Life Sciences Commons^™

Characterization Of The Alternative Oxidase From The Psychrophilic Green Alga Chlamydomonas Sp. Uwo241, Michael Sj Inman

Electronic Thesis and Dissertation Repository

The alternative oxidase (AOX) was studied in the psychrophilic green alga Chlamydomonas sp. UWO241. AOX is the sole component of the alternative pathway of mitochondrial electron transport and is present in all plant and algal species. In silico analysis of the deduced protein sequence of the cloned AOX cDNA showed that the UWO241 protein has lower amounts of proline and higher amounts of lysine and tryptophan compared to the AOX sequence of the mesophilic alga C. reinhardtii. These changes have been seen in other studies of cold-adapted enzymes. Interestingly, unlike C. reinhardtii, AOX transcript abundance in UWO241 …

The Role Of Choline Acetyltransferase Variants In Alzheimer's Disease Models, Shawn Albers

Electronic Thesis and Dissertation Repository

The primate specific 82-kDa choline acetyltransferase (ChAT) isoform is found in the nuclei of cholinergic neurons, with a disruption in the subcellular localization in aging and AD brain samples# The functional significance of this protein is poorly understood. Previous studies have revealed a potentially protective role for 82-kDa ChAT, mediated through a reduction in amyloid-!1-42 (A!1-42) release in APP/PS1 double transgenic primary cortical neurons. Here we examine the effect of 82-kDa ChAT expression in transgenic neurons, on the amyloidogenic processing of amyloid precursor protein (APP) and A! production. In this study we demonstrate 82-kDa ChAT transcriptionally increases golgi- localized "-ear-containing …

The Effect Of Testosterone On The Spring Migratory Phenotype Of A North American Songbird (Zonotrichia Albicollis), Caitlin L. Vandermeer

Electronic Thesis and Dissertation Repository

In passerines, the endocrine modulators responsible for seasonal changes in migratory behaviour and physiology are unclear. Spring photoperiods alter androgen levels, influencing muscle mass and fat deposition rates to power migration, as well as enhance nocturnal migratory restlessness activity (Zugunruhe). My study compared physiological indicators and migratory restlessness in castrated and intact white-throated sparrows (Zonotrichia albicollis) following photoperiod and hormone manipulation. Nocturnal restlessness activity was highest in migratory intact males or migratory castrated males that received testosterone replacement. Liver mass decreased in all photostimulated migratory groups regardless of testosterone treatment. Likewise, oxidative enzyme activity was unrelated to testosterone, …

Transforming Growth Factor-Beta Receptor Signalling Is Modulated By Integrin-Linked Kinase, Stellar H. Boo

Electronic Thesis and Dissertation Repository

Transforming growth factor-beta 1 (TGF-β1) modulates regeneration after injury through induction of fibroblast proliferation, migration, and differentiation into myofibroblasts. Induction of myofibroblast differentiation by TGF-β1 requires expression of integrin-linked kinase (ILK). I now show that ILK interacts with TGF-β receptor type II (TβRII) in primary dermal fibroblasts. Further, colocalization of ILK and TβRII can be observed at the cell membrane and in intracellular vesicles. The association of TβRII and ILK does not require TGF-β1 stimulation, kinase activity of TGF-β1 receptor type I or TβRII, and it does not involve interactions between ILK and focal adhesion-associated proteins. When this interaction is …

Expression Analysis Of Histone Acetyltransferases In Rice Under Drought Stress, Hui Fang

Electronic Thesis and Dissertation Repository

Histone acetylation is one of the vital reversible modifications in eukaryotes. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain the homeostasis of histone acetylation. HATs are associated with genome-wide transcriptional activation and various biological processes in response to various stresses. Drought stress causes a range of physiological and biochemical responses in plants. Eight HATs which belong to four different families (CBP, GNAT, MYST, and TAFII250 family) have been identified in rice. In this research, four OsHATs, one from each family, were chosen based on in silico domain and promoter analysis. The real-time qPCR analysis demonstrated that drought stress caused …

