Life Sciences Commons^™

Biochemistry Of Trehalose Accumulation In The Spring Field Cricket, Gryllus Veletis, Alyssa R. Stephens

Electronic Thesis and Dissertation Repository

The freeze tolerant spring field cricket, Gryllus veletis, accumulates trehalose in the blood and tissues during cold acclimation. Trehalose is the main blood sugar in insects, thus its blood concentration is tightly regulated, and trehalose is readily metabolized. How do crickets modify their metabolism to accumulate trehalose in their hemolymph and tissues? I hypothesized that trehalose production, transport, and consumption were modified during the cold acclimation to facilitate trehalose accumulation. Trehalose and the trehalose-specific transporter, TRET-1, are distributed among all tissues, and trehalose accumulates in the hemolymph, fat body, Malpighian tubules, and gut. Trehalose production increases during cold acclimation …

The Hydrophobic Residues In Amino Terminal Domains Of Cx46 And Cx50 Are Important For Their Gap Junction Channel Ion Permeation And Gating, Roa'a Jaradat

Electronic Thesis and Dissertation Repository

Connexins are ubiquitous transmembrane proteins that assemble to form intercellular channels, called gap junctions (GJs). The amino terminal (NT) domain and residues within this domain have been shown to be important for both V_j-gating and single channel conductance (γ_j) of several GJs including Cx46 and Cx50. High-resolution structures of Cx46 and Cx50 GJs were recently resolved showing that NT domain folds into the inner pore where hydrophobic residues are packed against M1/2 domains stabilizing open-state conformation. We studied functional properties of GJs formed by several point variants at NT (Cx46 L10I, N13E, A14V, Q15N and Cx50 …

The Impact Of Excess Nutrients, Fatty Acids Or Glucose, On Bewo Placental Trophoblast Metabolic Function, Zachary Jw Easton

Electronic Thesis and Dissertation Repository

An adverse intrauterine environment high in circulating nutrients in pregnancies complicated by maternal obesity and diabetes mellitus has been well linked with the development of metabolic health disorders in the exposed offspring. Impairments in placental development, mitochondrial respiration, and nutrient processing are thought to specifically underlie the in-utero programming of these metabolic diseases. In these “at-risk” pregnancies a maternal diet high in fat as well as poor glycemic control have been highlighted to be important regulators of placental metabolic functions (including the transport, storage, and oxidation of dietary nutrients), and consequently regulators of offspring metabolic health outcomes. However, the independent …

Collagen Iv-Β1integrin Regulation Of Exocytotic Machinery In Pancreatic Beta-Cell Insulin Secretion, Malina Barillaro

Electronic Thesis and Dissertation Repository

Diabetes is a prevalent metabolic disease characterized by impaired insulin secretion, action, or both. β1-integrin is a key receptor that regulates cell-ECM interactions and is important in maintaining beta-cell functions, including insulin secretion. However, little is reported about the relationship between β1-integrin and the exocytotic proteins involved in insulin secretion. This study examined the influence of ECM-mediated β1-integrin activation on exocytotic machinery involved in insulin secretion using rat insulinoma (INS-1) cells. Collagen IV (COL IV) promoted INS-1 cell adhesion, spreading, and insulin secretion. Additionally, these cells displayed changes in levels and localization of exocytotic proteins involved in insulin secretion. β1-integrin …

Targeting Ccn Proteins In The Treatment Of Dermal Fibrosis, Alex Peidl

Electronic Thesis and Dissertation Repository

Fibrotic disorders, including the inflammatory connective tissue disease systemic sclerosis (SSc), contribute to 45% of deaths in the Western world. Currently there is no universally agreed-upon treatment for fibrosis. The CCN family of matricellular proteins are tightly spatiotemporally regulated proteins involved in development and wound healing, and are aberrantly expressed in fibrotic disease, including in SSc. CCN1 and CCN2 are overexpressed in SSc and contribute to the progression of animal models of fibrosis. CCN3 is reciprocally regulated to CCN1 and CCN2 and has been shown to suppress CCN2-mediated fibrogenic activity in kidney fibrosis. This led to the development of therapeutic …

