Cell and Developmental Biology Commons^™

Rab35 Centered Membrane Trafficking Pathway Directs Apical Constriction During Drosophila Gastrulation, Hui Miao

Electronic Theses and Dissertations

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating high-tension contractile forces at the cell cortex before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be cellular processes to extract unidirectional changes that drive processive transformations in cell shape. During Drosophila melanogastergastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a …

Fxs-Causing Point Mutations In Fmrp Disrupt Neuronal Granule Formation And Function, Emily L. Starke

Electronic Theses and Dissertations

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by the disruption of Fragile X Mental Retardation Protein (FMRP) function in neurons, affecting nearly 1 in 7,500 individuals. Although FXS typically occurs from a complete loss of FMRP expression due to a CGG trinucleotide expansion within the 5’UTR of the FMR1 gene, single nucleotide polymorphisms (SNPs) within the KH domains of FMRP have been shown to severely disrupt FMRP function. FMRP is an RNA-binding translation repressor that interacts with ~4% of the neuronal transcriptome. Many target mRNAs encode for proteins important for regulating synaptic processes and modulate synaptic plasticity. It …

Electrical Sensing In Non-Excitable Cells To Promote Galvanotaxis And Tissue Survival, Anyesha Sarkar

Electronic Theses and Dissertations

Weak DC fields have been shown to induce polarity, cell migration and cell proliferation in 2D cultures in vitro. To understand the mechanism by which non-excitable cells sense such weak EFs, we have investigated the mechanism of cathode-directed water flow (electro-osmosis) in the boundary layer of cells by reducing it with neutral, viscous polymers. Our results indicate that low molecular weight polymers decrease cathodal migration and promote anodal migration in a concentration dependent manner. High molecular weight polymers do not affect directionality and can be explained using porosity and hydraulic permeability between the polymers. These results provide the first evidence …

A Novel Role For Rassf1a In The Regulation Of Ras Activation., Desmond Ramón Harrell Stewart

Electronic Theses and Dissertations

Ras is the most frequently activated oncogene in human cancer. It is not only the most frequently mutated oncogene, but is also rendered hyperactive in the wild-type form by aberrant regulation. Ras drives transformation and contributes to tumor aggressiveness by activating multiple downstream mitogenic effectors. Ras also possesses the paradoxical ability to induce apoptosis and senescence. Ras-induced apoptosis is not well understood, but has been largely attributed to the RASSF tumor suppressors, particularly RASSF1A. RASSF1A mediates Ras-induced apoptosis by activating pro-apoptotic proteins such as the MST kinases and BAX. RASSF1A is among the most frequently inactivated tumor suppressors in human …

Tissue-Resident Myeloid-Derived Suppressor Cells Modulate Immune Homeostasis In Healthy Adipose., Katlin Brooke Stivers

Electronic Theses and Dissertations

Our goal with this study was to gain insight into the homeostatic immune cell network in healthy adipose tissue. Specifically, we sought to determine if the immature myeloid cells found in healthy and inflamed adipose were, in fact, tissue-resident myeloid-derived suppressor cells (MDSCs). Using various in vitro and in vivo methods, we have uncovered a population of CD11b^Hi Ly6C^Hi Ly6G^- MDSCs resident in healthy adipose tissue. To the best of our knowledge this is the first time that these cells have been investigated and described in healthy adipose tissue. Systemic MDSC depletion lead to the accumulation of …

Development Of In Vitro Models To Study The Rapid Extraintestinal Dissemination Of Salmonella., Adarsh Gopinath

Electronic Theses and Dissertations

Salmonella appears in the bloodstream of mice in as little as 15 minutes after oral inoculation and establishes persistent colonies in the spleen and liver. While its pathway to blood is undetermined, this phenomenon is dependent on the activity of Salmonella pathogenicity island 2 (SPI-2) coded type III secretion system (T3SS) and CD18+ phagocytes. We hypothesize that dendritic cells associated with the basal face of the gut epithelium, that are naturally migratory and known to sample for luminal antigens directly transport Salmonella to the bloodstream. This process comprises of at least two phases, dissociation and reverse transmigration. We define dissociation …

