published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Glucagon and insulin play important roles in controlling blood glucose and energy metabolism in vertebrate species. Recent studies have identified large cohorts of genes that could be regulated by glucagon and insulin. Signal transducer and activator of transcription (STAT) is a group of signal mediators/inducible transcription factors functionally coupled to class I cytokine receptors through JAK activation. Although the involvement of JAK/STAT pathway has been reported in the physiological actions of insulin and glucagon, the effects of these pancreatic hormones on STAT expression have not been examined. Using grass carp (Ctenopharyngodon idellus) as an animal model, we have cloned the cDNAs for STAT1, STAT3 and STAT5 and confirmed that they are single copy genes in the carp genome. Tissue expression profiling using RT-PCR revealed that the three members of STATs were ubiquitously expressed in various tissues of the grass carp including the liver. Function expression of grass carp STAT1, STAT3 and STAT5 in mammalian cell lines also demonstrated that the STAT proteins of fish origin were all effective in transactivating the target promoters with STAT-binding sites. In grass carp, hepatocyte culture, glucagon and insulin treatment were both effective in increasing STAT1, STAT3 and STAT5 mRNA expression. Using a pharmacological approach, the stimulatory effect of glucagon on transcripts expression of the three forms of STATs were shown to be mediated through activation of the cAMP/PKA, PI3K/AKT and MAPK ( Erk1/2 and JNK) pathways. In the case of insulin stimulation, the PI3K/AKT and p38 MAPK but not JNK pathways were involved in STAT1, STAT3 and STAT5 mRNA up-regulation. Furthermore, insulin-induced STAT3 and STAT5, but not STAT1 mRNA expression, could be blocked by Erk1/2 inactivation, suggesting that the MEK1/2/Erk1/2 pathway might be differentially coupled to gene expression of the individual members of STAT family. These findings provide evidence for first time that glucagon and insulin can regulate STAT1, STAT3 and STAT5 gene expression at the hepatic level in fish model via overlapping and yet distinct signaling mechanisms. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

The global emergence of drug resistance is posing increasing difficulties in the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs) are regarded as having a pivotal role among the antimicrobial agents in multidrug regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of TB therapy. The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon the bactericidal effect and prevention of the emergence of resistance. In the first part of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90, AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as a promising agent for the treatment of MDR-TB. Molecular tests on resistance genes are reliable and rapid technology for diagnosis of drug-resistant TB which facilitates timely patient management and public health control of TB. In the second part of the study, the feasibility of a PCRsequencing assay for the examination of mutations in the quinolone-resistance-determining- region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr) Mycobacterium tuberculosis in direct clinical specimens was evaluated. As determined by gyrA QRDR DNA sequencing analysis, complete concordance of phenotypic and genotypic outcomes was demonstrated. The results indicate that the molecular assay is an accurate and effective method for the diagnosis of FQr TB and allows identification of mixed resistant variants in the same patient. GyrA mutations that associated with FQr in clinical isolates of M. tuberculosis were clustered in hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory specimens by using the PCR-DNA sequencing assay. In the third part of this study, the functional effect of the Ala74Ser mutant was verified through study of the DNA supercoiling inhibitory activities of OFX and MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations (IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than the wild-type H37Rv reference strain, and results correlated well with their phenotypic drug susceptibilities. Besides, a combination of gyrA mutations (Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3 helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer. Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative drug binding pocket, and leads to the reduction of the binding affinity of FQ due to the distance effect. This is the first report verifying that gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Tumor heterogeneity has long been observed and recognized as a challenge to cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses proposed to explain such a phenomenon. A specific cancer stem cell marker has not been determined for non-small cell lung cancers (NSCLC), preventing the definitive evaluation of whether the biology of NSCLC is governed by the CSC model. This study aimed to analyze the expression of candidate CSC markers and using the identified putative marker, to isolate CSC and determine the applicability of the CSC model