Characterizing The Structural, Biophysical And Functional Effects Of S-Glutathionylation On Stim1 Ca2+ Sensing, Christian Michael Sirko

Electronic Thesis and Dissertation Repository

Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca²⁺) sensing protein that initiates cytoplasmic Ca²⁺ influx via store-operated calcium entry (SOCE). STIM1, in conjunction with Orai, a plasma membrane (PM) protein, function as mediators of SOCE through the formation of calcium-release activated calcium (CRAC) channels. S-Glutathionylation of STIM1 at Cys56 has been shown to evoke constitutive Ca²⁺ entry in DT40 cells, however no studies have carefully investigated the biophysical and structural effects of this covalent modification to the luminal domain, which are critical for understanding the molecular mechanism underlying the regulation of …

Investigation Of The Hsp90 Co-Chaperone, Sti1, In Cellular Resilience And Neurodegenerative Diseases, Rachel E. Lackie

Electronic Thesis and Dissertation Repository

In neurodegenerative diseases, certain proteins misfold and form toxic aggregates that cause brain matter atrophy, leading to decline in motor and/or cognitive functions. To maintain cellular proteostasis and survival, molecular chaperones regulate protein maturation and help to prevent aberrant protein aggregation. The molecular chaperone Hsp90 regulates hundreds of proteins and interestingly, several of those are misfolded in neurodegenerative diseases. Stress inducible-phosphoprotein-1 (STI1, STIP1), an Hsp90 co-chaperone, orchestrates client protein transfer between chaperones Hsp70 and Hsp90 through physical interactions with both chaperones. Notably, previous work in yeast, worms, and mouse neurons all showed that STI1 protects organisms against stressors and amyloid-like …

Characterizing The Effects Of Pyrroloquinoline Quinone (Pqq) Supplementation On Skeletal Muscle Mitochondrial Function And Myogenesis During Oxidative Stress And Iugr., Allyson J. Wood

Electronic Thesis and Dissertation Repository

Intrauterine growth restriction (IUGR) affects 10-15% of births and is associated with placental insufficiency (PI), resulting in fetal oxidative stress (OS). This OS is a factor in the predisposition to postnatal noncommunicable disease (NCD) of which muscle mitochondrial dysfunctional is a key aspect. Pyrroloquinoline quinone (PQQ), an antioxidant-like compound, is capable of OS reduction and promotes mitochondrial function, though limited research has focused on its effects in in utero skeletal muscle. This study sought to investigate the impact of in vitro H₂O₂, a model of OS, and an in vivo model of OS associated IUGR, with …

Inwardly Rectifying Potassium Channels: Regulators Of Myogenic Tone In Cerebrovascular Smooth Muscle, Jacob Fletcher

Electronic Thesis and Dissertation Repository

In cerebral arteries, inwardly rectifying potassium channels (K_IR) contribute to smooth muscle hyperpolarization to control arterial diameter and tone. Emerging evidence highlighted their regulation by pressure, though the underlying mechanism remains unclear. This thesis explored this concept through examination of K_IR channels in mouse and rat cerebral vascular smooth muscle (VSM). Experiments progressed from isolated cells to whole animals, employing electrophysiology, immunocytochemistry, proximity ligation assay, and arterial spin-labelling MRI techniques. Initial experiments indicated that K_IR activity persists beyond K_IR2.1 knockout in smooth muscle, clarifying their molecular composition. Our subsequent study identified key structural components …

The Effects Of Maternal Delta-9-Tetrahydrocannabinol And Cannabidiol Exposure On Fetal Heart Development In Mice, Gregory Robinson

Electronic Thesis and Dissertation Repository

Up to 22.6% of pregnant women consume cannabis during pregnancy despite the uncertainty of teratogenicity of the main ingredients in cannabis, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). This study tested the hypothesis that gestational THC and CBD exposure leads to heart abnormalities. Daily, oral THC exposure induced heart abnormalities in 68% of offspring with three main phenotypes including thickened semilunar valves, ventricular myocardial hypertrophy and hypoplastic coronary arteries in fetuses, and postnatal cardiac dysfunction. Altered gene expression of key cardiogenic regulators, increased proliferation, and reduced epicardial epithelial-to-mesenchymal-transition were demonstrated implicating potential mechanisms responsible for these abnormalities. Also, maternal CBD exposure resulted …