Atrial Fibrillation-Linked Germline Gja5/Connexin40 Mutants Showed An Increased Hemichannel Function, Matthew D. Hills

Electronic Thesis and Dissertation Repository

Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40) have been linked to lone atrial fibrillation (AF). Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting AF. However, the effects of the AF-linked Cx40 mutants on hemichannel function has not been studied. Here we investigated two AF-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing …

Cyanobacteria Predominance In Alberta's Eutrophic Lakes Linked To Iron Scavenging Strategy That Uses Siderophores And Toxins, Xue L. Du

Electronic Thesis and Dissertation Repository

The role of the micronutrient iron in the regulation of cyanobacteria dominance and cyanotoxicity is poorly understood. Iron is required for important metabolic pathways, including both phosphorus (P) and nitrogen (N) assimilation, and low levels of this element may influence the assimilation of the macronutrients. The hypothesis tested was that cyanobacteria produce and utilize siderophores and toxins in low iron conditions to scavenge iron, when P and N are not limiting algal growth, providing some cyanobacteria with a competitive advantage over other algal species. Among the naturally eutrophic lakes studied, cyanobacteria were dominant at low iron (>pFe19) concentrations (Spearman …

Skin Diseases And Non-Syndromic Hearing Loss Linked To Cx30 Mutations Arise Through Several Distinct Mechanisms, Amy C. Berger

Electronic Thesis and Dissertation Repository

Connexin 30 (Cx30), a member of the large gap junction protein family, plays a role in the homeostasis of the epidermis and inner ear through gap junctional intercellular communication (GJIC). Here, we investigated four autosomal dominant Cx30 gene mutations linked to hearing loss and/or various skin diseases. First, the T5M mutant linked to non-syndromic hearing loss formed functional gap junction channels and hemichannels, similar to wild-type Cx30. The V37E mutant associated with keratitis-ichthyosis-deafness (KID) syndrome was retained in the endoplasmic reticulum (ER) and significantly induced unfolded protein response (UPR)-mediated apoptosis. The loss-of-function G59R mutant linked to Vohwinkel and Bart-Pumphrey syndromes …

Regulation Of Trpm2 Channels By Fyn Kinase, Matthew L. Johnston

Electronic Thesis and Dissertation Repository

TRPM2 is a calcium-permeable non-selective cation channel that acts as a mediator of cell death in response to oxidative stress. It has been shown that oxidative stress increases TRPM2 tyrosine phosphorylation and activation – an effect that is blocked by PP2, a non-specific inhibitor of Src family kinases. However, the kinase and target TRPM2 tyrosine residue(s) involved have not yet been identified. Here, we investigated the potential regulation of TRPM2 by Fyn. Intracellular application of recombinant Fyn potentiated TRPM2 currents in HEK293 cells expressing inducible TRPM2 (TRPM2-HEK293 cells). Further, a physical interaction between Fyn and TRPM2 was demonstrated by co-immunoprecipitation …

A C-Terminus Mitochondrial-Localization Region And Bh3 Domain Of Puma Are Required For Apoptotic Function, Liz A. Merwin

Electronic Thesis and Dissertation Repository

Apoptosis is known to contribute to the loss of neurological function in brain injury and several neurodegenerative diseases. The Bcl-2 family of proteins consist of pro-apoptotic and anti-apoptotic members that interact physically and functionally to regulate apoptosis in a cell type and stimulus specific manner. We have previously demonstrated that the Bcl-2 family member Puma plays a key role in triggering neuronal apoptosis under diverse stress conditions. However, the mechanism by which Puma mediates apoptosis remains unclear. Here we found that Puma contains two domains required for its apoptotic function: the BH3 domain and a region in the c-terminus that …

Ectopic Mineralization Of Spinal Tissues In Mice Lacking Equilibrative Nucleoside Transporter 1, Sumeeta Warraich

