Life Sciences Commons^™

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 …

Analysis Of Subcellular Localization Patterns Suggest Non-Enzymatic Roles For Select Arogenate Dehydratases, Travis R. Howes

Electronic Thesis and Dissertation Repository

The final step of phenylalanine biosynthesis in planta is catalyzed by arogenate dehydratases (ADTs). Previously cloned ADT-CFP fusion genes were used to provide an in depth study of the subcellular localization of all six ADTs from Arabidopsis thaliana. Through co-localization of ADT-CFPs with a stroma-marker it is shown that most ADTs localize to stroma-filled projections from chloroplasts called stromules. The localization of ADT5 and ADT2 provide evidence for additional, non-enzymatic roles. In the case of ADT5, it is found to localize to the nucleus, suggestive of an uncharacterized nuclear role. The localization patterns of ADT2 are suggestive of a …

P2x7 Nucleotide Receptor Signaling In Osteoblasts, Matthew W. Grol

Electronic Thesis and Dissertation Repository

Nucleotides are released from cells of the osteoblast lineage in response to mechanical stimulation, and signal through two families of P2 nucleotide receptors – G protein-coupled P2Y receptors and ligand-gated P2X cation channels. Nearly every cell-type expresses multiple P2 receptor subtypes. However, the significance of these networks of receptors in any system is unclear. In this thesis, we demonstrate that the endogenous network of P2 receptors expressed by osteoblasts permits graded increases in Ca²⁺ signaling over a million-fold range of ATP concentrations. P2Y receptors mediate transient activation of the Ca²⁺/NFATc1 pathway, whereas stimulation of P2X7 causes sustained …

Amyloid Beta Resistance And The Warburg Effect: Re-Examining Alzheimer's Disease, Jordan Taylor Newington

Electronic Thesis and Dissertation Repository

Alzheimer’s disease (AD) is characterized by deposition of the amyloid beta (Ab) peptide in the brain, an event which frequently but not universally correlates with nerve cell death. Although most nerve cells die in response to Ab, small populations of cells are able to survive by becoming resistant to Ab toxicity. Understanding the mechanisms by which cells become resistant to Ab may reveal novel treatments for AD. Interestingly, nerve cell lines selected for resistance against Ab exhibit increased glucose uptake and glycolytic flux. Here I show that these metabolic changes are mediated through an upregulation of pyruvate dehydrogenase kinase 1 …

Systematic Analysis Of Residues In Conserved Region 3 Of The Human Papillomavirus 16 E7 Oncoprotein, Biljana Todorovic

Electronic Thesis and Dissertation Repository

Although remarkable biological diversity is exhibited by viruses, as obligate intracellular parasites, they rely on host cell functions. As such, viruses typically must overcome a set of host barriers that prevent infection. For human papillomaviruses (HPV) one of these barriers is the state of terminal differentiation of the host cell. For that purpose HPVs encode two major oncoproteins, E6 and E7, which combine their efforts to effectively uncouple cellular differentiation from the cell cycle arrest. The E7 proteins have no intrinsic enzymatic activity or DNA binding ability, but they bind and manipulate numerous host proteins. E7 is a modular oncoprotein …

Subcellular Analysis Of The Disulfide Proteome In P66shc Expressing Nerve Cells, Tyler Cann

Electronic Thesis and Dissertation Repository

The longevity associated protein p66Shc has been suggested to regulate organismal lifespan through initiation of apoptotic pathways. Following stress-induced translocation into the mitochondria, p66Shc promotes increased reactive oxygen species (ROS) production and triggers poorly defined downstream signaling events that lead to decreased cell viability. Protein disulfide bonding has recently emerged as aROSdependent post-translational modification that regulates protein function and signaling processes. Using the mouse hippocampal HT-22 cell line, I sought to determine the changes in the disulfide proteome associated with p66Shc mediatedROSproduction. Through Redox 2D-SDSPAGEanalysis of mitochondrial and cytosolic extracts, redox sensitive proteins altered by p66Shc mediatedROSformation were identified. Of …

Characterization Of The Circadian Properties Of Runt-Related Transcription Factor 2 (Runx2) And Its Role In The Suprachiasmatic Nucleus, Meghan E. Reale

Electronic Thesis and Dissertation Repository

Circadian rhythms orchestrate physiological, behavioral and cognitive processes in order to anticipate and adapt organisms to key environmental cues. These endogenously driven oscillations are generated by a network of interlocked auto-regulatory transcriptional-translational feedback loops driven forward by the Bmal1/Clock heterodimer transcription factor. Given the ubiquitous and dynamic quality of circadian rhythms, the identification of factors involved in the coordination and regulation of the endogenous oscillations is central in broadening our understanding of biological timing systems. In an examination of gene expression in the mammalian central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), revealed a previously unreported rhythmic expression of runt-related …