An Approach For The In-Vivo Characterization Of Brain And Heart Inflammation In Duchenne Muscular Dystrophy, Joanne Tang

Electronic Thesis and Dissertation Repository

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by dystrophin loss—notably within muscles and CNS neurons. DMD presents as cognitive weakness, progressive skeletal and cardiac muscle degeneration until pre-mature death from cardiac or respiratory failure. Innovative therapies improved life expectancy, but this is accompanied by increased late-onset heart failure and emergent cognitive degeneration. Thus, there is an increasing need to both better understand and track disease pathophysiology in the dystrophic heart and brain prior to onset of severe degenerative symptoms. Chronic inflammation is strongly associated with skeletal and cardiac muscle degeneration, however chronic neuroinflammation’s role is largely unknown in …

Characterization Of Mechanotransduction In Annulus Fibrosus Cells, Min Kyu M. Kim

Electronic Thesis and Dissertation Repository

IVD degeneration is a multifactorial pathological process associated with back pain. While biomechanical factors are important regulators of IVD homeostasis, mechanical loading also contribute to the onset of IVD degeneration. Importantly, the mechanotransduction pathways that mediate cell type-specific responses to mechanical loading are not well understood. Transient receptor potential vanilloid 4 (TRPV4) is a multimodally activated cell surface cation channel implicated as a mechanoreceptor regulating the mechano-response in other musculoskeletal cell types. Using both in vitro and in vivo models, the current study aimed to characterize the role of TRPV4 in annulus fibrosus (AF) cell mechanotransduction. Using a mechanically dynamic …

The Role Of Regulator Of G Protein Signaling 2 In Inflammatory Cytokine Release In Endotoxemia In Mice, Xin Tong Ma

Electronic Thesis and Dissertation Repository

In sepsis, lipopolysaccharide (LPS) activates toll-like receptor 4 to stimulate the release of inflammatory cytokines (e.g. tumor necrosis factor-alpha, TNF-α), leading to cardiac dysfunction. Regulator of G protein signaling 2 (RGS2) limits G protein-coupled receptor signaling by increasing the rate of G protein deactivation or inhibiting G protein-effector interactions. We hypothesized that RGS2 deficiency would enhance proinflammatory responses in endotoxemia. Adult wild-type and RGS2^-/- C57BL/6 mice and neonatal cardiomyocytes were treated with LPS and assessed for inflammatory responses and cardiac function. Myocardial TNF-α expression was higher in RGS2^-/- mice during endotoxemia. Additionally, cardiac function was impaired in …

Regulators Of Ectopic Calcification In A Mouse Model Of Dish: A Multi-Omics Perspective, Matthew A. Veras

Electronic Thesis and Dissertation Repository

Diffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory spondyloarthropathy and the second most common form of arthritis characterized by formation of ectopic mineral along the spine. Pathological findings in DISH include regional calcification of the anterior longitudinal ligament, paraspinal connective tissues, and annulus fibrosus (AF) of the intervertebral disc (IVD). Clinical symptoms of DISH include increased spine stiffness, decreased spinal range of motion, and in severe cases dysphagia and spinal cord/nerve root compression. The molecular pathways responsible for DISH have not been delineated and as such, there are no disease-modifying treatments. Clinical treatment for DISH is limited to surgical resection …

The Amino Terminal Domains Of Sheep Cx46 Or Cx50 Determine Their Gap Junction Channel Open Stability And Unitary Channel Conductance, Benny Yue

Electronic Thesis and Dissertation Repository

Connexins form intercellular communication channels known as gap junctions (GJs), which are found throughout the vertebrate species. GJs formed by different connexins harbor unique channel properties that have not been fully defined. High-resolution structures of native Cx46 and Cx50 GJs from sheep (sCx46 and sCx50) were recently resolved. Molecular dynamics studies identified the NT domains, especially the 9^th position, as key determinants in the differences of energetic barrier to K⁺ permeation in sCx46 and sCx50 GJs. We studied functional properties of GJs formed by sCx46, sCx50, NT domain swapped chimeras (sCx46-50NT and sCx50-46NT), and point variants at the …