Electronic Thesis and Dissertation Repository

Equilibrative nucleoside transporter 1 (ENT1) regulates the bi-directional transfer of hydrophilic nucleosides, such as adenosine, across the plasma membrane. Mice lacking ENT1 developed calcified lesions resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. By 12 months of age, ENT1^-/- mice exhibit spine stiffness, hind limb dysfunction, and paralysis. Histological examination of ENT1^-/- mice revealed irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels. Analysis of intervertebral disc tissues of 6 month old ENT1^-/- and wild-type mice revealed reduced expression …

Elongation Factor 1a-1 And Hepatocyte Response To Fatty Acid Excess, Alexandra M. Stoianov

Electronic Thesis and Dissertation Repository

Obesity is associated with elevated levels of serum fatty acids, which accumulate in nonadipose tissues including the liver. Elongation factor 1A-1 (EF1A-1) has previously been shown to participate in the cell stress and death response of cardiomyocytes to excess saturated fatty exposure, and in steatotic mouse myocardium. In this thesis, the hypothesis that the hepatocyte response to fatty acid overload involves EF1A-1 was tested. EF1A-1 expression was induced in the livers of obese mice in association with severe hepatic steatosis, and in HepG2 human hepatoma cells in response to excess palmitate. Partial translocation of EF1A-1 from the ER to polymerized …

The Distribution And Functional Properties Of Pmld-Linked Cx47 Mutations On Cx47/Cx47 Homotypic And Cx47/Cx43 Heterotypic Gap Junctions, Mi Seong Kim

Electronic Thesis and Dissertation Repository

Gap junctions (GJ) allow direct intercellular communication, and consist of connexins (Cx). In the mammalian central nervous system, oligodendrocytes express Cx29, Cx32 and Cx47, whereas astrocytes express Cx26, Cx30 and Cx43. Cx47/Cx47 GJs couple oligodendrocytes, and Cx47/Cx43 channels are the primary heterotypic GJs at oligodendrocyte/astrocyte junctions. Interestingly, mutations in Cx47 have been linked to central hypomyelinating Pelizaeus Merzbacher-like Disease (PMLD). My aim is to determine the cellular distribution and functional properties of PMLD-associated Cx47 mutants (I46M, G149S, G236R, G236S, M286T, T398I). All mutants were detected at the cell-cell interface. These mutants, except G149S and T398I, showed no electrical coupling in …

Post-Transcriptional Regulation Of Telomerase Reverse Transcriptase (Tert) In Human Embryonic Stem Cells Cultured Under Varying Oxygen Microenvironments, Lida Radan

Electronic Thesis and Dissertation Repository

Recent evidence points to extra-telomeric roles of telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% oxygen for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% oxygen cultured hESCs despite no significant difference in telomerase activity. Pharmacological inhibition of TERT caused spontaneous differentiation while inhibition of the telomerase RNA component (TERC) did not. RT-PCR analysis revealed variations in transcript levels of full-length and …

Human Cerebrovascular Endothelial Cell Responses In Severe Sepsis, Christopher J. Blom

Electronic Thesis and Dissertation Repository

Sepsis-associated encephalopathy (SAE) is an independent predictor of mortality in severe sepsis patients. Mechanisms of SAE are poorly characterized. Pro-inflammatory mediators are up-regulated in blood plasma of severe sepsis patients and may lead to activation/dysfunction of cerebrovascular endothelial cells (CMEC) of the blood-brain barrier (BBB) – potentially contributing to brain dysfunction. The objective of the present study was to determine the effects/mechanisms of both human severe sepsis blood plasma (hSSP; 20% v/v) and a mixture of 8 cytokines/chemokines that mimicked physiological concentrations of cytokines/chemokines in hSSP (cytomix; SSCM) on human-derived CMEC (hCMEC/D3) activation/dysfunction

in vitro. hSSP-stimulation up-regulated hCMEC/D3 pro-adhesive …

Kiss1r Induces Human Mammary Epithelial Cell Invasiveness And Promotes Breast Cancer Cell Migration And Invasion Via Iqgap1, Donna Cvetkovic

Electronic Thesis and Dissertation Repository

Kisspeptins (KP), peptide products of the kisspeptin-1 (KISS1) gene are the endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in human breast tumors correlates with higher tumor grade and metastatic potential. We have previously shown that KP-10, the most potent KP, stimulates invasion of estrogen receptor (ER)-negative breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, I report that KP-10 treatment of the ER-negative non-malignant mammary epithelial MCF10A cells, or stable …

