published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy

Infundibular (arcuate) neurokinin B (NKB) neurons play a critical role in neuroendocrine control of reproduction. Specifically, a local network of arcuate neurons that co-express kisspeptin, neurokinin B, and dynorphin (so-called, KNDy neurons), has emerged as a potential pacemaker driving the pulsatile secretion of gonadotropin-releasing hormone (GnRH) that is required for normal reproduction. These neurons are the target of estrogen and may be an important link in estrogen negative feedback on GnRH functioning. KNDy neurons respond to estrogen withdrawal with dramatic changes in gene expression and somatic hypertrophy, an effect that is reversible by estradiol replacement. Studies addressing the effects of estrogen withdrawal and replacement on morphological and electrophysiological features of KNDy neurons have been hindered by the inability to target this subpopulation of neurons in the live tissue. This dissertation examines estrogen-induced changes in arcuate NKB circuitry and excitability and discusses its implications in reproductive axis. First, the novel Tac2-EGFP transgenic mouse model was characterized. The reproductive function, EGFP-ir distribution in the brain, and co-localization of EGFP with proNKB in the arcuate nucleus were examined and compared to littermate controls. Indices of reproductive function (puberty onset, estrous cyclicity, and LH pulsatility) were comparable between Tac2 and wildtype mice, suggesting that the transgenic animals have preserved estrogen negative feedback. The long-term estrogen withdrawal via ovariectomy and estradiol replacement model was used to examine electrophysiological and morphological changes in arcuate NKB neurons. We found that low-dose chronic estradiol replacement results in decreased excitability of arcuate NKB neurons, a finding that is consistent with the proposed role of this neuronal population in estrogen negative feedback on reproductive axis. Changes in excitability were seen despite the overall similarity in intrinsic properties of estradiol-treated and untreated ovariectomized mice. We also demonstrated for the first time that single arcuate NKB neurons form a local network by way of recurrent collaterals. Axonal targets of single NKB neurons included the internal zone of the median eminence, ependymal layer of the 3rd ventricle, and sites lateral and dorsal to the borders of the arcuate nucleus. Long-term treatment with estradiol resulted in decreased somatic volume and decreased dendritic spine density. Together, these data demonstrate that low-dose chronic estradiol replacement in ovariectomized mice resulted in morphological plasticity of arcuate NKB neurons that was accompanied by changes in excitability of this neuronal population, supporting the role of these neurons in estrogen negative feedback on GnRH secretion.

Refuges of non-Bt cotton are used to delay Bt resistance in the pink bollworm (Pectinophora gossypiella, Lepidoptera: Gelechiidae), a pest that eats cotton seeds. Contamination of refuges by transgenic Bt cotton could threaten the efficacy of such refuges by increasing the relative survival of larvae that carry alleles for Bt resistance. Here I compared contamination levels in refuges of varying configuration and distance from Bt. I found two types of contamination at low rates in refuges: outcrossing by Bt pollen and adventitious Bt plants. Unexpectedly, outcrossing did not differ between refuge configurations, and did not decrease as distance from Bt fields increased, perhaps because Bt plants in refuges acted as the main Bt pollen source. Bioassays, conducted to evaluate the impacts of contamination on pink bollworm resistance, indicated that Bt plants in refuges may increase the frequency of resistance alleles at a higher rate than outcrossing by Bt plants.

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. The role of cystein-x-cystein (CXC) chemokine ligand 10 (CXCL10, formerly interferon-y-inducible protein 10) in CVB3-induced myocarditis is unknown. To explore the contribution of CXCL10 to CVB3-induced myocarditis, we performed functional analyses using newly generated transgenic mice with cardiac-specific CXCL10 overexpression (Tg) and CXCL10 knock out (KO) mice. The mRNA levels of CXCL10 peaked in the myocardium at day 3 post-infection prior to immune infiltration, suggesting that mainly resident cells of the heart are the sources of CXCL10. Indeed, we showed that CXCL10 can be induced by IFN-y but not by CVB3 infection in cultured cardiomyocytes. Further, a transgenic mouse model with cardiac-specific overexpression of CXCL10 was generated. CXCL10 Tg mice had spontaneous infiltrations of mononuclear cells with limited mRNA upregulation of IFN-y and IL-10, which were not sufficient to cause the impairment of cardiomyocyte or cardiac function. Following CVB3 infection, the viral titre in the mouse hearts inversely correlated with the levels of CXCL10 at day 3 post-infection. Further, the decreased virus titers in the CXCL10 Tg mouse hearts led to reduced cardiac damage indicated by low serum cTnI levels and improved cardiac functional performance and vice versa in the KO mice. This antiviral ability of CXCL10 may be through increased recruitment of natural killer (NK) cells to the heart and increased IFN-y expression early post-infection. At days 7 and day 10 post-infection with massive influx of mononuclear cells, the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells as well as their associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines did not alter the viral clearance and mouse survival. Our data demonstrate for the first time that CXCL1 0 confers the protection to the heart during the early course of CVB3 infection, which may be primarily attributed to NK cell recruitment to the site of infection. This data suggest that CXCL10 is an important player in the orchestrated action of a group of cytokines and chemokines in combating against the CVB3-induced myocarditis in the early phase of infection.

Heart failure (HF) is a prevalent disease in society which is associated with decreased cardiac output. This thesis describes the proteomic analysis of cardiac tissue obtained from HF patients and a transgenic animal model of HF. Initial experiments optimized one proteomic technology, 2-dimensional gel electrophoresis (2-DE), to maximize the number of proteins which could be observed / resolved from human cardiac tissue. Protein abundance changes in cardiac tissue between normal patients and those with a diagnosis of ischemic cardiomyopathy were determined by performing 2-DE and identifying proteins by mass spectrometry. HF patients had a reduced abundance of proteins involved in energy production and the sarcomere. Sarcomeres contain the myofilament subproteome consisting of thick and thin filaments with the thick filaments primarily myosin. Thick and thin filament undergo Ca2+ induced ATP hydrolysis to form crossbridge cycles, resulting in muscle contraction. An assembly chaperone for myosin, UNC-45B, was found to be increased in HF patients. Western blot analysis confirmed that the abundance of UNC-45B was increased in different etiologies of heart failure. Follow up physiological measurements demonstrated that UNC-45B is most likely a protein necessary for transcriptional control of the α-isoform of myosin heavy chain. In a second proteomics study, abundance changes occurring in a pacing induced model of HF in wild-type (WT) and transgenic (TG) rabbits with increased expression of the α-isoform of myosin heavy chain were examined. WT and TG rabbits had a different response in their myofilament and intermediate filament abundance changes following induction of HF. TG rabbits had a decreased abundance of heat shock proteins and non-sarcomeric associated desmin. As well, TG rabbits had an increased ratio of actin:myosin heavy chain and UNC-45B suggesting an altered ratio of thick to thin filaments. In conclusion, an altered abundance of contractile proteins, regulated in part by UNC-45B, may be one cause of the contractile dysfunction which occurs in HF. / Thesis (Ph.D, Physiology) -- Queen's University, 2007-12-21 09:19:45.172