Cell and Developmental Biology Commons^™

The Role Of Bone Sialoprotein In Periodontal Tissue Development And Bone Repair, Yohannes Soenjaya

Electronic Thesis and Dissertation Repository

Bone development and repair involve complex processes that include interaction between cells and their surrounding matrix. In the body, bone sialoprotein (BSP) expression is up-regulated at the onset of mineralization. BSP is a multifunctional acidic phosphoprotein with collagen-binding, hydroxyapatite nucleating, and integrin recognition (RGD sequence, which is important for cell-attachment and signaling) regions. Mice lacking BSP expression (Bsp^-/-), exhibit a bone phenotype with reductions in bone mineral density, bone length, osteoclast activation, and impaired bone healing. This thesis examined the role of BSP in tooth development and also its potential use as a therapeutic reagent for bone …

Investigation Of Cell-Penetrating Peptide Transformation In Two Regenerable Tissue Culture Systems, Atiyyah S. Ferouz

Electronic Thesis and Dissertation Repository

The genetic engineering of plants allows for the modification of plant genomes, subsequently improving plant traits. There are, however, plants recalcitrant to established transformation methods, requiring the implementation of novel techniques. This study investigates the viability of cell-penetrating peptide (CPP) transformation using Tat₂, a CPP with demonstrated transformation efficiency in plant and animal systems, in two regenerable tissue culture systems, soybean somatic embryos and Arabidopsis protoplasts. Assessments of complex formation, Tat₂ cellular translocation, complex uptake, and CPP-mediated transformation were carried out. The results show the formation of a Tat₂-plasmid complex and the uptake of Tat …

Hd2d Is A Regulator Of Abscisic Acid Responses In Arabidopsis, Joshua A. Farhi

Electronic Thesis and Dissertation Repository

Histone deacetylases have important roles in development and stress response in plants. To further investigate their function, the HD2D gene, of the plant specific HD2 family, was studied. An hd2d-1 mutant and two HD2D overexpression lines were used in this study. Germination was delayed in hd2d-1 and HD2D overexpression seeds only in the presence of ABA. HD2D was found to positively regulate the expression of members of the ABA-response pathway (ABI1, ABI5, and RD29A) leading to increased resistance to drought and salinity treatments. Furthermore, HD2D expression delayed flowering by positively regulating FLC expression. Using bimolecular fluorescence complementation, the HD2D protein …

Mechanisms Of Neuroprotection Against Ischemic Insult By Stress-Inducible Phosphoprotein-1, Jason Xu

Electronic Thesis and Dissertation Repository

Stress-inducible phosphoprotein-1 (STI1) levels are increased in the brain following ischemia. STI1 is a co-chaperone for Hsp70/Hsp90 modulating protein folding. STI1 can also be secreted by a number of cells and function to activate extracellular signalling by the prion protein (PrP^C) and type-I bone morphogenetic protein receptor ALK2. However, the mechanisms by which STI1 can protect neurons against ischemia are currently unknown. A caspase-3 reporter mouse line was used to evaluate the consequences of increased extracellular STI1 levels. Neurons were treated with recombinant STI1 and specific agonists/antagonists for PrP^C, α7nAChR, and ALK2 prior to oxygen-glucose deprivation …

Cardiomyocyte Polarity And Embryonic Heart Development: Role Of Rac1, Carmen Leung

Electronic Thesis and Dissertation Repository

Congenital heart defects (CHDs) are the most common human birth defect and the leading cause of death from a birth defect in the first year of life. Thus, a further understanding of the mechanisms underlying CHDs, which could lead to improved diagnosis and treatment, is crucial. The small GTPase, Rac1, acts as a pleiotropic effector of numerous cellular processes; however, little is known about its role in embryonic heart development. The aim of this thesis was to investigate the role of Rac1 signaling in cardiac development. Using the Cre/loxP system, mouse models with an anterior second heart field (SHF) or …

Application Of 3d Printing Technology In Porous Anode Fabrication For Enhanced Power Output Of Microbial Fuel Cells, Bin Bian

Electronic Thesis and Dissertation Repository

Microbial fuel cells (MFCs) are widely researched for application in wastewater treatment. However, the current anodes used in MFCs often suffer from high fabrication cost and uncontrollable pore sizes. In this thesis, three-dimensional printing technique was utilized to fabricate anodes with different micro pore sizes for MFCs. Copper coating and carbonization were applied to the printed polymer anodes to increase the conductivity and specific surface area. Voltages of MFCs with various anodes were measured as well as other electrochemical tests such as linear sweep voltammetry and electrochemical impedance spectroscopy. 3D copper porous anode produced higher maximum voltages and power densities …