The Role Of Transforming Growth Factor Alpha In Osteoarthritis And Skeletal Development, Shirine E. Usmani

Electronic Thesis and Dissertation Repository

Osteoarthritis (OA) is the most common joint disease and a major cause of physical disability, however no disease modifying treatments are available. To better understand the molecular mechanisms involved, my colleagues previously performed microarray studies on a surgical rodent model of OA. Transforming growth factor alpha (TGFa) was identified as a novel growth factor involved in cartilage degeneration and subsequent experiments revealed that it induced an OA-like phenotype in articular chondrocytes. These findings lead to the overarching hypothesis for my thesis: TGFa promotes OA.

I began by studying TGFa’s interaction with endothelin receptor A (ET(A)R) as this signaling system is …

Uncovering Dual Roles For Perk Signaling During Experimentally Induced Pancreatitis, Elena Fazio

Electronic Thesis and Dissertation Repository

Pancreatitis is characterized by inappropriate activation of digestive enzyme

precursors, or zymogens, local and systemic inflammation, dysregulation of

cellular calcium (Ca2+), and induction of the unfolded protein response (UPR).

The UPR consists of three distinct pathways all of which are activated during

pancreatitis. However, the molecular roles of each remain unclear. The

protein kinase RNA (PKR)-like ER kinase (PERK) pathway reduces general

protein translation by phosphorylating eIF2!, and is activated within minutes

of initiating pancreatic damage. Microarray analysis carried out by our lab

revealed robust upregulation of the PERK pathways members Activating

Transcription Factor (ATF) 3 and stanniocalcin (STC) 2. …

Identification And Characterization Of The Arabidopsis Homolog Of The Yeast Trex-2 Complex, Gang Tian

Electronic Thesis and Dissertation Repository

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast Thp1-Sac3-Cdc31-Sus1 complex, also known as the TREX-2 complex, is anchored to the NPC via the nucleoporin Nup1, and is essential for mRNA export. In this study, the Arabidopsis homolog of the yeast TREX-2 complex was discovered. Physical and functional evidence support the identification of the Arabidopsis orthologs of the yeast Thp1 and Nup1. Of three Sac3 Arabidopsis homologs, two are putative TREX-2 components. Surprisingly, none are required for mRNA export as is the yeast Sac3. Physical association with TREX-2 was observed for the two Cdc31 homologs, but …

The Developmental And Adaptive Role Of Mitogen Activated Protein Kinase Pathways During Preimplantation Development, Christine E. Bell

Electronic Thesis and Dissertation Repository

The preimplantation period of development represents the highest interval of embryonic loss throughout pregnancy. It is therefore imperative that we elucidate the mechanisms involved in regulating preimplantation embryonic responses to stress and that govern development. The MAPK pathways are involved in both responding to environmental stress and regulation of development throughout embryogenesis, and are therefore good candidates to study the mechanisms involved in preimplantation embryonic adaptation to stress and development. The preimplantation embryo culminates in the development of a fluid filled structure called the blastocyst. It is at this stage the first differentiation events occur and the trophectoderm (TE), which …

Morpho-Physiological Analysis Of Interneuronal Populations In The Rat Piriform Cortex Before And After Kindling Induced Epilepsy, Cezar Gavrilovici

Electronic Thesis and Dissertation Repository

The piriform cortex (PC) is involved in olfactory sensory processing, associative learning tasks and is highly seizurogenic. Understanding how interneurons participate in these behaviours, especially their contribution in epileptogenic mechanisms, is hampered by an incomplete understanding of their functional and morphological diversity. The hypothesis in this work is that kindling-induced epilepsy alters the firing properties of PC interneuronal populations. Altered/impaired interneuronal firing could lead to abnormal processing in the PC and epileptogenesis. Therefore it was important to first identify and describe interneuronal morpho-functional properties in the unkindled brain and then to assess the electrophysiological parameters following kindling.

