The primary goal of the research presented in this thesis has been to establish the yeast Yarrowia lipolytica as a model genetic system for the study of peroxisome biogenesis. To facilitate this, three steps were necessary. First, mutant strains of Y. lipolytica that manifested peroxisomal defects were generated; second, a genomic DNA library was created to rescue the mutants and, hence, clone the gene(s) involved; and third, the parameters governing high efficiency DNA transformation of Y. lipolytica were defined to make complementation of mutants with the genomic library feasible. This work culminated in the cloning, sequencing and characterization of a gene, dubbed PAY 4, that is required for peroxisomal assembly in Y. lipolytica. Two of the mutant strains of Y. lipolytica isolated, ole 2 and ole 4, were identified as peroxisome assembly mutants as they; (i) grew in acetate medium but failed to grow on oleic acid medium, and thus were peroxisomal as opposed to mitochondrial mutants; (ii) lacked recognizable peroxisomal structures when observed by immunofluorescence using a polyclonal serum that recognized a number of peroxisomal proteins and; (iii) had several peroxisomal enzyme activities localized to the cytosol, i.e. they manifested mistargeting of otherwise lumenal peroxisomal proteins. These were complemented with a genomic library, which represented at least 19 000 independent recombinants (insert size ~5-7 kilobases) cloned into a vector (piNA445) capable of autonomous replication in Y. lipolytica. Estimations of the size of the Y. lipolytica genome indicate that probability of this library containing a given portion of the genome is .999. To utilize the library effectively, an electro-poration protocol was developed that could transform Y. lipolytica at high efficiency (>5·10^4 transformants·μg-^1 of DNA). Using these resources, the gene complementing the ole 4 mutation was cloned and sequenced. It encodes Pay4p, a protein of ~112 kDa, containing two putative ATPase modules. Preliminary studies indicate that expression of Pay4p is induced slightly by growth on oleic acid. / Thesis / Master of Science (MSc)

The regulation of gene expression at the level of transcription is an important mechanism for maintaining homeostasis. The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear hormone receptor superfamily that is involved in transcriptionally modulating such pathways as lipid and fatty acid metabolism. This receptor binds to enhancer elements, peroxisome proliferator-responsive elements (PPREs), upstream of a variety of target genes including those involved in β-oxidation of fatty acids, in the peroxisome and mitochondria, and ketogenesis. One such element has been identified upstream of the rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase (mtHMG-CoAS) gene. This enzyme has been shown to be one of the key regulatory points of ketogenesis. To learn how PPARα mediated transcriptional regulation occurs, this receptor was used as bait in a yeast dihybrid screen and was found to interact with human mtHMG-CoAS. Reproduction of this interaction 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 was performed by solid phase capture assays, using GST fusion proteins, and by co-immunoprecipitations. It was also ascertained that the synthase enzyme interacts with the retinoid X receptor α (RXRα). The hamster cytoplasmic form of the enzyme was chosen as a control and showed no binding capabilities to either nuclear hormone receptor. Interestingly, the mitochondrial enzyme contains a motif, LXXLL that has previously been shown to be important for binding between a transcriptional coactivator and a receptor. A site-directed mutant of the mitochondrial synthase sequence from LASLL to LASVL was made. The mutant showed a reduced ability to interact with both nuclear receptors. Consequently, the LXXLL motif is responsible, at least in part, for the interaction between mtHMG-CoAS and both PPARα and RXRα. The cytoplasmic synthase does not contain the motif; its corresponding sequence is LASVL. The effect ofthe mitochondrial synthase within a cell was then determined by transient transfection assays. It was discovered that on the HMG PPRE the mitochondrial synthase potentiated PPARα mediated transactivation while on the AOx PPRE the enzyme inhibited it. Thus, mtHMG-CoAS modulates PPARα activity in a PPRE dependent manner. The LXXVL mitochondrial mutant inhibited PPARα transactivation on both the HMG and AOx PPRE. Therefore, the mutant acts as a dominant-negative inhibitor of α mediated activity. The cytoplasmic control enzyme had no effect on either PPRE. To determine if the mitochondrial enzyme could be detected within the nucleus where it appeared to be modulating PPARα mediated transcription, localization studies were performed with the use of immunofluorescence. Immunofluorescence was done by utilizing hemagglutinin (HA) epitope tagged fusions of the mitochondrial, mutant and cytoplasmic enzymes. When the HA tag was placed at the carboxyl terminus of mtHMG-CoAS, the enzyme was localized to the mitochondria with no apparent nuclear staining. This is consistent with previous localization studies done with the rat mtHMG-CoAS. Also, the mutant enzyme, with the HA tag at the carboxyl terminus, was only detected in the mitochondria. However, under certain conditions, when PPARα and the mitochondrial synthase were co-transfected, the mitochondrial enzyme was detected within both the mitochondria and the nucleus. The mutant, on the other hand, when co-transfected with PPARα was found to remain non-nuclear. The HA tagged cytoplasmic control enzyme was also non-nuclear. Therefore, mtHMG-CoAS can be detected within the nucleus, it binds, due to at least in part a LXXLL motif, to nuclear receptors, and it is capable of modulating transcription in a PPRE dependent manner. Ketogenesis becomes an important mechanism for fuel production during starvation, prolonged exercise and diabetes. Perhaps, during extreme circumstances such as starvation, mtHMG-CoAS is involved in autoregulation which allows for an amplification of the transcription of its own gene. Also, mtHMG-CoAS appears to inhibit the transcription of the AOx enzyme which is involved in peroxisomal fatty acid β-oxidation. Again, perhaps under extreme conditions, the β-oxidation of fatty acids is concentrated within the mitochondria which allows for the production of acetyl-CoA that can subsequently be converted to ketone bodies that can then be utilized for fuel. / Thesis / Master of Science (MS)

