Life Sciences Commons^™

The Atp2c2 Gene As Transcribed From A Novel Transcriptional Start Site In Pancreatic Acinar Cells, Caitlin M. Sullivan

Electronic Thesis and Dissertation Repository

Strict regulation of cytosolic Ca²⁺ is essential to regulated exocytosis and proper pancreatic acinar cell function, controlled in part by pumps that shuttle Ca²⁺ out of the cytosol. Our laboratory identified a novel isoform of Secretory Ca²⁺ ATPase 2 (SPCA2) containing only the carboxy terminus. Pancreatic SPCA2, is an approximately 17-20 kDa, protein encoded by the Atp2c2 gene and is completely absent in Mist1^-/- acini.. The focus of this thesis was to understand transcriptional regulation of Atp2c2 in the pancreas. Pancreatic Atp2c2 appears to be transcribed from an alternative transcriptional start site (TSS) and is regulated …

Prenatal Programming Of Hepatic Glucose And Cholesterol Regulation In Male Rat Offspring By Chronic Intermittent Hypoxia, Waseem Iqbal

Electronic Thesis and Dissertation Repository

Obstructive sleep apnea (OSA) is a chronic disorder involving repetitive interruptions in breathing during sleep. Sufferers of OSA are exposed to chronic intermittent hypoxia (CIH), characterized by cyclical reductions in oxygen availability. A number of studies have established a link between OSA and various cardiovascular and metabolic comorbidities in adulthood, including hypertension, obesity, and type II diabetes. While the consequences of OSA in adults have been well described, the cross-generational impact of this condition and potential effects on fetal development are not known. Epidemiological and animal studies have demonstrated that physiological insults during pregnancy lead to diminished growth of offspring …

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 …

Developmental Characterization Of Tissue Inhibitor Of Metalloproteinase Domain Functions In Xenopus Laevis, Michelle A. Nieuwesteeg

Electronic Thesis and Dissertation Repository

During development the extracellular matrix is cleaved and remodeled to facilitate the large-scale cell rearrangements that are necessary for processes like gastrulation, neurulation, angiogenesis and organogenesis. ECM remodeling occurs primarily through secreted enzymes called matrix metalloproteinases (MMPs). Regulation of MMP activity is achieved through the tissue inhibitor of metalloproteinases (TIMPs), a small family of secreted proteins that bind MMPs in a 1:1 manner to inhibit their activity. Although TIMPs were originally characterized based on their MMP-inhibitory activities, in vitro studies have revealed that TIMPs are multifunctional proteins, with structurally and functionally distinct N- and C-terminal domains. TIMP N-terminal domains bind …

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 …

Cx43 Reduces Melanoma Growth Within A Keratinocyte Microenvironment And During Tumorigenesis In Vivo, Mark J. Ableser

Electronic Thesis and Dissertation Repository

Connexins have been frequently identified as tumor suppressors in many cancers, however, their role in melanoma tumorigenesis remains controversial. Here, we show that B16-BL6 mouse melanoma cells express low levels of Cx26 and Cx43, rendering them gap junctional intercellular communication (GJIC) deficient. Following ectopic expression of Cx26 and Cx43, gap junction-like plaques were evident at the cell surface and the incidence of dye transfer was significantly increased similar to connexin-rich keratinocytes. The expression of Cx43, but not Cx26, significantly reduced proliferation and anchorage-independent growth relative to controls, whereas migration was unaffected. Additionally, Cx43-expressing melanoma cells displayed significantly reduced growth amongst …

Shroom3 Deficient Mice Show Congenital Heart Defects, Rami R. Halabi

Electronic Thesis and Dissertation Repository

Congenital heart defects (CHDs) are associated with a number of genetic and environmental risk factors affecting approximately 1% of newborns. Shroom3 is an actin binding and microtubule organizing protein essential for neural tube closure in mouse, Xenopus and chick. In Xenopus shroom3 expression is found within the forming heart and loss of activity results in malformed hearts. In addition, SHROOM3 has recently been associated with heterotaxy in a human patient. Mice homozygous for the Shroom3 gene trap die at birth due to exencephaly and here, I provide evidence that the majority of these mice have CHDs, including septal defects, semilunar …

Characterization Of A Putative Activation Domain In The Hulk Gene Family, Christopher Doan

Electronic Thesis and Dissertation Repository

The HULK gene family participates in regulation of both flowering time and development in the plant Arabidopsis thaliana. The proteins encoded by these genes share conserved domain structures including a proline-rich region (PRR) in the carboxyl-terminus. Based on sequence analysis and the presence of a proline-rich domain, it has been suggested that the HULKs are putative transcription factors in which HUA2 is known to regulate several late-flowering genes: FLC, FLM and MAF2.

