Under challenge by a dietary soybean cysteine protease inhibitor (scN), cowpea bruchids overcome the inhibitory effects by reconfiguring the expression profiles of their major digestive enzymes, the cathepsin L-like cysteine proteases (CmCPs). In addition, cowpea bruchids activate transcription of the counter-defensive cathepsin B-like cysteine protease (CmCatB). I undertook an interest in understanding the molecular mechanisms utilized by bruchids to differentially regulate cysteine proteases in response to plant inhibitors. First, to investigate the functional significance of the differential regulation of CmCPs, I expressed CmCP proprotein isoforms (proCmCPs) in E. coli, and characterized their activities. Among proCmCPs, proCmCPB1 exhibited the most efficient autocatalytic processing, the highest proteolytic activity, and was able to degrade scN in the presence of excessive CmCPB1. Second, to dissect the molecular mechanisms behind the differential function of CmCPs, I swapped domains between two representative subfamily members B1 and A16. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- or C-terminal mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16. Bacterially expressed isolated propeptides (pA16 and pB1) showed that pB1 inhibited B1 enzyme less than pA16 due to its protein instability. Taken together, these results suggest that cowpea bruchids selectively induce specific cysteine proteases for their superior autoprocessing, proteolytic efficacy, and scNdegrading activities, and modulate proteolysis of their digestive enzymes by controlling cleavage and stability of propeptides to cope with plant inhibitors. Third, to understand the transcriptional regulatory mechanisms of CmCatB hyperexpression that underlies bruchid adaptation, I cloned a portion of its promoter and demonstrated its activity in Drosophila S2 cells using a CAT reporter system. Gel shift assays identified cowpea bruchid Seven-up (CmSvp, chicken ovalbumin upstream promoter transcription factor homolog) in scN-unadapted insect midgut, and cowpea bruchid HNF-4 (CmHNF-4, hepatocyte nuclear factor 4) in scN-adapted insect midgut. When transiently expressed in S2 cells, CmSvp repressed, while CmHNF-4 activated CmCatB expression. CmSvp antagonized CmHNF-4-mediated transactivation when they were present simultaneously in the cell. Thus, the data suggest that transcriptional regulation of CmCatB in response to plant inhibitor depends, at least partly, on the cellular balance between positive and negative regulators.

No description available.

The phosphoinositide species phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes involved in cell growth, survival, and metabolism. The class I PI3Ks are responsible for production of PIP3, and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110, p110, p110) and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Growth of all cell types is dependent on PI3K signalling, and development of immune cells relies on a precise balance of PIP3 production. Activating mutations in the genes encoding the catalytic and regulatory subunits of PI3K lead to cancer and immunodeficiencies. The PIK3CA gene encoding the p110 catalytic subunit of class IA PI3K is one of the most frequently mutated genes in cancer, and mutations in the PIK3CD gene encoding the p110 catalytic subunit lead to primary immunodeficiency. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits (PIK3R1, PIK3R2, PIK3R3) have been identified in both cancer and primary immunodeficiencies. By asking how these mutations mediate activation and disease phenotypes, we can identify the natural regulatory molecular mechanisms of class IA PI3Ks. Fundamentally understanding how mutations in PI3K subunits mediate human disease will expand our knowledge of PI3K biology and is essential to the development of novel therapeutics. To identify the molecular mechanisms of class IA PI3K activating mutations, I employed a sophisticated combination of hydrogen-deuterium eXchange mass spectrometry (HDX-MS) with biochemical activity assays to probe the regulatory mechanisms of PI3Ks. HDX-MS measures the exchange rate of amide hydrogens in solution, which in turn can provide information on protein conformation and conformational changes between different states. By comparing PI3K mutants identified in primary immunodeficiency and cancer patients to wild-type enzymes, I have identified dynamic conformational changes induced by activating mutations. Biochemical and biophysical analysis of these mutants led us to generate a panel of engineered mutations to further characterise molecular mechanisms by which class IA PI3Ks are regulated. This thesis will consist of an introduction to class IA PI3K signalling and an introduction to the method of HDX-MS, followed by two data chapters wherein I investigate the mechanisms of activating mutations in PIK3CD followed by an investigation into activating mutations in PIK3R1. A conclusion and discussion of future directions will be presented in the final chapter. This work provides novel insight into the complex regulatory mechanisms of the class IA PI3Ks, which may lead to better understanding of human diseases that activate these enzymes. / Graduate / 2020-04-06

Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion (from < 30 repeats in normal individuals to ~ 11,000 for the full mutation in certain patients) of the repeating tetranucleotide CCTG•CAGG sequence in the intron of the zinc finger protein 9 (ZNF9) gene. The genetic instabilities of the CCTG•CAGG repeats were investigated to evaluate the molecular mechanisms responsible for these massive expansions. The effects of replication, recombination, repair and transcription on the genetic instabilities have been investigated in COS-7 cells and E. coli model systems. A replication assay was established in COS-7 cells wherein the CCTG•CAGG repeats cloned proximal to the SV40 origin of replication resulted in expansions and deletions in a length and orientation-specific manner, whereas the repeats cloned distal to the same origin were comparatively stable. These results fit with our data obtained from biochemical studies on synthetic oligonucleotides since these biochemical studies revealed that the d(CAGG)26 oligomer had a marked propensity to adopt a hairpin structure as opposed to its complementary d(CCTG)26 that lacked this capacity. Furthermore, a genetic assay in E. coli was used to monitor the intramolecular frequency of recombination. This assay revealed that the tetranucleotide repeats were indeed hot spots for recombination. Moreover, studies conducted in SOS-repair mutants showed that recombination frequencies were much lower in a SOS¯ strain as compared to a SOS+ strain. However, experiments conducted to ascertain the level of induction of the SOS response revealed that the SOS pathway was not stimulated in our studies. These results revealed that although breaks may occur within the repeats, the damage is most likely repaired without induction of the SOS response contrary to previous beliefs. Thus, a complex interplay of replication, recombination, and repair is likely responsible for the expansions observed in DM2.

<p> It is well understood that compared with men, women are better able to withstand starvation, have better ultra-endurance capacity, oxidize more fat during endurance exercise, and are more resistant to fat oxidation defects i.e. diet-induced insulin resistance. However, the mechanism(s) for the observed sex differences are unknown. It was my hypothesis that women have greater fat oxidation capacity in skeletal muscle than men.</p> <p> The objectives of my thesis were to determine the mechanism(s) by which women oxidize more lipids; including the role of estrogen as a possible regulator. The most significant findings were that: 1) mRNA for fatty acid oxidation genes are higher in women compared with men, which was confirmed by Stringent Affymetrix GeneChip array analysis, combined with RT-PCR (chapter 2); 2) long-chain acyl-CoA dehydrogenase in human skeletal muscle is not quantifiable despite the majority (90%) of fatty acids oxidized during exercise are long-chain fatty acids (chapter 3); 3) β-oxidation enzymes: tri-functional protein alpha, very long chain acyl-CoA dehydrogenase, and medium chain acyl-CoA dehydrogenase are significantly higher in women compared with men (chapter 4); 4) Acute (8 days) 17β-estradiol supplementation in men significantly increased protein content of β-oxidation enzymes in skeletal muscle, possibly through the regulation of PGC-1α and microRNA (chapter 5).</p> <p> In conclusion, my data provided novel insights into the enhanced ability of women to oxidize fat under periods of metabolic stress by showing that: 1) women are transcriptionally (mRNA) "primed" for known physiological differences in metabolism; 2) women have more protein content of the major enzymes involved in long and medium chain fatty acid oxidation; 3) E2 partially regulates lipid metabolism in skeletal muscle by pre-translational modifications of factors involved in β-oxidation. These findings contribute to the molecular understanding of sex differences in substrate utilization.</p> / Thesis / Doctor of Philosophy (PhD)