Efficient Conditional Inactivation Of Rdh10 Reveals Important Role Of Retinoic Acid During Lung Branching Morphogenesis., Nhut Quang Huy Tran

Electronic Theses and Dissertations

Birth defects are complicated pathological processes. Many birth defects can be traced back to disrupted regulation of molecular signaling pathways during embryogenesis. One of the most important gene regulatory factors during embryo development is retinoic acid (RA). However, studying the specific roles of RA in each organ during embryogenesis in vivo is challenging. The Sandell laboratory has a conditional mutant mouse model that can potentially be used for studying the role of RA during embryogenesis by inducing stage-specific RA deficiency. This model allows retinol dehydrogenase 10 (Rdh10), a gene required for RA production, to be inactivated at a …

Quantification Of Mitochondrial Zinc Homeostasis And Analysis Of Zinc And Polyamine-Mediated Axonal Trafficking, Dylan Hunt Fudge

Electronic Theses and Dissertations

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors …

Cellular And Developmental Insights Into The Early Evolution Of Muscle, Jeffrey J. Colgren

Electronic Theses and Dissertations

Whereas a great deal has been learned about the molecular underpinnings of morphological evolution in animals, much less is known about the origin of novel cell and tissue types. During the time in which the earliest animal lineages were diversifying, fundamental cell and tissue types, such as muscles, arose. Sponges are one of two animal lineages that lack muscles, yet they undergo coordinated full body contractions. Whereas the signaling processes have been studied, the physical mechanisms of contraction are completely uncharacterized. The main purpose of this work is to understand the primary contractile tissue of the sponge Ephydatia muelleri, …

Ehbp1 And Ehd2 Regulate Dll4 Caveolin-Mediated Endocytosis During Blood Vessel Development, Amelia Margaret Webb

Electronic Theses and Dissertations

During angiogenesis, the development of new blood vessels from existing vessels, there must be tight regulation between a leading tip cell and trailing stalk cells during vascular patterning. Lateral inhibition produced by Notch signaling is central to the specification and maintenance of tip and stalk cells in growing angiogenic sprouts. The Notch pathway is activated by the ligand Delta at adherens junctions between tip and stalk cells. Despite the centrality of Delta/Notch signaling to angiogenesis, relatively little is known about the mechanism of this signaling event. It has been proposed that the force generated by the Notch bound Delta ligand …

Sorting Of Cargo Proteins Within The Regulated Secretory Pathway: The Peripheral Membrane Protein Hid-1 As A Sorting And Vesicle Biogenesis Factor, Blake H. Hummer

Electronic Theses and Dissertations

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize …

Quantification Of Dynamic Epithelial Sheet Architecture In Botryllus Schlosseri Using 2-D & 3-D Image Analysis, Roopa Madhu

Electronic Theses and Dissertations

Epithelial tubules form critical structures in various body tissues; how- ever, since they are difficult to access experimentally, their architecture and dynamics are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel and uniquely accessible model system: the extracorporeal vasculature of Botryllus schlosseri (sea squirt). In Botryllus, massive retraction of blood vessels can be triggered without loss of barrier function, through (i) disrupting collagen crosslinking in the basement membrane using β-aminopropionitrile (BAPN); or (ii) disrupting the integrin pathway through inhibition of focal adhesion kinase (FAK). We performed stereographic projections of 3-dimensional …

Cellular And Organismal Ramifications Of De Novo Purine Synthesis Dysregulation, Randall Craig Mazzarino

Electronic Theses and Dissertations

Purines are a class of nitrogenous bases and are essential small molecules to life. Purines are used within the cell as genetic information carriers, energy currency, signaling molecules, and cofactors for multiple processes. They are formed through de novo and salvage pathways found in cells across the phylogenetic tree. The substrates of enzymes within de novo purine synthesis are known to influence other processes within the cell, such as energy homeostasis. In humans, de novo purine synthesis disorders are rare, with around 100 people identified. These patients exhibit a range of phenotypes, with varying degrees of mental retardation, seizure activity, …

