Relevant Thesis-Based Degree Programs
Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The precise coordination of inflammatory signaling, oxidative stress responses, steroidogenesis, and steroid metabolism are required in all aspects of pregnancy, from fertilization and implantation to parturition. Dysfunction in any of these processes can lead to unfavourable pregnancy outcomes and complications requiring obstetric interventions. As the use of assisted reproductive technologies (ART) has increased in recent decades, it has become evident that these pregnancies are associated with increased risks compared to unassisted pregnancies. However, the underlying reason for these increased risks is unknown. Preliminary studies from our laboratory using a mouse model of ART provide evidence that inflammatory, antioxidant, or steroid pathways could be dysregulated in ART. In this thesis, we investigated the levels inflammatory mediators, measures of antioxidant defense, and steroid hormones in pregnancy to determine any associations with pregnancy outcome. Beginning with an investigation of non- steroidal anti-inflammatory drug (NSAID) use in pregnancy (Chapter 2), we determined the prevalence of NSAID use close to labour. Next, to investigate the differences between ART and unassisted pregnancies, we used a cohort of placentas from ART pregnancies and unassisted pregnancies matched for gestational age, maternal age, ethnicity, and singleton/twin pregnancy. In this cohort, we evaluated the levels of cytokines and vascular endothelial growth factors and identified associations with pregnancy outcome using the accompanying clinical chart profiles (Chapter 3). Further, the activity of antioxidant enzymes and levels of antioxidants were measured in these placentas (Chapter 4). In Chapter 5, we measured the levels of steroid hormones and steroid sulfates in maternal, placental, and fetal compartments, again comparing these results between ART and unassisted pregnancy and identifying associations with pregnancy outcome. No widespread dysregulation of these processes was found between ART and unassisted pregnancies. However, when stratified for pregnancy outcome, two significant associations were found. In pregnancies complicated by chorioamnionitis and in twin pregnancy, there was dysregulation across all three signalling processes. These signalling pathways are complex and interconnected, but the data generated here adds novel findings to the ART field, and identifies potential pathways for future investigation to resolve the etiology of increased pregnancy complications in ART.
PURPOSE Uridine diphosphate glucuronosyltransferases (UGTs) are Phase II conjugation enzymes that are critical for vertebrate detoxication. They catalyze the glucuronidation biotransformations of numerous endo- and xenobiotics to facilitate their clearance and elimination and are important for hormonal regulation. Glucuronidation-related disorders include congenital syndromes, excessive chemical toxicities, adverse drug reactions, and dysregulated hormonal status. Since the 1980s it has been known that UGT proteins can be expressed but not fully active, yet little is known about how the UGT protein structure regulates its activity and function, because of the lack of a complete structure. We hypothesized that translational and post-translational mechanisms may play a role in regulating the activity and function of human UGTs. Aim 1 and 2 focused on UGT isoform 1A6, one of the most important isoforms in endo- and xenobiotic metabolism. Aim 3 studied the co-expression of major human hepatic UGT isoforms to gain an insight on their co-regulation in vivo.METHODSN-glycosylation variants of human UGT1A6 were established to study the influence of N-glycosylation on their expression, activity, cellular function and localization. HEK293 expressed UGT1A6-(His)6 and Sf9-insect expressed soluble UGT1A6 were purified and their activity and latency were characterized. The co-expression between 7 UGTs was investigated based on a western blot database collected from healthy liver donors, aged fetal (20 weeks) – 87 years.GENERAL CONCLUSIONSN-glycosylation is an important regulator of human UGT1A6 in expression, activity, and cellular function. Different forms of N-glycosylation were observed for UGT1A6 between HEK293 cells and human liver microsomes, showing a caveat of using recombinant cell lines to study UGT structure/function. Sf9-insect expressed soluble UGT1A6 is a good candidate for structure analysis due to sufficient yield and activity after purification. The influence of membranes on UGTs may be different from traditional beliefs as the data suggested alamethicin and Brij 58 can directly activate the enzyme. Lastly, expression of major human hepatic UGTs was significantly correlated and correlations were affected by age. The UGTs may be co-regulated in the human liver so single-isoform systems may have limitations on reflecting UGT activity in vivo.