Philosophiae Doctor - PhD / Cancer is a group of diseases that arises from irreversible genomic and epigenomic alterations that result in unrestrained proliferation of abnormal cells. Detailed understanding of the molecular mechanisms underlying a cancer would aid the identification of most, if not all, genes responsible for its progression and the development of molecularly targeted chemotherapy. The challenge of recurrence after treatment shows that our understanding of cancer mechanisms is still poor. As a contribution to overcoming this challenge, we provide an integrative multi-omic analysis on glioblastoma multiforme (GBM) for which large data sets on di erent classes of genomic and epigenomic alterations have been made available in the Cancer Genome Atlas data portal. The rst part of this study involves protein network analysis for the elucidation of GBM tumourigenic molecular mechanisms, identification of driver genes, prioritization of genes in chromosomal regions with copy number alteration, and co-expression and transcriptional analysis. Functional modules were obtained by edge-betweenness clustering of a protein network constructed from genes with predicted functional impact mutations and differentially expressed genes. Pathway enrichment analysis was performed on each module to identify statistical overrepresentation of signaling pathways. Known and novel candidate cancer driver genes were identi ed in the modules, and functionally relevant genes in chromosomal regions altered by homologous deletion or high-level amplication were prioritized with the protein network. Co-expressed modules enriched in cancer biological processes and transcription factor targets were identified using network genes that demonstrated high expression variance. Our findings show that GBM's molecular mechanisms are much more complex than those reported in previous studies. We next identified differentially expressed miRNAs for which target genes associated with the protein network were also differentially expressed. MiRNAs and target genes were prioritized based on the number of targeted genes and targeting miRNAs, respectively. MiRNAs that correlated with time to progression were selected by an elastic net-penalized Cox regression model for survival analysis. These miRNA were combined into a signature that independently predicted adjuvant therapy-linked progression-free survival in GBM and its subtypes and overall survival in GBM. The results show that miRNAs play significant roles in GBM progression and patients' survival finally, a prognostic mRNA signature that independently predicted progression-free and overall survival was identified. Pathway enrichment analysis was carried on genes with high expression variance across a cohort to identify those in chemoradioresistance associated pathways. A support vector machine-based method was then used to identify a set of genes that discriminated between rapidly- and slowly-progressing GBM patients, with minimal 5 % cross-validation error rate. The prognostic value of the gene set was demonstrated by its ability to predict adjuvant therapy-linked progression-free and overall survival in GBM and its subtypes and was validated in an independent data set. We have identified a set of genes involved in tumourigenic mechanisms that could potentially be exploited as targets in drug development for the treatment of primary and recurrent GBM. Furthermore, given their demonstrated accuracy in this study, the identified miRNA and mRNA signatures have strong potential to be combined and developed into a robust clinical test for predicting prognosis and treatment response.

No description available.

<p> The objective is to investigate the molecular mechanisms leading to neurodegeneration in Tay Sachs disease, a lysosomal storage disorder caused by a deficiency in the enzyme hexosaminidase A. This was accomplished using microarray analysis of normal and Tay Sachs neuroglia. Microarray data analysis was performed using Onto Express, PANTHER software, and cluster analysis. The expression levels of selected genes were validated using Real Time PCR. To establish a physiological relationship between GM2 accumulation and the expression of these genes, their expression was assessed after treatment with an inhibitor of ganglioside synthesis, nbutyldeoxynojirimycin (NBDNJ). Neuronal pentraxin 1 (NPTX1), potassium channel, subfamily K, member 2 (KCNK2), and prostaglandin synthase 1 (PTGS 1) were found to be upregulated in Tay Sachs neuroglia while heme oxygenase (HMOX1) was downregulated. The mRNA levels ofNPTX1, KCNK2, PTGS1, and HMOX1 all reverted to normal levels in response to ganglioside synthesis inhibitor. Pentraxin RNA levels were also reduced in response to sialidase overexpression, another method of GM2 reduction. Pentraxin protein levels were also increased in Tay Sachs cells in response to u-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA), and were attenuated upon treatment with either NBDNJ or AMPA antagonist 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo [f]quinoxaline-2,3-dione (NBQX). Strong colocalization was seen between NPTX1 and the AMPA receptor as well as the glutamate transporter EAACI. AMP A receptor function was also enhanced as illustrated by an increase in calcium influx upon stimulation. This stimulation also resulted in increased apoptosis during AMPA receptor stimulation. In this study, our genetic profiling experiment led to the identification of NPTXl as a marker of pathogenesis in Tay Sachs Cells. The role of NPTXl in excitotoxicity implicates the latter in disease mechanism associated with Tay Sachs disease. Furthermore, this study provides evidence that TSD cells undergo programmed cell death in response to increased intracellular calcium as a result of increased glutamate receptor stimulation. </p> / Thesis / Master of Science (MSc)