Investigating The Role Of Nuclear Receptor Peroxisome Proliferator-Activated Receptor Δ In Obesity-Associated Osteoarthritis, Bethia C. To

Electronic Thesis and Dissertation Repository

Osteoarthritis (OA) is a heterogeneous disease, differentiated based by risk factors that drive joint damage. Peroxisome proliferator-activated receptor delta (PPARd) is a nuclear receptor previously implicated in cartilage damage in OA. Given its deleterious role in post-traumatic OA and in regulating metabolism, we hypothesized that PPARd inhibition will protect against obesity-associated OA. Diet-induced obesity was used to induce OA in mice. Mice fed the western diet for 40 weeks exhibited mild OA, with subchondral bone remodelling occurring alongside cartilage damage. Cartilage-specific Ppard knockout (KO) mice were generated to study its role in metabolic OA. Both wildtype and PPARd KO mice …

The Secretory Pathway Calcium Atpase 2c Promotes Increased Cytosolic Calcium Levels, Melissa Fenech

Electronic Thesis and Dissertation Repository

Pancreatic acinar cell exocytosis requires precise calcium (Ca²⁺) signals. When cytosolic Ca²⁺ levels remain high, cellular functions are disrupted, which is associated with initiation of pancreatitis. Ca²⁺ signals are achieved through regulating endoplasmic reticulum (ER) stores, Ca²⁺ATPases, and store-operated Ca²⁺ entry (SOCE). However, how these pathways interact to create precise signals is not well understood. In a pancreatic model of dysregulated Ca²⁺ homeostasis, secretory pathway Ca²⁺ATPase 2 (SPCA2) expression is significantly decreased. In the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. Recent studies indicate a role …

Excess No Stabilizes The Luminal Domain Of Stim2 In A Cys-Specific Manner Thereby Regulating Basal Calcium Homeostasis And Store-Operated Calcium Entry, Matthew Novello

Electronic Thesis and Dissertation Repository

Stromal-interaction molecule 2 (STIM2) is an endoplasmic reticulum (ER) membrane-inserted Ca²⁺-sensing protein which, together with the plasma membrane Ca²⁺ channel Orai1, regulates basal Ca²⁺ homeostasis and store-operated Ca²⁺ entry (SOCE). Recent evidence suggests that S-nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to a cysteine thiol, can attenuate the function of the paralog STIM1 protein. Compared to STIM1, STIM2 also functions as a basal Ca²⁺ homeostatic feedback regulator. Therefore, the objective of my study was to evaluate the susceptibility of STIM2 to S-nitrosylation and the effects that this …

Assessing The Structure-Function Relationships Of The Apolipoprotein(A) Kringle Iv Sub-Type 10 Domain, Matthew J. Borrelli

Electronic Thesis and Dissertation Repository

Elevated plasma lipoprotein(a) (Lp(a)) is the most prevalent heritable risk factor in the development of cardiovascular disease. The apolipoprotein(a) (apo(a)) component of Lp(a) is strongly implicated in the pathogenicity of Lp(a). It is hypothesized that the inflammatory potential of Lp(a)/apo(a) is mediated by the lysine binding ability of the apo(a) kringle IV10 (KIV10) domain, along with its covalently bound oxidized phospholipid (oxPL). Using targeted mutagenesis, two novel null alleles for the LPA gene that generate non-secretable apo(a) species have been identified, resulting from amino acid substitutions in the KIV10 domain. A potential mechanism by which KIV10 oxPL modification is enriched …

Mapping And Functional Characterization Of Proteolytic Cleavage Of Murine Kidney Injury Molecule-1, Saranga Sriranganathan

