Biochemistry, Biophysics, and Structural Biology Commons^™

A Small Molecule Screen On Zebrafish Embryos Identifies Pathways Vital To Hypaxial Muscle Precursor Migration, Teresa Easterbrooks

Electronic Theses and Dissertations

Hypaxial muscles form through long-range migration of muscle precursor cells (MMPs) from the somites. In zebrafish, the MMPs migrate in three compact streams to generate four muscles - the sternohyoideus muscle, homologous to mammalian neck and tongue; the posterior hypaxial muscle; and the two pectoral fin muscles, homologous to mammalian limbs. Several factors, such as Six1/4, are known to promote this migration; however, many aspects of guidance, pathing, and modulation of these streams are still unknown. To fill this gap, we conducted a small-molecule screen. This pharmacological approach allows us to identify molecules that cause pronounced changes in the normal …

Endothelial Interleukin-17 Receptor D (Il17rd) Promotes Western Diet-Induced Aortic Myeloid Cell Infiltration, Shivangi Pande

Electronic Theses and Dissertations

The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor), belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent monocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83% compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 cells …

The Effects Of Resistance Exercise Training On Insulin Resistance Development In Female Rodents With Type 1 Diabetes, Mitchell James Sammut

Electronic Thesis and Dissertation Repository

The etiology of insulin resistance (IR) development in type 1 diabetes mellitus (T1DM) remains unclear; however, impaired skeletal muscle metabolism may play a role. While IR development has been established in male T1DM rodents, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1DM compared to aerobic exercise. Additionally, the molecular mechanisms mediating RT-induced improvements in insulin sensitivity remain unclear. Therefore, the purpose of this study was to investigate the effects of RT on IR development in female …

Rural Science Teachers Social Network Analysis Matrices, Rebecca L. Sansom, Tracy Poulsen

ScholarsArchive Data

These are the social network matrices for our study of rural science teachers. Each matrix is located on a separate tab on the spreadsheet.

Towards Clinical Microscopic Fractional Anisotropy Imaging, Nico Jj Arezza

Electronic Thesis and Dissertation Repository

Microscopic fractional anisotropy (µFA) is a diffusion-weighted magnetic resonance imaging (dMRI) metric that is sensitive to neuron microstructural features without being confounded by the orientation dispersion of axons and dendrites. µFA may potentially act as a surrogate biomarker for neurodegeneration, demyelination, and other pathological changes to neuron microstructure with greater specificity than other dMRI techniques that are sensitive to orientation dispersion, such as diffusion tensor imaging. As with many advanced imaging techniques, µFA is primarily used in research studies and has not seen use in clinical settings.

The primary goal of this Thesis was to assess the clinical viability of …

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 …

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 …

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 …

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 …

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 …

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 …

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 …