No / BACKGROUND: Androgenetic alopecia, or male pattern baldness, is a common, progressive disorder where large, terminal scalp hairs are gradually replaced by smaller hairs in precise patterns until only tiny vellus hairs remain. This balding can cause a marked reduction in the quality of life. Although these changes are driven by androgens, most molecular mechanisms are unknown, limiting available treatments. The mesenchyme-derived dermal papilla at the base of the mainly epithelial hair follicle controls the type of hair produced and is probably the site through which androgens act on follicle cells by altering the regulatory paracrine factors produced by dermal papilla cells. During changes in hair size the relationship between the hair and dermal papilla size remains constant, with alterations in both dermal papilla volume and cell number. This suggests that alterations within the dermal papilla itself play a key role in altering hair size in response to androgens. Cultured dermal papilla cells offer a useful model system to investigate this as they promote new hair growth in vivo, retain characteristics in vitro which reflect their parent follicle's response to androgens in vivo and secrete mitogenic factors for dermal papilla cells and keratinocytes. OBJECTIVES: To investigate whether cultured dermal papilla cells from balding follicles secrete altered amounts/types of mitogenic factors for dermal papilla cells than those from larger, normal follicles. We also aimed to determine whether rodent cells would recognize mitogenic signals from human cells in vitro and whether factors produced by balding dermal papilla cells could alter the start of a new mouse hair cycle in vivo. METHODS: Dermal papilla cells were cultured from normal, balding and almost clinically normal areas of balding scalps and their ability to produce mitogenic factors compared using both human and rat whisker dermal papilla cells as in vitro targets and mouse hair growth in vivo. RESULTS: Normal scalp cells produced soluble factors which stimulated the growth of both human scalp and rat whisker dermal papilla cells in vitro, demonstrating dose-responsive mitogenic capability across species. Although balding cells stimulated some growth, this was much reduced and they also secreted inhibitory factor(s). Balding cell media also delayed new hair growth when injected into mice. CONCLUSIONS: Human balding dermal papilla cells secrete inhibitory factors which affect the growth of both human and rodent dermal papilla cells and factors which delay the onset of anagen in mice in vivo. These inhibitory factor(s) probably cause the formation of smaller dermal papillae and smaller hairs in male pattern baldness. Identification of such factor(s) could lead to novel therapeutic approaches.

The whitefly Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) is a serious pest of protected vegetable and ornamental crops in most temperate regions of the world. Neonicotinoids, pymetrozine (a feeding blocker), spiromesifen (a tetronic acid derivative), bifenthrin (a pyrethroid), and pyriproxyfen (a juvenile hormone mimic) are among the most important insecticides used to control this species. Bioassays were used to quantify responses of recently-collected strains of T. vaporariorum to three neonicotinoids (imidacloprid, thiamethoxam, and acetamiprid), pymetrozine, spiromesifen, bifenthrin, and pyriproxyfen. 454 pyrosequencing was exploited to generate the first transcriptome for this species. PCR-sequencing was used to identify mutations in the target proteins of spiromesifen and bifenthrin potentially associated with resistance to these compounds. Microarray sequencing technology was employed to investigate differences in gene expression associated with pyriproxyfen resistance. Resistance to neonicotinoids was age-specific in expression and consistently associated with resistance to pymetrozine, supporting a hypothesis of metabolic resistance analogous to that in the tobacco whitefly, Bemisia tabaci. Bioassays also showed moderate to high level resistance to spiromesifen, bifenthrin and pyriproxyfen in some strains. Analysis of the transcriptome identified genes encoding enzymes involved in the detoxification of xenobiotics (cytochrome P450s, carboxyl/cholinesterases, and glutathione-s transferases) and ones encoding insecticide targets: acetyl-coA carboxylase (ACCase), the target of spiromesifen and the voltage-gated sodium channel protein targeted by pyrethroids. PCR-sequencing revealed a single nucleotide polymorphism in the ACCase gene, which was consistently associated with spiromesifen resistance. Three amino-acid substitutions in the sodium channel of pyrethroid-resistant T. vaporariorum were found in positions previously implicated in pyrethroid resistance in B. tabaci. Microarray sequencing disclosed that a cytochrome P450 gene (CYP4G61) was overexpressed in a strain selected for increased pyriproxyfen resistance. The implications of these results and opportunities for further work are discussed.