To investigate the putative transcriptional activation domain in the carboxyl-terminus of the HULKs, full-length HULKs and deletion constructs were 3-AT titrated in yeast-one hybrids. It …

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 …

Characterizing The Role Of The Retinoblastoma Protein Lxcxe Binding Cleft In Cellular Senescence And Tumor Suppression, Srikanth Talluri

Electronic Thesis and Dissertation Repository

The Retinoblastoma protein (pRB) is a key regulator of the cell cycle and is functionally inactivated in most cancers. pRB has been proposed to utilize simultaneous interactions with E2F transcription factors and chromatin regulatory proteins to repress transcription and block cell cycle progression. The goal of this study is to characterize the physiological role of pRB interactions with chromatin regulatory proteins. I used gene targeted mice carrying point mutations in the murine Rb1 gene (Rb1^∆L) that specifically disrupt pRB’s LXCXE binding cleft, and thereby its ability to interact with chromatin regulatory proteins while leaving its ability to …

Features Of Dormancy In Metastatic Ovarian Cancer Cells, Rohann Jm Correa

Electronic Thesis and Dissertation Repository

The most prevalent subtype of ovarian cancer – high-grade serous (HGS) carcinoma – is also the most lethal, since the majority of cases are characterized by advanced-stage (metastatic) presentation. Metastasis of this cancer proceeds by an intra-peritoneal route, involving detachment of cells from the primary tumour and dissemination throughout the peritoneal cavity as multicellular aggregates, or spheroids. Herein, we demonstrate that HGS patient-derived tumour cells cultured to form in vitro spheroids exhibit features of cancer dormancy, a cellular state known to promote therapeutic resistance and disease recurrence. We discovered that upon spheroid formation, cells became non-proliferative, exhibiting a cell cycle …

Molecular Mechanisms Underlying The Early Life Programming Of The Liver, Gurjeev Sohi

Electronic Thesis and Dissertation Repository

Clinical studies have demonstrated that intrauterine growth restriction (IUGR) offspring, faced with a nutritional mismatch postpartum, have an increased risk of developing the metabolic syndrome. The maternal protein restriction (MPR) rat model has been extensively studied to investigate the adverse effects of a nutritional mismatch in postnatal life of IUGR offspring. Previous studies have demonstrated that MPR leads to impaired function of the liver, an important metabolic organ. However the underlying mechanisms which predispose these offspring to the metabolic syndrome remain elusive. In the following studies, low protein diet during pregnancy and lactation led to IUGR offspring with decreased liver …

The Chromatin Remodeling Protein Atrx In Development And Maintenance Of Mouse Skeletal Tissues, Lauren A. Solomon

Electronic Thesis and Dissertation Repository

Alpha-thalassemia X-linked mental retardation (ATR-X) syndrome is a rare genetic disorder associated with severe developmental delay, mental retardation and craniofacial dysmorphism. This syndrome is caused by mutations in the ATRX gene which encodes a member of the SWI/SNF family of chromatin remodeling proteins. ATR-X patients exhibit dwarfism and skeletal defects, including hand and foot deformities. I hypothesized that the skeletal deformities in ATR-X syndrome are due to a direct role of ATRX in the development of the skeleton. My objective was to characterise skeletal phenotypes observed in three animal models conditionally deficient for ATRX in different skeletal tissues. Mice lacking …

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 …

Elucidating The Role Of Menin During Islet Cell Development In The Human Fetal Pancreas, Jessica L. Dubrick

Electronic Thesis and Dissertation Repository

Studies show that Menin, a tumour suppressor encoded by the multiple endocrine neoplasia type 1 (Men1) gene, is required during murine pancreatic development. In humans, mutation results in the MEN1 tumourigenic syndrome; however, knowledge of menin in human pancreatic development is limited. This study examined the expression pattern and functional role of menin during human fetal islet development. Immunostaining revealed the presence of nuclear menin within pancreatic cells from the 1^st through 2^nd trimester co-localizing with various developmental factors. Knockdown of MEN1 in human fetal islet-epithelial cells significantly increased apoptosis, reduced proliferation, and decreased SOX9, NKX2.2, …

Investigating The Interplay Between Protein Kinases And Caspases, Jacob P. Turowec

Electronic Thesis and Dissertation Repository

The balance between cell survival and death is a crucial process in human development and tissue homeostasis, but is also misregulated in disease. In large part, apoptosis is controlled by caspases, a hierarchical series of cysteine aspartic acid proteases that demolish the cell by cleaving key structural and enzymatic proteins, but emerging paradigms have highlighted the ability of kinases to regulate caspase activity. One way in which kinases can control the progression of apoptosis is through phosphorylation of caspase substrates, which acts to prevent caspase cleavage of that target.