In Situ Analysis Of Line-1 Promoter Activity Using Lacz Transgenic Mice, Partha Sarathi Saha

Electronic Theses and Dissertations

Apart from an evolutionary role, transposable elements have been implicated in animal development and also in pathophysiology. Non-LTR retrotransposons– LINE-1, Alu and SVA - are responsible for over 120 cases of human genetic diseases as heritable insertions, and are emerging as an important etiological factor for cancer and neurological disorders as somatic mutations. It is estimated that among the total number of 500,000 LINE-1s presents in the human genome, 80-100 LINE-1s remain competent for retrotransposition. Retrotransposition is only possible when LINE-1 is expressed. Because LINE-1 transcription is regulated by its 5’UTR promoter, it is essential to understand the spatiotemporal control …

The Role And Molecular Mechanisms Of Wss1 In Preserving Genomic Stability, Daniel Kwesi Sam

Electronic Theses and Dissertations

Cells are constantly under threat from both exogenous and endogenous sources of DNA damage. Eukaryotic organisms, however, possess conserved mechanisms that accurately and faithfully respond to DNA damage. The inability to effectively remove DNA lesions can lead to an accumulation of mutations which can compromise cellular viability. The DNA damage response is conserved from bacteria to eukaryotic organisms and have been well characterized, however, how covalently crosslinked proteins are removed from DNA remains enigmatic This thesis provides genetic and biochemical evidence implicating Wss1, a yeast metalloprotease in genome maintenance. We have identified SUMOylation to be an important signal that mediates …

Exploring The Properties Of Chitin For Promoting Tissue Engineering And Repair, M. Jahir Raihan

Electronic Theses and Dissertations

No abstract provided.

Crispr/Cas9 Whole Genome Screens Reveal Novel Regulators Of Endocytic Processes In Macrophages, Jared Wollman

Electronic Theses and Dissertations

Macrophages maintain tissue homeostasis by identifying and eliminating threats within their tissue microenvironment. Pattern recognition receptors allow macrophages to recognize and internalize specific ligands while macropinocytosis allows the internalization of all extracellular solutes from their environment. Without pattern recognition receptors, pathogens may grow unchecked if they cannot be detected, and without macropinocytosis, macrophages struggle to detect and move around their environment. This thesis presents the results of CRISPR/Cas9 whole-genome screens used to identify the regulators of both endocytosis (Chapter 2) and macropinocytosis (Chapter 3). In Chapter 2, we report genes regulating dextran uptake in primary murine macrophages and reveal Mrc1-mediated …

Actin Regulation And Furrow Dynamics During Early Drosophila Embryogenesis, Yi Xie

Electronic Theses and Dissertations

Drosophila embryogenesis starts with a single nucleus undergo 13 rounds of nuclear divisions called syncytial cycles. Staring at cycle 10 when nuclei migrate to the surface of the embryo, massive and dynamic cortical actin structures and cleavage furrow formations occur. How actin regulators coordinate into an organized network directing three-dimension actin structures in the developing organisms is an unsolved question. Here, I present an in-depth characterization of actin cap dynamics: the actin caps go through expansion, stabilization, elongation and fragmentation phases in each cycle. Arp2/3 is the major contributor to actin cap formation. The functions of 7 different actin and …

Genetic Analysis Of A Novel Ftsk Homolog, Hfka, In Streptomyces Coelicolor Development-Associated Chromosome Segregation, Sumedha Sethi

Electronic Theses and Dissertations

A quintessential phenomenon occurring during prokaryotic development is accurate segregation of the replicated genomes into the daughter cells. Key energy-dependent processes like chromosome condensation and subcellular partitioning of the genomes are driven by conserved proteins like SMC, ParB, FtsK. During its complex developmental cycle, Streptomyces coelicolor segregates its genomes into chains of unicellular spores when its multigenomic syncytial aerial hyphae undergo division.

