Biochemistry, Biophysics, and Structural Biology Commons^™

Dynamics Of Site Search Process And Formation Of Synaptosome Assembly Characterized By Single Molecule Approaches, Sridhar Vemulapalli

Theses & Dissertations

Genome dynamics and integrity are the two crucial features defining the successful functioning of cells and their maintenance and evolution. The genetic processes in the cell require distant communications between the regulatory regions controlled by specific proteins. Mistakes in this interaction process will lead to termination of the genetic process may lead to the cell damage, disease development or the cell death. Similar distant regulatory process is required for numerous genome integration systems such as Variable Diversity Joining (V(D)J) recombination system resulting in the specificity of the immunoresponse, a defining property of the adaptive immune system. A common feature of …

Gap Junctional Intercellular Communication: Role Of Cx43 Phosphorylation By Tyrosine Kinases, Ishika Basu

Theses & Dissertations

Phosphorylation of Cx43 is a process that regulates various functionalities of the gap junction including assembly, stability, internalization, turnover, channel permeability and channel gating. Src has been shown to phosphorylate Cx43 at Y247, Y265, Y313 and regulate gap junctions. Intercellular communication in B and T cells is necessary to mediate adaptive immune response. However, there is little-to-no expression of Src in these lymphocytes. An in vitro kinase screen identified Bruton’s tyrosine kinase (BTK) and Interleukin 2-inducible T-cell kinase (ITK) to phosphorylate Cx43. Mass spectrometry identified Tyr residues to be phosphorylated by BTK and ITK, and these residues are similar to …

Exploiting Vulnerabilities In The Ras-Rac Signaling Pathway For The Selective Targeting Of Pancreatic Cancer Cells, Neha Chaudhary

Theses & Dissertations

Deregulation of the KRas (Kirsten rat sarcoma virus) GTPase is one of the early hallmarks of Pancreatic Cancer (PC). The most common genetic alteration found in PC are mutations in the KRas protein that block its ability to hydrolyze GTP to GDP and resulting in higher levels of GTP-bound KRas, its active form. Pancreatic tumors driven by oncogenic mutants of KRas tend to be addicted to the oncogene, to the extent that its repression leads to the induction of cell death. This addiction to the KRAS oncogene makes the KRas protein an ideal target for cancer therapy. However, the globular …

Rhythms In Rna And Proliferation In Intestinal Stem Cell Organoids, Kristina Skurvidayte

Electronic Theses and Dissertations

The circadian clock is a transcription-translation feedback loop that controls our body’s daily rhythms. Previous work in our lab has shown that the circadian clock controls regeneration in the small intestinal epithelium and that the Hippo signaling pathway may serve as a link between regeneration and the circadian clock. In order to elucidate if the circadian clock directly controls the Hippo pathway as well as the Wnt pathway, both vital for intestinal homeostasis, intestinal stem cell-derived organoids, a 3D culture system that is able to recapitulate the cell biology of the intestinal epithelium in vitro, were used to examine the …

Profiling And Verifying The Substrates Of E3 Ubiquitin Ligase Rsp5 In Yeast Cells, Shuai Fang, Geng Chen, Yiyang Wang, Rakhee Ganti, Tatiana A Chernova, Li Zhou, Savannah E Jacobs, Duc Duong, Hiroaki Kiyokawa, Yury O Chernoff, Ming Li, Natalia Shcherbik, Bo Zhao, Jun Yin

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.

Effect Of Tamoxifen On Proteome Expression During In Vitro Myogenesis In Murine Skeletal Muscle C2C12 Cells, Emily A. Morris, Ahlenne Abreu, S. P. Scordilis

Biological Sciences: Faculty Publications

Tamoxifen (TMX), a selective estrogen receptor modulator, is commonly used in the treatment of hormone-responsive cancers. However, the effects of TMX in anabolic tissues harboring estrogen receptors, such as skeletal muscle, are poorly understood. We report a tandem mass-tag approach to TMX-treated myogenesis in C₂C₁₂ cells, a well-characterized model of in vitro murine skeletal muscle differentiation. A longitudinal analysis of >10,000 proteins identified in untreated C₂C₁₂ myogenesis revealed a novel subset of 1,062 myogenically regulated proteins. These proteins clustered into five distinct longitudinal expression trends which significantly overlap those obtained in similar analyses performed …

Upland Cotton And Nematodes: An Analysis Of Historical Resistance, Upcoming Threats, And Co-Inoculation Effects, Amanda Gaudin