In this thesis, we develop new strategies to study this regulatory …

Functional Characterization Of The Hua2 Gene Family In Arabidopsis Thaliana, Preetam Janakirama

Electronic Thesis and Dissertation Repository

HUA2 encodes a key developmental regulatory protein implicated in the coordination of induction and the maintenance of floral state in Arabidopsis. To gain further insight into the function of HUA2, I have conducted a series of studies aimed at elucidating the molecular function(s) of its individual domains. I show that the PWWP, RPR and CT-proline rich domains within HUA2 are required for the proper regulation of the flowering time phenotype. I also establish that HUA2 interacts with characterized splicing factors (FCA, AtPRP40, RBP45 and UBP1) through its CT-proline rich domain. In addition, I examine the overlap in function between HUA2 …

Sunergos1, A Lotus Japonicus Gene Required For Proper Accommodation Of Rhizobial Infection, Hwi Joong Yoon

Electronic Thesis and Dissertation Repository

Here, I characterize a symbiotic mutant of Lotus japonicus, called sunergos1 (suner1), which originated from a har1-1 suppressor screen. I have shown that suner1 supports epidermal infection by rhizobia and initiates nodule primordia organogenesis as in wild-type. However, the infection process is temporarily stalled, such that infection threads fail to ramify within the root cortex and timely release of bacteria inside the nodule primordia cells does not occur. This symbiotic defect is ephemeral and with additional time, functional nodules are formed. Using a combined approach involving map-based cloning and next-generation sequencing, I have shown that the suner1 …

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 …

Nucleoredoxin And Wnt Signaling In F9 Cells, Leanne Sandieson

Electronic Thesis and Dissertation Repository

Nucleoredoxin is a redox sensitive protein recently shown to be involved in the Wnt/β-catenin pathway through binding dishevelled. When Wnt is present, dishevelled prevents a destruction complex from degrading β-catenin. This facilitates the translocation of β-catenin into the nucleus where it binds to TCF-LEF to impart changes in gene expression. This study used mouse F9 cells as a model to examine whether or not nucleoredoxin is involved in augmenting the Wnt/β-catenin signaling required for extraembryonic endoderm differentiation. It was found that nucleoredoxin and three dishevelled isoforms were present in F9 cells. Furthermore, nucleoredoxin was found to bind to dishevelled-2, and …

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 …

Cloning And Analysis Of Reck During Early Xenopus Laevis Development, Jessica Willson

Electronic Thesis and Dissertation Repository

Extracellular matrix (ECM) remodeling is crucial for the development and maintenance of multicellular organisms. Degradation of ECM components occurs through the activity of matrix metalloproteinases (MMPs) and their inhibitors. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene encodes a membrane-anchored protein and plays an important role in mediating ECM remodeling by inhibiting MMPs. To date, few in vivo studies exist examining RECK during development. The present study focuses on cloning and examining the expression of RECK during early Xenopus laevis development. A mature cDNA clone of the RECK gene was generated. RT-PCR, in situ hybridization, and immunohistochemistry were used to …

Characterization Of A Tumour Suppressor Function Of Ranbpm, Elnaz Atabakhsh

Electronic Thesis and Dissertation Repository

Ran-binding protein M (RanBPM) is an evolutionarily conserved nucleocytosolic protein that has been proposed to regulate various cellular processes, including protein stability, gene expression, receptor-mediated signalling pathways, cell adhesion, development, and apoptosis. Despite the multitude of functions attributed to RanBPM however, little is known regarding the precise mechanisms by which RanBPM executes these cellular roles. In this work, we seek to address this matter by describing functions for RanBPM in the regulation of apoptotic and pro-survival signalling pathways, and in cellular transformation.

We first identify RanBPM as a pro-apoptotic protein that regulates the activation of the intrinsic apoptotic signalling pathway …

Har1 And Ljamp1 Dependent Regulation Of Root Architecture In Lotus Japonicus, Chong Sung Kim

Electronic Thesis and Dissertation Repository

Here, I characterize a root-branching hypermorph of Lotus japonicus, called cluster root-like1 (crl1), which originated from a har1-1 suppressor screen. I have shown that the crl1 root phenotype is determined by two independently segregating recessive mutations, har1-1 and Ljamp1-1, with corresponding HAR1 and LjAMP1 encoding an LRR‑receptor-like kinase and a predicted homologue of the Arabidopsis ALTERED MERISTEM PROGRAM 1 protein, respectively. Unlike har1-1, the Ljamp1 mutation does not affect the symbiotic properties of L. japonicus Gifu but exerts a pleiotropic effect on shoot development. Root architecture, however, is regulated by a synergistic action of HAR1 …

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 …

Characterization Of A Negative Regulatory Domain In Fushi Tarazu, A Pair-Rule Protein In Drosophila Melanogaster, Elyse Susanne Burlingham

Electronic Thesis and Dissertation Repository

Fushi tarazu (FTZ) is a pair-rule protein important for the development of the anterior-posterior axis during embryogenesis. Low level ectopic expression of FTZ^Δ148-206 from a Tubulin α1 promoter, but not FTZ^1-410 (full length FTZ), results in the anti-ftz phenotype in developing Drosophila melanogaster larvae, indicating that FTZ^Δ148-206 is a hyperactive FTZ protein. Through deletion analysis, using a high level ectopic expression system and assaying survivorship, I narrowed the location of the negative regulatory domain (NRD) to the 178-206 amino acid region of FTZ. Mutations that mimic both constitutive phosphorylation and dephosphorylation in the …

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 …