Functional Significance Of Ilk-Elmo2 Interactions In Epidermal Keratinocytes, Ernest Ho

Electronic Thesis and Dissertation Repository

The epidermis consists of a basal layer of undifferentiated keratinocytes and multiple suprabasal layers of differentiated keratinocytes. Undifferentiated keratinocytes are adherent and highly motile, whereas differentiated keratinocytes are comparatively less motile and downregulate cell attachments to the extracellular matrix. Integrin-linked kinase (ILK) is a scaffold protein implicated in the regulation of many cellular functions in keratinocytes, including cell attachment, migration, phagocytosis, and protein trafficking. To determine the mechanisms by which ILK is involved in these processes, I sought to identify other proteins which may interact with ILK in keratinocytes. I identified Engulfment and Cell Motility 2 (ELMO2) to interact with …

Timp-2 Decreases The Invasive Potential Of Mcf-7 And Mdamb-231 Cells Independent Of Mmp Inhibition, Mario Cepeda

Electronic Thesis and Dissertation Repository

Tissue Inhibitors of Metalloproteinases (TIMPs) are the natural inhibitors of Matrix Metalloproteinases (MMPs), a family of proteins primarily responsible for Extracellular Matrix (ECM) remodeling. TIMP-2 is a special member of the TIMP family as it is both an inhibitor and promoter of MMP activity, and can also bind the cell surface and signal inside the cell to influence cell behavior. In this study, MDAMB-231 and MCF-7 breast cancer cells were treated with physiological concentration of TIMP-2, which decreased the invasive potential of both cell lines. This was independent of MMP inhibition, and instead a decrease in the expression and secretion …

Phenotypic Analysis Of Schizosaccharomyces Pombe Strains Bearing Site-Directed Mutations In The Carboxy Terminal Domain Of The Largest Subunit Of Rna Polymerase Ii, Kyle S. Hoffman

Electronic Thesis and Dissertation Repository

The phosphorylation status of the largest sub-unit of RNA polymerase II (Rpb1p) is crucial to the control of transcription in eukaryotes. The domain subject to this phosphorylation is known as the carboxyl terminal domain (CTD) and consists of multiple repeats (from 20 to 52 copies depending on the species in question) of the heptad sequence Y₁S₂P₃T₄S₅P₆S_7. Interestingly, differential phosphorylation of S₂, S₅, and S₇ residues is known to play an important role in the control of pre-mRNA processing. To determine the number …

The Effects Of Telomerase Inhibition On Cellular Senescence In Fetal Guinea Pig Muscle Cells, Stephanie E. Hallows

Electronic Thesis and Dissertation Repository

Low birth weight infants have a higher risk of developing metabolic syndrome, including Type II Diabetes. Fetal muscle is growth restricted in low birth weight infants and is the main tissue for determining insulin resistance. Recent studies indicate premature senescence in low birth weight rodents, which could lead to adult disease. In utero environments may play a role in the development of senescence through increased reactive oxygen species (ROS). Telomerase is present at high levels during development and protects cells from cellular stress and apoptosis. We postulate that telomerase protects cells from DNA damage and premature senescence. A primary culture …

The Embryonic Protein Nodal Supports Metastatic Phenotypes In Breast Cancer, Daniela F. Quail

Electronic Thesis and Dissertation Repository

Metastasis is the process by which tumour cells disseminate to distant organ sites. Aberrant expression of stem cell-associated proteins within tumours is associated with metastasis and poor patient prognosis. One example of a stem cell factor that is associated with cancer progression is Nodal, a member of the TGF-β superfamily. Nodal is normally limited to pluripotent stem cells during embryonic development, and to specialized dynamic adult tissue (such as the cycling endometrium), but is aberrantly re-expressed in multiple cancer types, including melanoma, glioma, prostate cancer, and pancreatic cancer. The central objective of this thesis is to determine the role of …

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. …

Create The Scene And Watch The Show Unfold: Following Vegetative To Embryonic Developmental Transitions By Over-Expressing Lec2 In Leaves Of Arabidopsis Thaliana, Mistianne Feeney

Electronic Thesis and Dissertation Repository

During seed development, the lytic vacuole (LV) is replaced by a protein storage vacuole (PSV) which specializes in accumulating seed storage proteins (SSPs). As seed protein reserves are mobilized upon germination, the PSV is once again replaced by the LV which takes on different roles in vegetative tissues. Cellular events occurring during these developmental transitions are not well understood, particularly, the transition between vacuole types. This research investigates whether PSVs can exist in leaves. To study vacuole transitions in leaves, an Arabidopsis thaliana line over-expressing the LEAFY COTYLEDON2 (LEC2) transcription factor was used. LEC2 is a master regulator of embryogenesis …

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 …

Modulation Of G Protein-Coupled Receptor Intracellular Trafficking And Signal Transduction, Jessica L. Esseltine