Based on interneuronal …

Trace Metal Limitation And Its Role In Oxidative Stress Of Coral Algal Symbionts; Implications For Thermally Induced Coral Bleaching Events., Katrina Lynn Iglic

Electronic Thesis and Dissertation Repository

Coral bleaching, the process in which corals expel their photosynthetic symbionts (Genus Symbiodinium), is caused by high temperature and irradiance stress. The synergistic effect of each stressor is the generation of damaging reactive oxygen and nitrogen species (ROS and RNS), which are normally mitigated physiologically by antioxidant enzyme activity. Antioxidant enzymes require iron, copper, manganese and zinc in their structure and the limited nature of these trace metals in reef environments may enhance ROS production under elevated temperature and irradiance. The hypothesis tested within this thesis was that a limited availability of the trace metals Fe, Mn, Cu and …

Purine Transport And Metabolism In Microvascular Endothelial Cells, Derek B J Bone

Electronic Thesis and Dissertation Repository

The microvascular endothelium serves as the barrier between the blood and perfused tissues. Proper function of the endothelium is dependent on the ability of the endothelial cells to produce nitric oxide and form tight junctions between themselves. Dysfunction occurs when stresses overwhelm the endothelial cell, with oxidative stress being a leading cause. Intracellular metabolism of purine nucleosides and nucleobases has been implicated in the production of oxidative stress. Nucleosides (e.g. adenosine) and nucleobases (e.g. hypoxanthine) are moved across cell membranes by a specialized family of proteins called equilibrative nucleoside transporters (ENTs).

We characterized primary human cardiac microvascular endothelial cells (MVECs) …

Role Of Rac1 In Myocardial Tnf-Alpha Expression In Sepsis, Ting Zhang

Electronic Thesis and Dissertation Repository

Tumor necrosis factor-a (TNF-alpha) is a pro-inflammatory cytokine and its high levels of expression in the heart leads to cardiac dysfunction in sepsis. However, the underlying molecular mechanisms of regulating myocardial TNF-alpha expression are not fully understood. The aim of this thesis was to investigate the role of Rac1 in myocardial TNF-alpha expression and cardiac dysfunction during sepsis. Studies were performed using cultured neonatal cardiomyocytes and a mouse model of endotoxemia.

PI3K-mediated Rac1 activation is required for induction of TNF-alpha mRNA and protein expression in cardiomyocytes and cardiac dysfunction during endotoxemia. Rac1 promotes TNF-alpha mRNA expression via NADPH oxidase/ERK1/2 and …

Cardiac Adaptation To Chronic Blockade Of Voltage-Gated, L-Type Calcium Channels In The Sarcolemma, Ji Zhou

Electronic Thesis and Dissertation Repository

L-type Ca²⁺ channels (dihydropyridine receptors, DHPRs) in the sarcolemma are essential to cardiac excitation-contraction (E-C) coupling. Thus, Ca²⁺ influx through DHPRs upon cardiomyocyte excitation triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) to initiate myofilament activation and muscle contraction. Muscle relaxation occurs upon sequestration of Ca²⁺ back into the SR lumen by sarco/endoplasmic reticulum calcium-ATPase (SERCA) in the SR. As a treatment option for hypertension, long-term use of DHPR blockers is associated with increased risk of heart failure; underlying mechanisms are unknown. This research used male Wistar rats treated with verapamil (subcutaneously, …

The Roles Of Nitric Oxide Synthases (Nos) In Endochondral Bone Formation, Qian Yan

Electronic Thesis and Dissertation Repository

Longitudinal growth of endochondral bones is controlled by the cartilage growth plate. Chondrocyte proliferation and hypertrophy, vascular invasion, formation of ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. These biological processes have to be tightly regulated or disturbances will lead to skeletal diseases. A large number of genes, growth factors and hormones have been implicated in the regulation of growth plate biology, however, less is known about the intracellular signaling pathways involved. Nitric oxide (NO) has been identified as a regulator of cellular proliferation, differentiation, migration, survival and metabolism in multiple cell …