A novel ftsK-like gene, hfkA (Homolog of FtsK protein A), was examined for function and localization during development-associated chromosome segregation. Individual deletions did not affect segregation, but a ΔhfkA ΔftsK mutant exhibited 8% anucleate …

Characterization Of A More Clinically Relevant Human Leukemia Xenograft Model To Examine Perturbation Of Met/Sam Metabolism As A Novel Therapeutic Paradigm For Mll-R Leukemia In Vivo., Aditya Barve

Electronic Theses and Dissertations

Acute myeloid leukemia (AML), is a heterogeneous clonal disorder characterized by an accumulation of malignant myeloid progenitors in the bone marrow (BM), hindering normal hematopoiesis. AML exhibits dramatic heterogeneity in terms of cytogenetics, morphology, and chemotherapeutic sensitivity. Therefore, the investigation of novel, efficacious AML therapeutics will require advanced preclinical in vivo model systems, capable of recapitulating patient specific disease heterogeneity, and induction chemotherapy outcomes. A major focus and eventual outcome of this work was the establishment and development of a more clinically relevant mouse xenograft model of patient AML, that efficiently harbors patient derived xenografts (PDXs), and unlike more prevalent …

The Distinct Expressions Of Integrins Αdβ2 And Αmβ2 Differently Regulate Macrophage Migration In 3d Matrix In Vitro And In Tissue During Inflammation, Kui Cui

Electronic Theses and Dissertations

Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins α_Dβ₂ and α_Mβ₂ to macrophage migration and development of chronic inflammation.

We found that integrin α_Dβ₂ is upregulated on M1-macrophages in vitro and …

Defects In Fetal Mouth Movement And Pharyngeal Patterning Underlie Cleft Palate Caused By Retinoid Deficiency., Regina Friedl

Electronic Theses and Dissertations

Cleft palate is a common birth defect. Etiologic mechanisms of palate cleft include defects in palate morphogenesis, mandibular growth, or spontaneous fetal mouth movement. Cleft palate linked to deficient fetal mouth movement has been demonstrated directly only in a single experimental model of loss of neurotransmission. Here, using retinoid deficient mouse embryos, we demonstrate directly for the first time that deficient fetal mouth movement and cleft palate occurs as a result of mis-patterned development of pharyngeal peripheral nerves and cartilages. Retinoid deficient embryos were generated by inactivation of retinol dehydrogenase 10 (Rdh10), which is critical for production of …

Exploring The Role Of Ptn1 In Ustilago Maydis., Lalu Murali Krishna Vijayakrishna Pillai

Electronic Theses and Dissertations

Signal transduction is a key aspect of biological life. Cell signaling involves a series of biochemical and molecular steps that regulate the normal cellular functions, and any disruption at any of these steps is likely to have substantial impacts on cell physiology. The current study was aimed at the characterization of ptn1 of Ustilago maydis, a basidiomycete fungus. When the U. maydis, ptn1 gene was either deleted or overexpressed, it was found that the deletion of this gene lead to reduced virulence, spore production, and germination rate; effects of the overexpression were more subtle or not discernable. Previous studies …

Paraoxonase 2 Is Critical For Non-Small Cell Lung Carcinoma Proliferation., Aaron Whitt

Electronic Theses and Dissertations

Non-small cell lung carcinoma (NSCLC) comprises 85% of lung cancer diagnoses and is plagued by drug resistance. Thus, elucidating the underlying mechanisms of NSCLC is paramount to expand future treatment options. Paraoxonase 2 (PON2), an intracellular enzyme with arylesterase and lactonase functions, has well-established anti-atherosclerotic activity. Recent studies show PON2 is overexpressed in a variety of tumors and confers drug resistance, although these interactions have not been thoroughly examined in NSCLC. Thus, we sought to investigate the role of PON2 in cellular proliferation using PON2-knockout mice, primary mouse cells, and NSCLC cell lines. Using these approaches, we demonstrate that PON2 …

New Insights Into Mycofactocin Biosynthesis, Structure And Function, Richard Selorm Ayikpoe