No description available.

No description available.

Peroxisome proliferator-activated receptor (PPAR)γ plays a role in tumorigenesis. Previous studies with PPARγ(+/-) mice suggest PPARγ normally suppresses dimethylbenz[a]anthracene (DMBA)-induced breast, and other, tumor progression. Since many cell types associated with the mammary gland express PPARγ, each with unique signaling pathways, the present study aimed to define which tissues are required for PPARγ-dependent anti-tumor effects. Conditional adipocyte and endothelial cell-specific PPARγ knockout mice (PPARγ-A KO and PPARγ-E KO respectively) were used to evaluate whether PPARγ signaling normally acts to prevent DMBA-mediated breast tumour progression in a stromal cell-specific manner. Twelve week old PPARγ KO mice and their congenic wildtype (WT) controls were randomly assigned to one of two treatment groups. All mice were treated by gavage once/week for 6 weeks with 1 mg DMBA and maintained on a normal chow diet. At week 7, mice in each group were divided into those continuing normal chow, and those receiving a PPARγ ligand (ROSI, 4 mg/kg/day) supplemented diet for the duration of the 25 week study, and monitored weekly. Tumour and tissue samples were collected at necropsy, and portions of each were fixed and frozen for future analysis. In both PPARγ-A KOs and PPARγ-E KOs versus PPARγ-WT mice, malignant mammary tumor incidence was significantly higher and mammary tumor latency was decreased. DMBA+ROSI treatment reduced average mammary tumor volumes by 50%. Gene expression analyses of mammary glands by qRT-PCR and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared to PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. In the PPARγ-E KO mice, both treatment groups saw a significant increase in thymic tumour incidence, a finding not established before with the study of other stromal cell knockout mice. These studies provide the first direct in vivo evidence that PPARγ signalling in stromal adipocytes and endothelial cells attenuates DMBA-mediated breast tumourigenesis. This study supports a protective effect of activating PPAR gamma as a novel chemopreventive therapy for breast cancer. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-24 11:28:17.668