in NSCLC. The expression or activities of four putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours. Markers with increased expression after treatment were considered potential CSC markers and used for isolating tumor cell subpopulations from the untreated cell lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further characterized by functional and phenotypic studies. Flow cytometry showed amongst the 4 markers, only ALDH1 expression was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise, higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells formed tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH- cells with even ten times the number of cells. Also, expression analysis revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view of previous studies reporting CD44 as a CSC marker in lung cancer, double marker selection of putative CSC was performed to compare ALDH1+CD44+ and ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation possessed higher self-renewal ability and chemo-resistance. Cell migration and invasion assays showed differences between the ALDH1+CD44+ and ALDH1- CD44+ subpopulations. The significance of these observations require further investigation. In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem cells as evidenced by enhanced self-renewal and differentiation abilities in ALDH1+ subpopulation. Furthermore, this study observed the presence of at least two potential stem cell subpopulations in NSCLC cells with differential selfrenewal, chemotherapy resistance and cell mobility properties. Further investigations are required to validate these observations and to investigate the underlying mechanisms. Better understanding of these issues would help to solve the challenges brought by tumor heterogeneity in lung cancer therapy. / published_or_final_version / Pathology / Master / Master of Philosophy

Recently, progress was made in collecting, classifying, and characterizing the genetic diversity of type 2 porcine reproductive and respiratory syndrome virus (PRRSV) using all known and publically available sequencing information. Despite this voluminous attempt, these analyses were largely na?ve of the Canadian contribution to circulating viruses. This represented a vital omission in the study of molecular epidemiology due to the fact that Canada had recorded the earliest evidence of the existence of type 2 PRRSV. To this end, the genetic diversity and evolutionary aspects of PRRSVs distributed in the Province of Ontario in Canada were characterized to abridge this existing knowledge gap on type 2 PRRSV. Genotyping of type 2 strains is primarily based on either a phylogenetic or restriction fragment length polymorphism (RFLP) approach. Classification of Ontario PRRSV field isolates (n = 505) from 1999 to 2010, based on a global type 2 PRRSV ORF5 phylogenetic framework, revealed genetic diversity comparable to PRRSV in the USA, with sequences assigned to five of nine lineages (1, 2, 5, 8 and 9). A majority (~85%) of these isolates were typed to the first two lineages (1 and 2). Despite a relatively smaller sample size to the USA, the topology of the phylogenetic tree indicated Canadian origins of these two lineages. Mapping RFLP patterns of Ontario isolates onto the phylogenetic tree revealed numerous examples of different patterns located within the same phylogenetic cluster. Examples of the non-specificity of RFLP patterns to any particular lineage or sub-lineage were abound. Statistical analysis showed occurrences where similar RFLP patterns masked diverse genetic distances and instances of close genetic proximity with divergent RFLP patterns. An examination of the most abundant 15 RFLP patterns revealed that the discrepancy between RFLP typing and genetic distances was not attributable to a single or few patterns but was rather a permeating feature. Importantly, the tree topology also indicated a Canadian ancestry for the highly virulent MN184-related strains that first emerged in 2001 in the USA. Selective pressure analyses highlighted a handful of positively selected sites most of which were located in the ORF5 ectodomains of outbreak strains, implicating the host immune system as the possible selective agent. This was in contrast to the closely-related Ontario strains which were subject to strong purifying selection. A broader survey of transmission dynamics in North America unveiled a higher virus flow from Canada to the USA with the primary targets being the Lake States and Corn Belt. In turn, these regions served to disseminate viruses to other swine production regions in the USA. Virus flow from the USA to Canada occurred on a much smaller scale. Collectively, extensive genetic diversity prevails in type 2 PRRSV in one region of the North American swine industry and it is not described adequately by RFLP typing which might have some value in differentiating strains at the local farm level, instead. For diagnostic and research purposes, phylogenetic typing should be the preferred method. Finally, stronger surveillance needs to be adopted to minimize cross-border virus transmission. / published_or_final_version / Biological Sciences / Master / Master of Philosophy