Wnt Signalling During F9 Cell Differentiation, Gregory M. Golenia

Electronic Thesis and Dissertation Repository

Mouse F9 cells differentiate into primitive endoderm (PrE) when treated with retinoic acid (RA) and this is accompanied by the upregulation of Wnt6 and activation of the canonical WNT/β-catenin pathway. Previous studies have demonstrated the necessity of β-catenin-TCF/LEF transcription for primitive endoderm differentiation, however the Frizzled (FZD) receptor responsible for binding WNT6 and activating the canonical WNT pathway is not known. It was hypothesized that FZD7 is responsible for binding and transducing the WNT6 signal. Fzd7 mRNA was detected in undifferentiated and primitive endoderm cells, and its expression does not change significantly in response to RA. Moreover and contrary to …

Characterizing The C-Terminal Region Of Human Adenovirus E1a: An Undiscovered Country, Michael J. Cohen

Electronic Thesis and Dissertation Repository

Human Adenovirus (HAdV) E1A is the first protein expressed during viral infection. The primary function of E1A is to reprogram the cell for viral replication, but it is additionally capable of transforming primary rodent cells in co-operation with other oncogenes such as HAdV E1B. Despite extensive study, little is known about the function and cellular targets of the C-terminal region of E1A. Importantly, this region is required for the transforming ability of E1A with E1B, but can also suppress transformation with Ras. Previous studies showed that interaction with the C-terminal Binding Protein (CtBP) plays a role in both functions described …

Clinical And Experimental Importance Of Circulating Tumor Cells In Prostate Cancer, Lori Lowes

Electronic Thesis and Dissertation Repository

Prostate cancer (PCa) remains a leading cause of death in men, primarily due to ineffective treatment in the metastatic setting. During this phase of PCa, circulating tumor cells (CTCs) are shed into the bloodstream and their presence and number are important in patient prognosis. The CellSearch® system (CSS) is the only U.S. Food and Drug Administration (FDA) and Health Canada approved instrument for detection of CTCs, making it the current clinical gold standard in CTC technology. Although the CSS provides a minimally invasive means of patient monitoring in the metastatic setting, little is known about the role of CTCs in …

Investigating Hox Protein Requirement For Tarsus Determination In Drosophila Melanogaster, Samantha Koot

Electronic Thesis and Dissertation Repository

Generally, all bilaterans examined have similar conservation of HOX protein structure, function, expression, and requirement. However, at the level of being the same, it is unknown whether the HOX protein, Antennapedia, is required for tarsus determination in Drosophila melanogaster as in Tribolium casteneum, or whether the requirement of HOX proteins in determination of body parts diverges in insects. I proposed to use a heat shock-inducible nanobody (UAS- NSlmb-vhhGFP4 driven by hsp-GAL4) activated during the third larval stage in all cells to degrade thoracically expressed HOX proteins (Sex combs reduced, Antennapedia, and Ultrabithorax) tagged with green fluorescence protein …

The Role Of Connexins And Pannexins In Mammary Gland Development And Tumorigenesis, Michael K. G. Stewart

Electronic Thesis and Dissertation Repository

The identification of key regulators of breast cancer onset and progression is critical for the development of targeted therapies. Connexins and pannexins are characterized by their ability to form large-pore channels and are frequently dysregulated in cancer. However, their role in breast cancer progression remains poorly understood due to a lack of in vivo models capable of assessing the proposed and opposing roles of connexins and pannexins as both tumor suppressors and/or facilitators in multiple stages of the disease. Using 2 previously uncharacterized genetically-modified mice, connexin43 (Cx43) and connexin26 (Cx26) were evaluated for their role in normal mammary gland development …

Maternal Nutrient Restriction In Pregnant Guinea Pigs Impacts Fetal-Placental Growth And Oxygenation, Alexander Elias

Electronic Thesis and Dissertation Repository

Maternal nutrient restriction (MNR) in guinea pigs results in placental structural abnormalities that reduce nutrient transport contributing to fetal growth restriction (FGR). However, whether fetal oxygenation is also reduced as a further mediator of altered growth and development remains unknown. Guinea pig sows were fed ad libitum (Controls) or 70% of the control diet pre-pregnant switching to 90% at mid-pregnancy (MNR). Animals were necropsied near term for fetal-placental growth measures, blood metabolites, and markers of tissue hypoxia and oxidative stress. MNR resulted in FGR with brains that are large and livers that are small relative to body weight which suggests …

Purification Of Recombinant Proteins In Plants Using Small-Molecule Dependent Inteins Fused To Elp Or Hfbi, Kira Liu