Endometrial cancer is a common gynaecological cancer. Improving outcomes for women with advanced disease remains a challenge and there is also a need to develop preventative strategies in those women at highest risk of developing disease. Peroxisome proliferator-activated receptors (PPARs) comprise of a group of transcription factors belonging to the nuclear hormone receptor subfamily. PPAR sub-types are involved in metabolic homeostasis and have been implicated in malignancy, particularly breast and colo-rectal malignancies both of which are associated with obesity. Endometrial cancer is also closely associated with both obesity and insulin resistance. The work described in this thesis examined the expression of PPARs in endometrioid endometrial cancer and investigated their effects on key pathways implicated in this disease. Immunoblotting revealed over expression of PPARα and loss of PPARγ in human endometrioid endometrial cancer tissues. Pull-down assays also demonstrated differential selectivity of different PPARs for heterodimerisation with different isoforms of the RXR family of transcription factors. PPARα was localized to tumour cells and vascular endothelium and ELISA demonstrated an increase in VEGF-A in PPARα silenced cells suggesting that PPARα may promote tumour angiogenesis. PPARγ was largely seen in epithelial cells and also macrophages within benign endometrium. Reduction of PPARγ expression in cultured endometrial cells led to increased proliferation and decreased apoptosis. Loss of PPARγ was correlated with a loss of the tumour suppressor PTEN in endometrial tissues. Furthermore, PPARγ silencing led to diminished expression of PTEN and a concomitant increase in phosphorylated AKT suggesting that PPARγ is protective against deregulated growth within the endometrium. Synthetic PPAR-specific ligands reduced proliferation and increased apoptosis in endometrial cell lines. These effects were present in PPAR-silenced cells too although reduced in magnitude, indicating that the actions of specific PPAR ligands are mediated via both receptor dependent and receptor independent pathways.In conclusion, this work has demonstrated the differential expression of PPARs and RXRs in endometrial cancers and identified possible mechanisms, both direct and indirect, by which these may modulate endometrial cancer growth. Different PPAR family members may provide targets for therapeutic intervention in endometrial cancer care and require further study in this regard.

We have inestigated the expression of the genes hydratase-dehydrogenase-epimerase (HDE), acyl-CoA oxidase (AOX) and catalase (CATL) of the diploid yeast Candida tropicalis. These genes encode enzymes which are localized to the peroxisome. Expression of each gene was monitored by immunoblot analysis of yeast lysates using antibodies directed against each protein. We demonstrate that carbon sources influence expression of these genes, and do so in a coordinate fashion. We expressed C. tropicalis HDE in Saccharomyces cerevisiae and demonstrate that this trifunctional enzyme can be regulated by S. cerevisiae in a fashion that closely resembles that of C. tropicalis. Expression of constructs containing deletions in the upstream region of the HDE gene allowed us to localize regions responsible for regulating the expression of this gene. Regions were identified that are responsible for both repression by glucose and induction by oleic acid. A glucoseresponsive region lies between nucleotides -466 and -334. An oleic acid-responsive region lies between nucleotides -333 and -281. An additional region controlling derepression by nonfermentable carbon sources is located downstream of nucleotide -281. Comparison of the upstream nucleotide sequences of HDE, AOX and CATL both to each other, and to upstream regions of other oleic acid-responsive genes of C. tropicalis has identified possible consensus nucleotide sequences for glucose-and oleic acid-responsive upstream elements. Since the regulation of the HDE gene in S. cerevisiae closely resembles that of C. tropicalis, this implies that similar mechanisms of transcriptional control operate in both yeasts. / Thesis / Master of Science (MS)

Peroxisomes are ubiquitous organelles found in almost all eukaryotes. They are sensitive to changes in cellular homeostasis and involved in various metabolic processes. Deficiencies in peroxisome function cause severe neurological problems. Here I report, investigation of peroxisome motility and its relation to peroxisomal functions in the fungal model system Ustilago maydis. Peroxisomes are mostly motile in Ustilago maydis. Motile peroxisomes show different motility patterns: short-range pulse type movements and long range bidirectional motility. Motility behaviour is not static as oscillating peroxisomes may start long-range motility. Here, I present evidence that long-range bidirectional peroxisome motility is an energy driven process and is essential for homogeneous distribution of peroxisomes. Similar to early endosomes and endoplasmic reticulum, microtubule motors kinesin-3 and dynein are responsible for long-range peroxisome transport. In addition to using the same molecular motors for transport, early endosomes, endoplasmic reticulum and peroxisomes have the same transport velocity. Interestingly, motile peroxisomes and endoplasmic reticulum tubules co-localize with early endosomes. Functional investigation of early endosome mutants, Δrab5a and Yup1ts has revealed a novel transport mechanism where endoplasmic reticulum and peroxisomes hitch hike on early endosomes. Additionally, I report functional characterization of an AAA-ATPase, um05592, which has high homology to human protein NP_055873. Altogether these results reveal molecular mechanism of peroxisome transport in Ustilago maydis. Similarities in transport machinery illustrate Ustilago maydis as a model system to study peroxisome function in mammalian cells.