Electronic Theses and Dissertations

Mycofactocin is a putative ribosomally synthesized and post-translationally modified peptide (RiPP)-derived redox cofactor. Its biosynthesis is accomplished through the dedicated actions of the products of six conserved genes, mftABCDEF. The mycofactocin pathway is one of the most widely distributed RiPP systems in bacteria however, this distribution is heavily skewed towards the Mycobacteria genus including human pathogenic variants such as M. tuberculosis and M. ulcerans. Gene expression studies have demonstrated the essentiality of the pathway in the ability of M. tuberculosis to utilize the host's cholesterol as sole carbon source during latency. However, the biosynthesis, structure and physiological function …

Transgenerational Effects Of Maternal Age On Offspring Fitness In Crickets, Jacob D. Wilson

Electronic Theses and Dissertations

Advanced parental age is an important aspect of parental condition that can have both positive and negative effects on offspring fitness, and thus, parental age can be considered a parental effect. As a parental effect, parental age may affect a variety of offspring traits and may cascade to influence several generations of offspring. Given the complexities of studying both paternal and maternal age, we studied the effects of maternal age only. Using the Pacific field cricket, Teleogryllus oceanicus, we asked 1) does maternal age have influences over several generations of offspring and 2) does maternal age influence the reproductive …

Investigation Of Intergroup Bias In Two Neuromaturationally Distinct Age Cohorts: An Erp Study, Reuven M. Hanna

Electronic Theses and Dissertations

Currently, sociological investigation of adolescent behavior focuses on the intersection of biography, history, and structure to explain adolescent risk-taking, reward-seeking, impulsivity, novelty-seeking and peer-salience. However, the preponderance of the evidence points away from social ecology and to a significant neuromaturational restructuring event between the 12th and 25th years of life as the root of adolescent behavioral tendencies. As a result, sociological social psychology can benefit from engaging in basic research using neuroscience methods. The present study expands the dual systems model of brain development to account for maturational changes in the social brain network as a way to explain social …

Identifying Critical Genes For Cholesterol Metabolism In Macrophages Using Crispr-Cas9 Whole-Genome Screens, Kevin Wanniarachchi

Electronic Theses and Dissertations

Macrophage foam cells contribute to atherosclerotic plaque formation, a pathology that underlies heart disease, peripheral arterial disease and stroke. Foam cells form when macrophages take up excessive amounts of low-density lipoprotein (LDL) leading to elevated cellular levels of neutral lipids, which are packaged into lipid droplet organelles. Despite the high public health priority motivating research of cardiovascular disease processes, current understanding of macrophage cholesterol metabolism including mechanisms of LDL uptake, cholesterol trafficking, lipid droplet biogenesis, lipid droplet degradation and cholesterol efflux is limited. Here, we implemented a CRISPR-Cas9 whole genome screening strategy to identify critical genes regulating macrophage cholesterol metabolism. …

Epidermal Growth Factor-Like Ligands Regulate Dimer Selection., Jamie S. Rush

Electronic Theses and Dissertations

There are thirteen known endogenous EGF-like ligands. We previously reported that Betacellulin (BTC) increases ligand-mediated corneal wound healing more than Epidermal Growth Factor (EGF) [Peterson et al. (2014) IOVS 55(5):2870-80], although the molecular reason for this is unknown. Despite being better at promoting wound healing via enhanced cell migration, BTC has reduced receptor affinity and weaker induction of EGFR phosphorylation. These data indicate that BTC’s response is not due to enhanced affinity or EGFR-kinase activity. Receptor phosphorylation and proximity ligation assays indicate that BTC treatment significantly increases ErbB3 phosphorylation and EGFR:ErbB3 heterodimers. BTC traffics EGFR at a faster rate than …

Egfr Signaling From The Early Endosome., Julie A. Gosney

Electronic Theses and Dissertations

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR dimerizes with another ErbB family receptor, leading to kinase domain activation and transphosphorylation of C-terminus tyrosine residues. These phosphotyrosines act as crucial regulators of EGFR signaling as effector proteins dock to the receptor at these sites. The receptor undergoes clathrin-mediated endocytosis into early endosomes, where it can then be trafficked to a lysosome for degradation. However, the kinase domain of EGFR retains its activity during trafficking, suggesting that EGFR can continue …