Molecular Biology Commons^™

The Identification Of Small Molecule Inhibitors To Candida Albicans Phosphatidylserine Synthase, Yue Zhou

Doctoral Dissertations

Candida albicans phosphatidylserine (PS) synthase, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis due to its importance in virulence, absence in the host and conservation among fungal pathogens. This dissertation is focused on the identification of inhibitors for this membrane enzyme. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif present within Cho1, and here we revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1. For serine, we have predicted a …

Protein Trafficking In The Endoplasmic Reticulum Mediated By A Tpr-Containing Adapter Protein, Nathan P. Canniff

Doctoral Dissertations

The endoplasmic reticulum (ER) is a large, multifunctional organelle that acts as the entrance into the secretory pathway, which accounts for the folding and maturation of approximately one third of the human proteome. It is the largest organelle in most cell types and is comprised of a single lumen and a contiguous membrane. The ER is responsible for a multitude of roles including protein translocation, folding, maturation, quality control, and glycosylation to name a few. These processes are buoyed by a large collection of chaperones and cochaperones, the largest subfamily of which is characterized by the presence of tetratricopeptide repeat …

A Shift In Rna Fate: Investigating The Role Of C19orf66 During Kshv Lytic Replication, William Rodriguez Jr.

Doctoral Dissertations

During viral infection, virus and host clash for control of the cell in a conflict that ultimately drives the evolution of both sides and has lasting consequences in the form of pathogenesis. At the heart of this struggle is a contest for control of cellular gene expression, a struggle epitomized by an evolutionary tug-of-war for supremacy over RNA fate. During lytic replication, Kaposi’s sarcoma-associated herpesvirus (KSHV) triggers a massive RNA decay event known as viral “Host Shutoff” which decimates greater than 70% of the host transcriptome, simultaneously suppressing the cellular anti-viral response and freeing host resources for viral replication. However, …

Modulation Of Plant Immunity During The Establishment Of The Nitrogen-Fixing Symbiosis, Miriam Hernandez-Romero

Doctoral Dissertations

Nitrogen is essential for plant tissue growth but is often a limited resource in soils. Many legumes overcome this limitation by entering a symbiotic association with soil microbes, called rhizobia, which provide nitrogen to the plant while rhizobia receive fixed carbon. To successfully form a symbiosis, the host and symbiont exchange a series of molecular signals. One major obstacle during this interaction is the host's innate immune system, which becomes active upon rhizobial detection. It is therefore the main focus of this thesis to identify the mechanisms that modulate host immunity. In the subsequent chapters, we focus on a rhizobial …

A Novel Transmembrane Ligand Inhibits T Cell Receptor Activation, Yujie Ye

Doctoral Dissertations

T lymphocytes (T cells) play essential roles in the adaptive immune system. Each mature T cell expresses one type of functional T cell receptor (TCR). The TCR recognizes antigens bound to the major histocompatibility complex (MHC) in antigen presenting cells. The resulting stimulation signal crosses the transmembrane domain of TCR and initiates downstream signaling cascades. The human immune system relies on TCRs to recognize a variety of pathogens. Normally, TCR can distinguish the self-antigens from pathogenic antigens. However, dysfunction or aberrant expression of TCRs causes different inflammatory and autoimmune diseases, which afflict millions of people annually (Chapter I). Current treatments …

The Sos Response In Escherichia Coli K12: An Exploration Of Mutations In Lexa And Reca Using Fluorescence Microscopy, Steven Van Alstine

Doctoral Dissertations

Faithful replication of the genome is paramount for maintaining the fitness of an organism. Therefore, life has evolved inducible mechanisms to be able to repair damaged DNA and maintain evolutionary fitness. The SOS response is a highly conserved DNA damage inducible response that is tightly regulated. Multiple factors contribute to the ability of the cell to perform proper DNA repair and induction of the SOS response including the amount of RecA, mutations in RecA that affect competition for DNA, and other proteins that interact with the RecA filament. The complex relationship between RecA and LexA is the subject of this …

Chemical Biology Approaches For Tracking And Manipulation Of Macrophage Phenotypes, Javier A. Mas Rosario