Peroxisome proliferator-activated receptor alpha (PPARa) and delta (PPARd) belong to the nuclear receptor superfamily. PPARa is a target of lipid-lowering drugs and PPARd promotes fatty acid utilization and is a promising anti-diabetic drug target. However, evidence is growing that PPARd-agonism can stimulate fat accumulation in liver, which may aggravate the toxic situation in diabetics. The aim of the study was to characterise the hepatic transcriptional and lipid response of humanized mouse models to PPARd-agonists. In our studies of mice conditionally-expressing human PPARd (hPPARd), or the dominant-negative derivative of hPPARd (hPPARd?AF2) or wild-type animals, we demonstrated that GW501516, a potent PPARd activator, promoted up-regulation of the genes involved in lipid turnover, stimulated significant weight loss and promoted hepatic steatosis in these mouse models. There was time-dependent accumulation of hepatic triglycerides observed in wild-type and in conditionally-expressing hPPARd mice fed a diet containing PPARd synthetic ligand. This was not seen in animals conditionally-expressing hPPARd?AF2, neither in PPARa-KO or PPARd-KO animals. Concurrently, activation of PPARd in humanised animals caused significant depletion, as compared with controls, of adipose tissue deposits when fed normal or high fat diet. This effect was completely absent in PPARa-KO or PPARd-KO mice, fed diet containing GW501516. Genome-wide transcriptional profiling of GW501516 effects in the livers of these different mouse strains was performed. In PPARa-KO mice fed PPARd-agonist, some direct PPARd target genes were still up-regulated, demonstrating that they are not sufficient for the observed phenotype. In addition the blood HDL-raising effects of GW501516 were preserved in the PPARa-KO mice. This suggests a novel finding that both PPARd and PPARa receptors are essential for GW501516-driven weight loss and hepatic steatosis, with PPARa working downstream of PPARd.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by lesions that form within the central nervous system which induce symptoms such as muscle weakness and paralysis. Many aspects of MS, ranging from causation to immunopathology, are currently under investigation as little is known of the factors that contribute to and exacerbate this disease. Presently, evidence suggests MS to be an inflammatory disease mediated by an autoimmune response to an unknown antigen. Results from clinical studies as well as animal models such as experimental autoimmune encephalomyelitis (EAE) suggest MS is initiated and maintained by immune cells such as Th1 lymphocytes. As a result, therapeutics prescribed to MS patients’ focus on modulating the inflammatory response so as to minimize myelin loss and CNS damage. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors that show promise as potential targets for MS therapeutics. The PPAR sub-types, PPARα and PPARγ, have been shown to inhibit the propagation of inflammatory pathways and decrease the activity of pro-inflammatory cells in a number of inflammation driven diseases including rheumatoid arthritis and atherosclerosis. The anti-inflammatory role of PPARβ/δ is less well known, although preliminary studies suggest activation of this receptor may potentiate the activity of other transcription factors involved in inhibiting inflammatory pathways. As the PPAR family of transcription factors exhibit similar functions, it is hypothesized that the PPARβ/δ sub-type may have immunomodulatory effects that are comparable and complimentary to PPARα and –γ. This thesis describes a novel model of relapsing-remitting EAE (RR-EAE) that presents a disease course where EAE relapses are followed by periods of recovery that are characterized by the absence of clinical symptoms. Furthermore, a therapeutic intervention study carried out using this model demonstrates that the PPARγ agonist pioglitazone can decrease the severity of a relapse episode when drug treatment begins prior to a predicted relapse event. The inhibition of immune cell infiltration into the CNS and decreased immune cell activity mediated by pioglitazone, suggests that this ligand modulates the immune response. These results indicate that pioglitazone may be an effective treatment for relapsing-remitting MS. To examine the role of PPARβ/δ in RR-EAE and explore its effect on the activity of inflammatory cells, PPARβ/δ knockout mice were used due to the current lack of specific antagonists for this receptor. PPARβ/δ wild-type mice developed RR-EAE when immunized using protocol intended to induce this disease course. PPARβ/δ knockout mice however, developed chronic EAE when immunized in the same manner. Consistent with sustained clinical symptoms, CNS immune cell infiltration and activity was maintained throughout the disease in PPARβ/δ knockout mice. In contrast, the presence of immune cells within the CNS and consequent activity fluctuated according to the relapse and recovery pattern of disease in PPARβ/δ wild-type mice. PPARβ/δ appears to modulate inflammation by potentiating the apoptosis of activated T cells. Therefore, PPARβ/δ agonists may be potential candidates for MS treatment.