Theses and Dissertations

Upland cotton (Gossypium hirsutum ) is an important fiber crop grown throughout the southern United States. Plant-pathogenic nematodes are worm-like animals that feed on the roots of most agronomic crops, including cotton. The southern root-knot nematode (Meloidogyne incognita, RKN) and the reniform nematode (Rotylenchulus reniformis, RN) cause significant yield losses in cotton every year. Current sources of resistance are effective but limited, therefore historical screenings of cotton accessions were revisited in search for novel resistance sources. None were identified but many of the screened accessions possessed markers of known root-knot nematode and reniform nematode resistance. …

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, …

Role Of Fat Content On The Structure And Function Of Human Skeletal Muscle, Joseph A. Gordon Iii

Doctoral Dissertations

Muscle size does not fully explain variations in muscle strength. Fat content has been implicated in muscle weakness, though this relationship remains unclear. The relationship between fat and strength may vary between scales (e.g., cellular, organ, and organism). The goal of this dissertation was to clarify the role of fat in the structure and function of muscle using in vitro and in vivo techniques across multiple scales in adults 21-45 years old. Study 1 tested the agreement of intramyocellular lipid (IMCL) content between oil red o (ORO) and magnetic resonance spectroscopy (MRS) techniques. These measures of IMCL were also compared …

Selective Activation Of Thrombin Activatable Fibrinolysis Inhibitor (Tafi) Attenuates Metastatic And Angiogenic Capabilities Of Melanoma And Lung Carcinoma In Vitro, Jacklyn Krizsan

Electronic Thesis and Dissertation Repository

Metastasis and angiogenesis are hallmarks of aggressive cancers, both depending on degradation of extracellular matrix by proteases such as plasmin. Plasmin activation is inhibited by thrombin-activatable fibrinolysis inhibitor (TAFI)-mediated cleavage of terminal lysine residues on plasminogen receptors. Activation of TAFI is most effectively done in complex with thrombomodulin (TM). TM is known to have anti-cancer properties, but it is not known if this is due to TAFI activation or an alternative substrate protein C (PC). We hypothesize that specific promotion of TAFI activation with TM treatment will attenuate metastatic and angiogenic capabilities of tumour cells.

Melanoma and lung carcinoma cells …

Structural Study Of Lsd13 And Lsd14 Type I Modular Polyketide Synthases From The Lasalocid A Biosynthesis Pathway, Dayan Viera

Open Access Theses & Dissertations

Bioactive natural products often possess a complex structure and have a large size which make their chemical synthesis highly challenging. To overcome this challenge, we are developing a biosynthetic method for production of natural products and their analogs. This technology is expected to decrease the synthesis time from years to weeks and reduce the cost of drug production by multiple orders of magnitude. We are investigating the biosynthesis pathway of lasalocid A, an anticancer antibiotic, to develop this technology. Specifically, we aim to characterize the enzymes structurally and biochemically in this pathway, thus paving the way for their rational engineering. …

Determining The Existence And Regulation Of Microlipophagy In Primary Brown And White Adipocytes, Sara C. Arenas De Leon

Biomedical Sciences ETDs

Lipids are one of life’s four main macromolecules and provide essential functions to cells.

The degradation of lipid droplets and mobilization of lipids occurs through lipolysis. Emerging evidence demonstrates evidence of a selective form of autophagy in lipolysis. The process of microlipophagy has only recently been described, and many features of its regulation are still poorly understood. Our recent study showed that inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway by depletion of Raptor in adipocytes led to an influx of lysosomes and accumulation of lipid droplets within lysosome. Here, we expand on these previous findings. We were able …

Utilizing Crispr Cas9 To Visualize Dopamine Receptors In Caenorhabditis Elegans, Lauren Michelle Velasquez

Electronic Theses, Projects, and Dissertations

Dopamine (DA) is a neurotransmitter with imperative implications in many functions including movement, reward, and cognition. Studying the pathways of dopaminergic neurons at multiple levels allows us to understand the ways in which these systems can go wrong. We study dopamine in a model system such as the worm Caenorhabditis elegans because of its relatively simple and well-characterized nervous system. DA is involved in regulating chemosensory behaviors in worms. The purpose of this research project is to definitively answer the following question: Are the dopamine receptors DOP-1 and DOP-4 expressed in chemosensory neurons? Previous reporter assays show that neither of …

