suggesting a complementary and independent function for these two regulators in HSC activity (Chapter 3). / The second part of this thesis highlights efforts that were made in order to get a better understanding of self-renewal mechanisms. We have identified potential new regulators of stem cell activity by characterizing a stem cell leukemia population (Chapter 4) and by assessing the expression of asymmetrical distributed factors (Chapter 5) and selected nuclear factors of the Hematopoietic Stem Cell Nuclear Factor Database (Chapter 6) in stem cell-enriched sub-fractions. / This project will lead to a better understanding of the cellular basis regulating self-renewal of both normal and cancer stem cells and potentially to the future identification of new self-renewal determinants.

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