Electronic Thesis and Dissertation Repository

Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein expressed apically on proximal tubule epithelia during acute kidney injury (AKI). KIM-1, as a phagocytic receptor, facilitates clearance of apoptotic cells (efferocytosis) from tubular lumen, thereby reducing inflammation and promoting repair. Human KIM-1 undergoes spontaneous and accelerated ectodomain shedding into urine and blood via metalloproteases; tumour necrosis factor-a converting enzyme (TACE/ADAM17) and a disintegrin and metalloprotease 10 (ADAM10). Blood and urine KIM-1 are clinical AKI biomarkers, however, biological significance of KIM-1 shedding is unknown. To study this in vivo in mice, I first aimed to identify murine KIM-1 cleavage site and study …

Pregestational Diabetes Induced Congenital Heart Defects And Coronary Artery Malformations; Mechanisms And Preventative Therapies, Anish Engineer

Electronic Thesis and Dissertation Repository

Congenital heart defects (CHDs) arise from perturbations in complex molecular and cellular processes underlying normal embryonic heart development. CHDs are the most common congenital malformation, occurring in 1 to 5% of live births, and are the leading cause of pediatric mortality. Adverse genetic and environmental factors can impede normal cardiogenesis and increase the likelihood of CHDs. Pregestational maternal diabetes increases the risk of CHDs in children by more than four-fold. As the prevalence of diabetes rapidly rises among women of childbearing age, there is a need to investigate the mechanisms and potential preventative strategies for these defects. The aim of …

Maternal Nicotine Exposure Induces Congenital Heart Defects In The Offspring Of Mice, Elizabeth Greco

Electronic Thesis and Dissertation Repository

Congenital heart defects are the most prevalent birth defect, and maternal cigarette smoking is a known risk factor. Nicotine replacement therapies are recommended to pregnant women who smoke to aid in smoking cessation, as this alternative is thought to be much safer compared to cigarette smoking. However, these products contain nicotine, and the safety of nicotine on the developing heart is not well known. In this thesis, a mouse model was used to test the hypothesis that maternal nicotine exposure (MNE) during pregnancy leads to congenital heart defects and coronary artery defects in the offspring of mice. MNE resulted in …

Oleic Acid Attenuates Palmitic Acid-Induced Impairments In Mouse Blastocyst Development, Maisoon Dafalla Yousif

Electronic Thesis and Dissertation Repository

Palmitic acid (PA) and oleic acid (OA) are the most abundant saturated and monounsaturated non-esterified fatty acids, respectively, in serum. The consequences of PA and OA treatment on preimplantation mouse embryo development were investigated. It was hypothesized that OA treatment from the two-cell stage onwards would attenuate PA-induced reductions in mouse blastocyst development. PA significantly reduced development to the blastocyst stage. Transcript analysis revealed that PA increased ER stress pathway markers activating transcription factor 3 and C/EBP homologous protein transcripts, and XBP1 splicing. Co-culture of PA-treated embryos with OA prevented this PA-induced ER stress and rescued blastocyst development. Cytoplasmic lipid …

Quantitative Proteome Analysis Of Alfalfa In Drought Stress Under The Influence Of Mir156, Alpa Puri

Electronic Thesis and Dissertation Repository

Medicago sativa is one of the most produced perennial forage crops in Canada. Drought stress is a major form of abiotic stress affecting the productivity and annual yield of alfalfa. A small noncoding RNA, miR156 has a role in abiotic stress tolerance, while regulating downstream SPL genes. In this study, whole leaf proteome and amino acid concentrations of miR156OE (A8) genotype of alfalfa under drought stress were quantified. In results, 3,000 proteins groups were identified using MaxQuant when searched against Medicago truncatula protein sequence database. Gene ontology analysis showed enriched proteins were involved in biological and molecular processes such as …

Effects Of Maternal Protein Restriction On The Pulmonary Surfactant System During The Early Life And Adulthood, Reza Khazaee

Electronic Thesis and Dissertation Repository

Fetal growth restriction (FGR) is defined by low birth weight and contributes to a variety of adult-onset diseases with different severities between males and females. However, the effects of FGR on the pulmonary surfactant are not fully elucidated. In this thesis, first, we investigated the FGR effects on the lung function and the surfactant system at the early postnatal life. It was hypothesized that FGR contributes to alterations of lung mechanics and the surfactant system during the neonatal period. Second, we assessed the FGR effects on the surfactant system in response to sepsis in adulthood. It was hypothesized that FGR …