Phenotypic And Transcriptomic Characterization Of Rice Snrk1 Mutants Developed By Crispr/Cas9 Mutagenesis, Maria Clara Faria Chaves

Graduate Theses and Dissertations

SnRK1 is a heterotrimeric protein kinase that is composed of a catalytic subunit (α) and two regulatory subunits (β and βγ), and it has a main role in regulating energy homeostasis in the plant by modulating anabolic and catabolic process. SnRK1 phosphorylates and alters the activities of enzymes involved in metabolism and regulates gene expression by altering the activity of chromatin-remodeling enzymes or the transcription factors. Rice contains three functional paralogs of SnRK1α: SnRK1αa (LOC_Os03g17980), SnRK1αb (LOC_Os08g37800), and SnRK1αc (LOC_Os05g45420). This study focused on the function of these SnRK1 paralogs by evaluating the phenotypic and transcriptomic characteristics and the disease …

Fbg Αc 389 – 402 Modulates Factor Xiii Crosslinking In The Fibrinogen Αc Region., Francis Dean Orlina Ablan

Electronic Theses and Dissertations

Fibrinogen (Fbg) is a coagulation protein critical for clot formation. Coagulation Factor XIII (FXIII) is a calcium-dependent transglutaminase that crosslinks reactive glutamines (Q) and lysines (K) between fibrin and other anti-fibrinolytic proteins. In the presence of Ca²⁺, FXIII could be activated non-proteolytically (FXIII-A°), or proteolytically by thrombin (FXIII-A*). Significant increases in clot stability and red blood cell retention are linked to FXIII activity in the fibrinogen αC region (Fbg Aα 221 – 610). This region contains several FXIII-reactive glutamines and lysines, as well as a binding site for FXIII-A* (Fbg αC 389 – 402) that includes a key …

Modeling Biphasic, Non-Sigmoidal Dose-Response Relationships: Comparison Of Brain- Cousens And Cedergreen Models For A Biochemical Dataset, Venkat D. Abbaraju, Tamaraty L. Robinson, Brian P. Weiser

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Biphasic, non-sigmoidal dose-response relationships are frequently observed in biochemistry and pharmacology, but they are not always analyzed with appropriate statistical methods. Here, we examine curve fitting methods for “hormetic” dose-response relationships where low and high doses of an effector produce opposite responses. We provide the full dataset used for modeling, and we provide the code for analyzing the dataset in SAS using two established mathematical models of hormesis, the Brain-Cousens model and the Cedergreen model. We show how to obtain and interpret curve parameters such as the ED50 that arise from modeling, and we discuss how curve parameters might change …

Fatty Acids And Parasitism: Towards A Better Understanding Of Lipid Metabolism In Trypanosoma Brucei, Joshua Saliutama

All Dissertations

Trypanosoma brucei is an extracellular eukaryotic parasite that causes sleeping sickness in humans and cattle. As an extracellular parasite, T. brucei relies on the host’s nutrients to satisfy its growth requirements. The parasite is unusual because it does not uptake most of the host’s lipid species. Instead, T. brucei prefers to perform de novo synthesis of most lipid species. One of the lipid species that T. brucei can both uptake and synthesize is fatty acids. In my thesis work, I investigated the dynamics of fatty acid uptake, metabolism, and utilization of T. brucei. My work starts by determining the …

X-Ray Crystal Structure Of E399q,E708q Ecm16 Double Mutant, Gileydis Guillama

Open Access Theses & Dissertations

The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes …

Epitranscriptomic Regulation In Breast Cancer And Pcb-Induced Liver Disease., Belinda Petri

Electronic Theses and Dissertations

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, …

The Inner Workings Of Thrombin-Activatable Fibrinolysis Inhibitor: A Study Of The Tm-Mediated Activation Of Tafi And Inactivation Of Tafia, Haley D N Marier

Electronic Thesis and Dissertation Repository

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a key regulator of hemostasis and inflammation. TAFI is activated by thrombin proteolysis, most effectively when in complex with cofactor thrombomodulin (TM). Soluble TM, encompassing EGF-like domains 3 through 6 is sufficient to promote TAFI activation, though the role of EGF 4 has not been assessed. Through rearrangement of the soluble TM domains, we determined that EGF 4 was acting as a spacer to promote TAFI activation. Activated TAFI (TAFIa) is a metastable enzyme, with a half-life of only 8-15 min at 37^oC. The mobile loop of TAFIa was expected to modulate stability; …