Electronic Thesis and Dissertation Repository

Elastin-like polypeptides (ELP) and hydrophobins (HFBI) are fusion tags which enhance the accumulation levels of recombinant proteins in plants and aid in the non-chromatographic purification of these proteins. An intein is inserted between the target protein and fusion tag to replace the protease cleavage site. In this study, transient expression of intein-GFP fused to ELP or HFBI in Nicotiana benthamiana yielded the full size fusion protein. The ELP-fused proteins were successfully purified using inverse transition cycling (ITC) with 1.0 M NH4(SO4)2 at room temperature. However, the purification using membrane ITC was unsuccessful due to clogged membrane pores. Purification of HFBI-fused …

Characterization Of The Nicotine-Induced Endoplasmic Reticulum Stress Response In The Rat Placenta In Vivo And In Vitro, Michael Ka Chun Wong

Electronic Thesis and Dissertation Repository

Nicotine exposure during pregnancy leads to adverse health outcomes, including compromised placental development. Although the molecular mechanisms remain elusive, recent studies identified that endoplasmic reticulum (ER) stress may underlie poor placentation. Therefore, we were interested in investigating the effects of nicotine exposure on the ER stress response in the placenta. A well-established maternal nicotine exposure rat model and Rcho-1 trophoblast giant cell model were utilized to address the research questions. Maternal nicotine exposure in vivo led to elevated ER stress in association with impaired disulfide bond formation and hypoxia. Nicotine exposure in vitro further differentiated that ER stress may be …

Analyzing A-Series Gangliosides In Neurons Following Exposure To Glutamate, Dae Hee Park

Electronic Thesis and Dissertation Repository

Neurons within different brain regions have varying levels of vulnerability to external stress and therefore respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell’s plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) technique and immunohistochemistry to evaluate the temporal changes in the expression profiles of various ganglioside species during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic …

Investigation Of Pancreatic Β-Cell Insulin Receptor Regulation Of Β-Cell Growth, Function, And Survival Via A Temporal Conditional Knockout, Liangyi Zhou

Electronic Thesis and Dissertation Repository

The expression of insulin receptor (IR) in β-cells suggests an autocrine role for insulin signalling in β-cell function and regulation. Studies have demonstrated that β-cell Ir knockout (βIrKO) mice develop age-dependent glucose intolerance. We investigated the temporal role of β-cell IR signaling in pre- and postnatal islet development and function, and under high-fat diet stress, using a tamoxifen-inducible Cre-recombinase Ir knockout mouse model.

Prenatal βIrKO mice exhibited increased mean islet area, β-cell area, and islet area percentage. Additionally, there was upregulation of insulin-like growth factor-2 levels, increased Akt activity, and increased proliferation in islets. Postnatally-induced βIrKO mice did not exhibit …

The Ctcf Chromatin Organizer Is Required For Hindlimb Development, Katherine L. Rabicki

Electronic Thesis and Dissertation Repository

Mutations in chromatin organizer CTCF were identified in patients with intellectual disability and skeletal defects. Previous studies demonstrated that depletion of CTCF in murine limb mesenchyme results in apoptosis in the forelimb. The role of CTCF in the hindlimb, however, is unknown. My objective was to investigate effects of CTCF deletion on chondrogenesis and skeletal development in the hindlimb. In vitro wild-type micromass cultures demonstrate that chondrocyte gene expression is delayed in the hindlimb when compared to forelimbs. Embryonic Ctcf^Fl/Fl;Prx1Cre mice were investigated, and qRT-PCR and histology were performed on limb buds and long bones. Results show that …

Underlying Mechanisms Regulating Gingival Healing And Fibrosis, Shawna Soheun Kim

Electronic Thesis and Dissertation Repository

Successful wound healing involves tightly controlled regulation of extracellular matrix (ECM) deposition and remodeling, and myofibroblast-mediated wound contraction, to restore normal tissue function. However, these events involved in healing are also associated with development of tissue fibrosis, which is characterized by excessive deposition of ECM and the presence of myofibroblasts. Increasing evidence suggests that periostin is a critical player in regulating skin healing and fibrosis. In contrast to most adult tissues, gingival connective tissue is known to heal more rapidly and with less scarring, yet is still associated with fibrosis, such as drug-induced gingival enlargement. The aim of this study …

The Lung Microenvironment Influences The Metastatic Behaviour Of Breast Cancer Cells In An Innovative 3d Ex Vivo Pulmonary Metastasis Model, Matt M. Piaseczny

Electronic Thesis and Dissertation Repository

Lung metastasis remains a leading cause of death in breast cancer patients. This study established an innovative 3D ex vivo pulmonary metastasis assay (PuMA) to test the hypothesis that the lung microenvironment promotes metastatic behaviour of whole population and stem-like ALDH^hiCD44⁺ breast cancer cells. Following in vivo delivery of breast cancer cells to mice, lungs were excised, maintained in culture and imaged to observe breast cancer growth over time. We observed metastatic progression of breast cancer cells in the PuMA, most notably of ALDH^hiCD44⁺ cells which progressed rapidly from single cells to multicellular colonies …