Electronic Thesis and Dissertation Repository

The seven transmembrane-spanning G Protein-coupled Receptor (GPCR) super family is the largest family of cell-surface receptors, comprising greater than 650 members. GPCRs represent the primary targets of most therapeutic drugs. Desensitization, endocytosis and recycling are major mechanisms of receptor regulation and intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. In the present study, we examined whether multiple Rab GTPase regulate receptor trafficking through endosomal cellular compartments as a consequence of their direct association with GPCRs. We find that Rab4, Rab7 and Rab11 all bind to the last 10 amino acid residues of the angiotensin …

The Role Of Pex11-Beta In Peroxisome Biogenesis, Intracellular Relationship To Reactive Oxygen Species Levels And Redox-Sensitive Cell Signaling, Mark A. Fox

Electronic Thesis and Dissertation Repository

Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species (ROS) elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms to peroxisome functioning, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as …

Molecular Mechanisms Of Cell Migration Inhibition By Synthetic Triterpenoids, Ciric Chi Wing To

Electronic Thesis and Dissertation Repository

Cell migration is an important mediator of cancer metastasis and invasion, which is responsible for 90% of cancer-related premature deaths in Canada. Synthetic triterpenoids are a class of promising anti-cancer compounds that have shown considerable efficacy in targeting various cellular functions including apoptosis, growth, inflammation and cytoprotection in both cell culture and animal tumor models. However, their effect on cell migration, an important event in metastasis, remains poorly understood. This thesis focuses on deciphering the molecular mechanisms whereby the synthetic triterpenoids affect cell migration. I observed that the imidazolide and methyl ester derivatives of the synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic aic acid …

The Intracellular Behaviour Of Burkholderia Cenocepacia In Murine Macrophages, Jennifer S. Tolman

Electronic Thesis and Dissertation Repository

Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in cystic fibrosis and other immunocompromised patients. The bacterium survives within macrophages by interfering with typical endocytic trafficking, resulting in delayed maturation of a B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia alters gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. Furthermore, we hypothesize that specialized bacterial secretion systems are involved in the interactions between intracellular bacteria and macrophages. In this work, we characterize later-stage infection of macrophages by B. cenocepacia, showing replication within an acidified endosomal compartment suggestive of a phagolysosome. …

Brassinosteroid-Mediated Stress Tolerance In Arabidopsis Thaliana, Tawhidur Rahman

Electronic Thesis and Dissertation Repository

Brassinosteroids (BRs) are a group of steroidal plant hormones that are essential for proper plant development and also promote stress tolerance. Without BRs, plants are dwarfs and infertile. To understand the molecular mechanisms underlying BR-mediated stress tolerance, global gene expression analysis of untreated and 24-epibrassinolide (EBR)-treated Arabidopsis thaliana seedlings under non-stress and heat stress (HS) conditions was carried out. Microarray data analysis indicated that stress-related genes were predominant within the EBR up-regulated gene data set. Furthermore, several of these genes were abscisic acid (ABA) and jasmonic acid (JA) related. Measurements of endogenous hormones showed significant increases in the levels ABA …

The Regulation Of Cell Division And Neurogenesis By The Chromatin Remodeling Protein Atrx, Kieran L. Ritchie

Electronic Thesis and Dissertation Repository

Mutations in the ATRX gene cause alpha thalassemia mental retardation X-linked syndrome (ATR-X). Individuals with ATR-X syndrome show a broad range of developmental abnormalities including severe cognitive, behavioural, and learning deficits, seizures, and microcephaly, indicating that ATRX is essential for normal development of the central nervous system. Conditional inactivation of Atrx in the developing mouse forebrain leads to a reduction in cerebral cortical size, elevated levels of p53-dependent neuronal apoptosis, and dysgenesis of certain subcortical structures, including the hippocampus and dentate gyrus, confirming a requirement for Atrx in mammalian brain development. The mammalian ATRX gene encodes a member of the …

The Life Cycle Of Pannexins: Trafficking, Cell Surface Dynamics, Turnover And Degradation, Ruchi Gehi

Electronic Thesis and Dissertation Repository

Pannexins (Panxs) are a recently identified family of channel-forming glycoproteins, comprised of three members: Panx1, Panx2 and Panx3. Panxs were classified as gap junction (GJ) proteins based on their shared sequence homology to the invertebrate GJ protein, innexins. While no sequence homology exist between Panxs and the GJ proteins, connexins (Cxs), they share similar topology with four transmembrane domains, two extracellular loops, an intracellular loop and cytoplasmic exposed amino and carboxy terminal tails. In this study, we investigated if Panx1 and Panx3 exhibit unique or shared properties of cell surface delivery, mobility, cytoskeletal dependency, internalization and degradation, to the well …

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 …