Doctoral Dissertations

Macrophages are white blood cells of the innate immune system that have the ability to change phenotypically depending on the stimuli present in their surroundings through a process commonly referred to as polarization. Macrophage phenotypes broadly range from pro-inflammatory, anti-tumor (M1) to immune-suppressing (M2). Of particular interest to this work, breast cancer progression and metastasis rely on the presence of M2-like tumor-associated macrophages (TAMs). While many studies have shown the involvement of macrophages in tumor progression and metastasis, there remains a need to further explore these interactions and the polarization process, including tracking of macrophage subtypes. Toward this end, I …

Characterizing The Multifaceted Roles Of The Proteasomal Deubiquitinase Uch37 In Proteostasis, Heather A. Bisbee

Doctoral Dissertations

Cellular protein pools are maintained through the biological processes of synthesis, degradation and quality control. As the dysregulation of these processes has been implicated in diseases such as neurodegeneration and cancer, understanding their functions is critical for drug development. Modification of proteins with ubiquitin may direct them to the proteasome, a large cellular protease complex, for degradation. Yet, the proteasome contains three deubiquitinating enzymes (DUBs) which remove ubiquitin from proteins, potentially altering their fate. As each DUB recognizes specific ubiquitin linkages and architectures, their activity may regulate how the proteasome handles substrates in dynamic cellular contexts. In this work, we …

Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward

Doctoral Dissertations

Molecular motors, such as myosin, have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction in these motors is a root cause of many pathologies necessitating the application of intrinsic control over molecular motor function. We hypothesized that altering the myosin’s energy substrate via minor positional changes to the triphosphate portion of the molecule will allow us to control the protein and affect its in vitro function. We utilized positional isomers of a synthetic non-nucleoside triphosphate, azobenzene triphosphate, and assessed whether myosin’s force- and motion-generating capacity could …

Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni

Doctoral Dissertations

Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …

Tracking And Modulating Circadian Rhythms In Cell Culture Models, Sujeewa Sampath Lellupitiyage Don

Doctoral Dissertations

Circadian rhythms are 24–hour activity cycles that exist for all organisms, from yeast and bacteria to mammals. Epidemiological data has shown that disruptions to circadian rhythms are associated with various diseases, including cancers. Circadian rhythms are regulated at the cellular level by a "molecular clock,” whose oscillations in protein transcription and translation control a range of downstream pathways. However, the connections between altered rhythms and diseases at the molecular level is unclear. Therefore, in this project, I tracked circadian rhythms in a high-resolution manner and thoroughly analyzed them to more completely reflect circadian behaviors and clock functioning at the molecular …

Mechanisms By Which Xenorhabdus Nematophila Interacts With Hosts Using Integrated -Omics Approaches, Nicholas C. Mucci

Doctoral Dissertations

Nearly all organisms exist in proximity to microbes. These microbes perform most of the essential metabolic processes necessary for homeostasis, forming the nearly hidden support system of Earth. Microbial symbiosis, which is defined as the long-term physical association between host and microbes, relies on communication between the microbial community and their host organism. These interactions among higher order organisms (such as animals, plants, and fungi) and their bacteria links metabolic processes between interkingdom consortia. Many questions on microbial behavior within a host remain poorly understood, such as the colonization efficiency among different microbial species, or how environmental context changes their …

Nodulin 26 Like Intrinsic Proteins: Structurally Similar Membrane Channels With Diverse Functions In Plant Hypoxia Stress, Metalloid Nutrition & Toxicity, Zachary Beamer

Doctoral Dissertations

Plant nodulin 26 intrinsic proteins are categorized into three groups (NIP I, II, and III) based on pore architecture. NIP II and III participate in metalloid nutrition, whilst the function of a third (NIP I) is less understood. Here we investigate the physiological function of one NIP I protein (Arabidopsis thaliana NIP2;1) as a lactic acid channel, and also explore the structural basis for metalloid and water permeability of NIP I and NIP II proteins in general. In addition, a strategy was developed for the purification and crystallization of soybean nodulin 26 as a step towards structure determination of a …

Characterizing Mechanical Regulation Of Bone Metastatic Breast Cancer Cells, Boyuan Liu

Doctoral Dissertations

Breast cancer most frequently metastasizes to the skeleton. Bone metastatic cancer is incurable and induces wide-spread bone osteolysis, resulting in significant patient morbidity and mortality. Mechanical stimuli in the skeleton are an important microenvironmental parameter that modulates tumor formation, osteolysis, and tumor cell-bone cell signaling, but which mechanical signals are the most beneficial and the corresponding molecular mechanisms are unknown. This work focused on bone matrix deformation and interstitial fluid flow based on their well-known roles in bone remodeling and in primary breast cancer. The goal of our research was to establish a platform that could define the relationship between …

Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri

Doctoral Dissertations

Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA …

Calmodulin Like 38 Is Required For Autophagy Of Hypoxia-Induced Cytoplasmic Rna Granules In Arabidopsis Thaliana, Sterling Field

Doctoral Dissertations

In response to the energy crisis resulting from submergence stress and hypoxia, the model plant Arabidopsis thaliana limits non-essential mRNA translation, and accumulates cytosolic stress granules. Stress granules are phase-separated mRNA-protein particles that partition transcripts for various fates: storage, degradation, or return to translation after stress alleviation. Another response by the plant cell to low oxygen stress is the induction of the turnover pathway autophagy. Stress granule regulation by autophagy occurs by a ‘granulophagy’ pathway in yeast and mammalian systems through which parts or whole stress granules are degraded. Whether this occurs in plants has not been investigated.

A connection …

Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro

Doctoral Dissertations

Plants are sessile and must adjust their organ growth to their environments. A reservoir of stem cells in the shoot apical meristem (SAM) supplies cells for differentiation into organs. The SAM must balance organ production with stem cell maintenance. The ERECTA family (ERfs) encodes the leucine-rich repeat receptor-like kinases ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2. ERf signaling regulates organ initiation and stem cell maintenance. Results presented in this work include the following:

1) WUSCHEL (WUS) and CLAVATA3 (CLV3) make up a negative feedback loop to maintain SAM size. WUS and CLV3 expression localization is critical for …

Microbial Community Dynamics Of A Microcystis Bloom, Helena Pound

Doctoral Dissertations

Harmful algal bloom events are notoriously associated with massive economic and environmental consequences, causing wildlife and human health risks. As these blooms increase in occurrence, duration, and severity around the world, it is essential to understand conditions leading to bloom formation and why they persist. Abiotic factors such as nutrients are commonly considered in bloom dynamics, but biotic interactions with co-occurring microbial species and viruses must also be taken into account. Harmful algal blooms dominated by the cyanobacterial genus Microcystis occur in bodies of water around the world and provide an ideal system in which to study top-down controls on …

Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo

Doctoral Dissertations

Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …

The Histone Variant H2av Regulates Stress Responses And Tissue Development Through Interactions With Chromatin Insulator Proteins In Drosophila Melanogaster, James Ryan Simmons

Doctoral Dissertations

The ability of a cell to sense and respond to various forms of stress is essential to maintain integrity of the genome. Numerous pathways have been implicated in cellular responses to environmental and genotoxic stresses, often involving proteins and complexes that bind DNA directly to orchestrate changes in transcription and genome organization. Chromatin insulators describe a class of protein complex that bind specific sequences in the genome and work through two classically described functions: to restrict communication between enhancers and promoters through physical separation into different genomic domains and to prevent the spread of heterochromatin into euchromatic regions of the …

Modulation Of Protein Dynamics By Ligand Binding And Solvent Composition, Richard J. Lindsay

Doctoral Dissertations

Many proteins undergo conformational switching in order to perform their cellular functions. A multitude of factors may shift the energy landscape and alter protein dynamics with varying effects on the conformations they explore. We apply atomistic molecular dynamics simulations to a variety of biomolecular systems in order to investigate how factors such as pressure, the chemical environment, and ligand binding at distant binding pockets affect the structure and dynamics of these protein systems. Further, we examine how such changes should be characterized. We first investigate how pressure and solvent modulate ligand access to the active site of a bacterial lipase …

Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac

Doctoral Dissertations

High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes …

Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu

Doctoral Dissertations

Human lysosomal neuraminidase 1 (hNEU1) is an exo-a-sialidase which cleaves a(2-3) and a(2-6) linked sialic acids on glycoproteins in the lysosome. Deficiency of hNEU1 in the lysosome results in sialidosis, a lysosomal storage disease. Currently there is no effective treatment for sialidosis, which leads to a rising interest in discovering potential therapies. Here we presented a small molecule, α-D-N-acetylneuraminic acid (NANA), increases the protein amount and activity of both wild-type hNEU1 and three different hNEU1 mutations found in sialidosis patients in our mammalian cell system, suggesting that NANA works as a potential pharmacological chaperone for hNEU1 and provides …