Endothelial Glycocalyx Shedding In Pediatric Diabetic Ketoacidosis, Rufina Ning

Electronic Thesis and Dissertation Repository

Diabetic ketoacidosis (DKA) is a state of severe insulin deficiency resulting in hyperglycemia, ketoacidosis, and inflammation. Children with DKA have an increased risk of microvascular complications (e.g. stroke). The glycocalyx maintains the anti-thrombotic properties of endothelial cells (ECs), however the glycocalyx status is unknown in DKA. We quantified major glycocalyx components: hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate (CS), and syndecan-1 in DKA plasma and correlated them with DKA severity. HA/HS were significantly increased in the plasma of DKA patients in comparison to insulin-controlled type-1 diabetics, but only HA correlated to DKA severity. Exogenous addition or removal of HA …

Mechanisms Underlying Freeze Tolerance In The Spring Field Cricket, Gryllus Veletis, Jantina Toxopeus

Electronic Thesis and Dissertation Repository

Freeze tolerance has evolved repeatedly across insects, facilitating survival in low temperature environments. Internal ice formation poses several challenges, but the mechanisms that mitigate these challenges in freeze-tolerant insects are not well understood. To better understand how insects survive freezing, I describe a novel laboratory model, the spring field cricket Gryllus veletis (Orthoptera: Gryllidae). Following acclimation to six weeks of decreasing temperature and photoperiod (mimicking autumn), G. veletis juveniles becomes moderately freeze-tolerant, surviving freezing at -8 °C for up to one week, and surviving temperatures as low as -12 °C. Acclimation is associated with increased control of the temperature and …

Beta-Cell Β1 Integrin Deficiency During Second Transition Stage Of Fetal Pancreas Development On Islet Growth, Phyo Win

Electronic Thesis and Dissertation Repository

β1 integrin has been shown to be important for pancreatic beta-cell growth and functioning. Congenital and postnatal studies have shown beta-cell mass reduction with β1 integrin knockout (β1KO). Using an inducible cre recombinase (CreERT) expressed from the mouse insulin promoter (MIP) crossed to a line in which Itgb1 (β1 integrin encoding gene) was flanked by loxP sites (MIP-CreERT+; β1itg designated as MIPβ1KO), we examined the in vivo temporal role of beta-cell β1 integrin effect on islet growth during the second transition stage of pancreas development.

Prenatal MIPβ1KO mice displayed decreased beta-cell area, mass, density and proliferation. In addition, a decrease …

Heterotypic Docking Compatibility Of Human Connexin 37 With Other Vascular Connexins, Nicholas Kim

Electronic Thesis and Dissertation Repository

Gap junction (GJ) channels provide direct intercellular communication. A GJ channel consists of two docked hemichannels and each hemichannel is a hexamer of six connexins. Human vascular connexins (Cx37, Cx40, Cx43, and Cx45) can form various types of gap junction channels to synchronize vasodilation/constriction to control local circulation. Most of our knowledge on heterotypic GJs of these vascular connexins comes from studies on rodent connexins. However it is not clear if these human connexins can also form heterotypic GJs. The present study used an in vitro expression system to investigate the coupling status and GJ properties of human heterotypic Cx37/Cx40, …

Investigating The Role Of Pannexin 3 In Intervertebral Disc Health And Disease, Meaghan E. Serjeant

Electronic Thesis and Dissertation Repository

Pannexin 3 (Panx3) is a mechanosensitive, channel-forming glycoprotein implicated in the progression of post-traumatic osteoarthritis. Despite evidence for Panx3 expression in intervertebral discs (IVDs), its function here remained unknown. Using Panx3^-/- mice, this study investigated the role of Panx3 in age-associated and injury-induced models of IVD degeneration. While loss of Panx3 did not significantly impact the progression of age-associated IVD degeneration, it was associated with a protective phenotype in an injury model of IVD degeneration, specifically in the annulus fibrosus (AF). Following IVD injury, WT mice showed an accumulation of enlarged cells that appeared to contribute to disrupted AF …