Investigating The Role Of Hydrogen Sulfide In The Survival, Growth And Angiogenic Potential Of Clear Cell Renal Cell Carcinoma Cell Lines And Xenografts, Eric Sonke

Electronic Thesis and Dissertation Repository

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel-Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H₂S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H₂S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism, and angiogenesis. Using H₂S-specific probes, it was found that H₂S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H₂S-producing enzymes could reduce the proliferation, metabolism …

Investigating The Fibroblastic Origin Of Skin-Derived Precursors And The Role Of Ccn2 As A Mediator Of Myofibroblastic Differentiation, Matthew Tsang

Electronic Thesis and Dissertation Repository

Skin-derived precursors (SKPs) are defined as multipotent spheroid-forming cells cultured from the dermis that express markers of neural crest origin. Recent evidence suggests that tissue fibrosis can occur through the differentiation of progenitor cells into smooth muscle-like myofibroblasts. CCN2, a marker and mediator of fibrosis, is highly expressed during myofibroblast differentiation and is required for skin fibrogenesis. Here, I clarify the cellular origin of SKPs, and the molecular contribution of CCN2 in the myofibroblastic differentiation of SKPs. Using lineage tracing, I show that SKPs originate primarily from Col1a2-expressing dermal fibroblasts. Furthermore, I show that differentiation of SKPs into myofibroblasts requires …

Diabetic Bone Marrow & Stem Cell Dysfunction, Meghan A. Piccinin

Electronic Thesis and Dissertation Repository

ii Abstract Abstract Defects in the proliferation, differentiation, and activity of bone marrow (BM)-derived vasculogenic/vascular stem cells (VSCs) have been observed in diabetes and contribute to the development of vascular complications. Diabetes leads to enhanced bone marrow adipogenesis, altering the composition of the BM stem cell (SC) niche and potentially disrupting the normal functioning of resident VSCs. Here, I establish that adipocytes have a negative influence on SC survival in culture. I also show that adipocytes and osteoblasts are responsible for the creation of distinct extracellular microenvironments, with unique expression patterns of several pro- and anti-angiogenic factors with known effects …

Translesion Synthesis And Mutations: On The Mutagenic Properties Of The Two Dna Lesions, 8-Oxo-G And Pt-Gg, And The Functions Of Y-Family Dna Polymerases And Rev3l On The Bypass Of Each Of The Dna Lesions In Mammalian Cells, Lizhen Guo

Electronic Thesis and Dissertation Repository

I studied the capabilities of the two DNA lesions 8-oxo-guanine and cisplatin intrastrand crosslinked 1,2-d(GpG) or Pt-GG to cause mutations in mammalian cells. Using isogenic cell lines generated from mice with selective gene knockouts of distinct DNA polymerases as models, I deduced the biological functions of the translesion DNA polymerases Pol eta, Pol kappa, Pol iota, Rev1 and Rev3L on bypassing each of the lesions 8-oxo-G and Pt-GG. My study takes advantage of the Next Generation Sequencing (NGS) technology to determine mutagenic effects of the DNA lesions in vivo and effects of translesion DNA polymerases on bypassing the lesions. Through …

Atrx Loss-Of-Function In Mouse Neuroprogenitor Cells As A Model Of Early Events In Gliomagenesis, Hannah E. Goldberg

Electronic Thesis and Dissertation Repository

ATRX is a chromatin remodeling protein important for neural development, and ATRX inactivation leads to genomic instability, mitotic defects and TP53-mediated apoptosis. In the last few years, ATRX mutations were identified in a large proportion of paediatric and adult gliomas that often coincide with mutations in the tumor suppressor TP53. The present work shows that combinatorial loss of ATRX and TP53 function in vitro causes genomic instability while improving cell viability, identifying potential early events in gliomagenesis. Furthermore, several gene transcripts associated with glioma development and known oncogenic pathways were significantly upregulated in the Atrx-null neonatal mouse forebrain. …

Soybean Isoflavonoid Biosynthesis: Constituents And Circumstance At The Transcriptomic And Molecular Levels, Mehran Dastmalchi

Electronic Thesis and Dissertation Repository

Isoflavonoids are specialized metabolites, almost exclusive to the legume family of plants. They are actors in symbiosis with nitrogen-fixing bacteria and in plant stress response. Isoflavonoids are noted for their human health benefits. Isoflavonoid content in legumes has proven to be a complex trait. The goal of the present research is to determine the mechanisms underlying isoflavonoid biosynthesis in soybean.

The first approach was to unravel the genetic factors of isoflavonoid biosynthesis. A branch-point enzyme of the phenylpropanoid pathway, chalcone isomerase (CHI), catalyzes the reaction producing flavanones, the nucleus for many downstream metabolites such as isoflavonoids. I identified twelve soybean …