Engineering Modularity Of Ester Biosynthesis Across Biological Scales, Hyeongmin Seo

Doctoral Dissertations

Metabolic engineering and synthetic biology enable controlled manipulation of whole-cell biocatalysts to produce valuable chemicals from renewable feedstocks in a rapid and efficient manner, helping reduce our reliance on the conventional petroleum-based chemical synthesis. However, strain engineering process is costly and time-consuming that developing economically competitive bioprocess at industrial scale is still challenging. To accelerate the strain engineering process, modular cell engineering has been proposed as an innovative approach that harnesses modularity of metabolism for designing microbial cell factories. It is important to understand biological modularity and to develop design principles for effective implementation of modular cell engineering. In this …

The Role Of Med31 And Med12 In Directing Adipogenesis Of Human Adult-Derived Mesenchymal Stem Cells, Joseph Straub

Doctoral Dissertations

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multisubunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules: head, middle, tail, and kinase. In our introduction, we show the state of the field of Mediator study with a focus on the critical kinase module. In the following chapters, we used siRNA knockdowns to investigate the roles of the highly-conserved core subunit MED31 and the kinase module …

Regulation Of Plasmodesmata By Specialized Metabolites Glucosinolates In Arabidopsis Thaliana, Jessica C. Fernandez

Doctoral Dissertations

Communication is an essential component to all living organisms. In

plants, the additional cell wall surrounding each cell adds a layer of complexity

not observed in animals. To overcome the literal wall separating cells, plants

have evolved specialized pores to connect adjacent cells. Plasmodesmata (PD)

allow plants to have a continuous cytoplasm between cells. Although

plasmodesmata may appear simple and lack regulation, their structural

components and their regulatory machinery is complex and not well understood.

Organelle-to-nucleus-to-plasmodesmata signaling (ONPS) have been worked as

a leading model for a possible regulatory mechanism. Many of the details of

organelle-to-nucleus retrograde signaling pathways have …

Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois

Doctoral Dissertations

Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient's tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor …

The Effect Of Nuclear Perturbations On The 3d Organization Of The Genome, Rosela Golloshi

Doctoral Dissertations

Cells in our body experience constant mechanical forces that influence biological functions such as growth and development. The nucleus has been implicated as a key mechanosensor and can directly influence chromatin organization and epigenetic alterations leading to gene expression changes. However, the mechanism by which such mechanical forces lead to genomic alterations and expression of mechanosensitive genes is not fully understood. The work presented in this dissertation investigates the effect of mechanical and epigenetic perturbations on the 3D genome organization. To investigate this 3D genome folding, we use Chromosome Conformation Capture followed by high throughput sequencing (Hi-C) (Chapter-1) which identifies …

Root Phosphomonoesterase As A Vital Component Of Increasing Phosphorus Availability In Tropical Forests, Kristine Grace Manno Cabugao

Doctoral Dissertations

Tropical forests, relative to other terrestrial ecosystems, exchange the largest amount of carbon with the atmosphere and also constitute a significant carbon sink. However, nutrient limitation, particularly of phosphorus (P), could limit growth of tropical forests and their function with the global carbon cycle. Thus, understanding root mechanisms to acquire P is necessary to representing the P cycle and corresponding interactions with plant growth. A large portion of total soil P in tropical forests occurs in organic forms, only accessible through root and microbial production of phosphatase enzymes. These phosphatase enzymes mineralize organic P into orthophosphate, the form of P …

Iojap: Morphological And Physiological Phenotype Characterization In Arabidopsis, Thomas Jay Payne

Doctoral Dissertations

IOJAP protein is found in all organisms that contain a ribosome of bacterial origin. The majority of studies suggest that IOJAP plays a role in translation, although this has yet to be thoroughly investigated in plants. Using Arabidopsis thaliana, an extensive phenotype characterization of iojap mutants was performed. Many processes of plant growth were slightly impaired at optimal temperature (22˚C) but became severely hindered at low temperature (12˚C and 4˚C). These cold temperature defects manifested in an overall reduction of plant growth as well as variegation, chlorosis, leaf hyponasty, as well as reduced maximum quantum yield (F_v/F …