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Phase II metabolism regulates endo- and xenobiotic levels through compound inactivation and elimination. The major phase II drug metabolizing enzyme family, UDP-glucuronosyltransferases (UGTs), conjugate a broad variety of substrates through glucuronidation. Historically, drug metabolism research focused extensively on UGT activity and compound elimination, with minimal comparative data investigating beta-glucuronidase (βG) catalyzing the reverse recycling reaction. Despite this, available research shows βG activity is upregulated in a variety of clinically relevant conditions including human immunodeficiency virus (HIV) and cancer. The purpose of this thesis is to characterize βG expression and activity in health and disease, providing novel insights into the role of recycling in chemical homeostasis in the body. In Chapter 2, the first comprehensive profile showing differential βG expression and activity across 14 distinct human tissues was developed. Greatest enzyme activity was observed in prostate, caecum, liver and adrenal, with intestinal tissues showing closest UGT:βG activity at pH 7.4 suggesting further investigation into the recycling role in the gut. Measuring E. coli and human βG at each respective optimal pH revealed that bacterial strains showed enzyme activity comparable or greater to that in human. However, human βG activity increased 13-fold from pH 7.4 to pH 5.4 (native lysosomal pH), suggesting that more acidic environments favour the human enzyme. These findings demonstrate that enterohepatic recycling is likely due to a combination of both bacterial and human βG. In Chapter 3, the role of βG in prostate cancer was assessed. Upregulated βG activity was related to disease progression, with greatest activity reported in PC3 cells and adenocarcinoma tissue. In androgen-sensitive models, elevated enzyme activity suggests a role for βG in locally recycling androgens whereas the recycling of other endobiotics may stimulate cancer growth in androgen-insensitive models. Total UGT activity was greatest in PNT1A cells and adenocarcinoma tissue, with UGT2B17 more commonly expressed in prostate cell lines and UGT2B15 in prostate tissues. Overall, βG is upregulated in androgen-sensitive and androgen-insensitive models and may represent a novel prostate cancer biomarker and/or therapeutic target.
Voltage gated potassium channels (Kv) belong to a larger family of proteins called ion channels. Kv channels occur ubiquitously in the body and play a range of essential roles in cellular processes such as signal propagation and cellular excitability. Kv are made up of four subunits, each of which is translated individually, followed by assembly and expression on the cell surface. Kv1.1 is a Kv family member capable of assembling as either a homotetramer or a heterotetramer. In particular, when Kv1.1 subunits assemble with other Kv1.x subunits, the former shape the activation threshold and kinetics of macroscopic current of a channel, and play an important role in the trafficking and surface expression of other Kv1.4 channels. Dysfunction of Kv1.1 has been linked to an autosomal dominant neurological disorder, episodic ataxia (EA-1), that affects both the central nervous system and peripheral nervous system. Patients with EA-1 are 10 times more likely to develop epilepsy than normal individuals. Kv1.1 channels have also been implicated in sudden unexpected death in epilepsy (SUDEP) and Alzheimer’s disease. Dysfunction of Kv1.1 channels has been characterized by studying specific mutations in KCNA1 genes. Current techniques for generating Kv1.1 channels in heterologous expression systems for subsequent biophysical characterizations include coexpression and dimer construction methods, and the creation of tandem dimer-linked concatemers. The latter provides the greatest control over stoichiometry and arrangement of subunits; however, generation of each concatemer is extremely labour- and time-intensive. This thesis focuses on the development of a new concatemer system built on an inhouse plasmid (pICDNA), with an intentionally designed linker sequence that physically concatenates four Kcna1 genes. The Kv1.1 homotetramer concatemer system has been developed to permit flexibility, such that each gene (or multiple genes) in the concatemer can be targets for future cloning. The development of the Kv1.1 homotetramer system will facilitate the examination of the role Kv1.1 channels play, independently of its main partners, Kv1.2 and Kv1.4. The Kv1.1 concatemer platform can be used in the future as a backbone upon which future Kv1.x heterotetramers can be developed more easily.