Biochemistry, Biophysics, and Structural Biology Commons^™

Room-Temperature Crystallography Reveals Altered Binding Of Small-Molecule Fragments To Ptp1b, Tamar Skaist Mehlman, Justin T. Biel, Syeda Maryam Azeem, Elliot R. Nelson, Sakib Hossain, Louise Dunnett, Neil G. Paterson, Alice Douangamath, Romain Talon, Danny Axford, Helen Orins, Frank Von Delft, Daniel A. Keedy

Advanced Science Research Center

Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of …

Cell Signaling And Stress Response In The Yeast Saccharomyces Cerevisiae: A Study Of Snf1, Scott E. Arbet Ii

Graduate Theses, Dissertations, and Problem Reports (ETD)

Saccharomyces cerevisiae are yeast that are unicellular eukaryotic organisms that are well studied as a model organism for understanding fundamental cellular processes. The ability of yeast to sense nutrient availability is crucial for their survival, growth, and reproduction. Yeast cells use various mechanisms to sense and respond to nutrient availability, including transporter-mediated uptake, receptor-mediated signaling, and sensing of metabolites. The subcellular localization of nutrient-sensing components is crucial for yeast function in nutrient sensing and signaling. Protein complexes, such as the AMP-activated protein kinase (AMPK) pathway, in nutrient sensing and response, as well as the downstream effects of these pathways …

The Affect Of Dha Concentration On Lipid Membrane Fluidity, Elise Rezabeck

Williams Honors College, Honors Research Projects

My proposed project will be creating large unilaminar vesicles with varying concentrations of DHA in the vesicles. The diffusion in and out of these vesicles will be tracked by using singular lipid molecules. These singular lipid molecules will be tagged with fluorescence and viewed using an internal reflection fluorescence microscope. The data collected over the course of this project will then be analyzed using Matlab. The result of the project will be to find the specific lipid diffusion constant of these unilaminar vesicles. The relationship between the concentration of DHA and lipid diffusion and membrane fluidity will be found as …

Dual Mechanisms Contributing To Pyruvate Dehydrogenase Activity Deficiency In A Barth Syndrome Cell Model, Zhuqing Liang

Wayne State University Dissertations

Barth syndrome (BTHS) is a rare genetic disease that results from mutations in the TAFAZZIN gene, which encodes the cardiolipin (CL) remodeling enzyme tafazzin (Taz). The mechanisms linking perturbation of CL remodeling and the pathological features of BTHS are not understood. We have recently reported that intermediary metabolism is perturbed in BTHS, and activity of the metabolic gatekeeper enzyme pyruvate dehydrogenase (PDH) is deficient in BTHS models. The mechanism whereby PDH is regulated by Taz is unknown, and this knowledge gap represents an obstacle to the development of therapeutics to treat BTHS. Using an established C2C12 myoblast model of BTHS, …

Biological Adaptations Leading To Metastatic Potential In Ovarian Cancer, Hussein Chehade

Wayne State University Dissertations

Ovarian cancer induces most deaths from gynecological cancers. Patients succumb due to heavy metastatic tumor burden, which accounts for 90% of cancer related deaths and is characterized by the dissemination of malignant cells from primary tumor to secondary site(s), partaking in cancer progression. Ovarian cancer progression is an extremely demanding process that requires cancer cells to be able to survive anoikis conditions and be metabolically flexible to survive microenvironmental changes during transit to a secondary site. Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1) is a bi-organellar protein primarily present in the mitochondria by which it can prevent apoptosis and/or enhance mitochondrial …

From Culture To Metagenomics: A Comprehensive Evaluation Of The Female Reproductive Tract Microbiome In Pregnancy, Jonathan Panzer

Wayne State University Dissertations

The advent of Next Generation Sequencing technologies prompted novel investigations of the microbiome. These revealed both increased diversity of known microbiomes such as those of the gut, oral cavity and vagina, but also bacterial DNA in tissues previously considered sterile such as the blood, liver, brain, and placenta. Thus, previous paradigms of sterility, based primarily on culture data, were called into question. However, interpretation of bacterial DNA signals from low-microbial biomass tissues is complex due to the strong potential for contamination and interference from the surrounding environment. Yet, many new methodologies are being developed to help isolate valid bacterial DNA …

Food Waste Storage Gaseous Emissions Detection And Quantification Using Infrared Spectroscopy, Ryley A. Burton-Tauzer

Cal Poly Humboldt theses and projects

A growing interest in sustainable waste management and the implementation of new policies have prompted a shift towards alternative resource recovery methods for organic waste, including food waste. To effectively assess alternative food waste treatment scenarios, it is important to evaluate the life cycle impacts associated with each scenario. The storage phase of food waste, encompassing its accumulation in kitchens, and storage in bins for collection and transportation, has been overlooked as a source of greenhouse gases in previous studies. This investigation aimed to identify the greenhouse gases emitted during the initial five-day period of low-oxygen storage. An open dynamic …

Characterizing Vhh Domains As Stabilization And Oligomeric Control Modules, Niyati Patel

Graduate Research Theses & Dissertations

The biophysical properties of the VHH domain from heavy-chain-only antibodies obtained from the Camelidae family has expanded opportunities for novel antibody applications. The small, single domain nature of VHH domains makes them attractive modules for fusion proteins. Here, two VHH fusion systems were evaluated, RNase A-anti-RNase A VHH and GFPMut3aR80Q-anti-GFP VHH, to investigate how antigen-VHH fusions may enhance protein thermostability. The introduction of various length flexible linkers was hypothesized to play a role in stability, as well as influence the oligomeric state. Bovine Pancreatic Ribonuclease A, or RNase A, is an enzyme belonging to the ribonuclease A superfamily, which is …

Role Of Micrornas In Intestinal Inflammation And Barrier Homeostasis After Alcohol And Burn Injury, Caroline J. Herrnreiter

Dissertations

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression. Within the intestinal epithelium, miRNAs play a critical role in gut homeostasis and aberrant miRNA expression has been implicated in various disorders associated with intestinal inflammation and barrier disruption. In this study, we sought to profile changes in intestinal epithelial cell miRNA expression after alcohol and burn injury and elucidate their impact on inflammation and barrier integrity. In a more targeted approach, we began by focusing on anti-inflammatory miRNAs that, when downregulated, could exacerbate inflammation and result in intestinal barrier disruption. Using a mouse model of acute ethanol intoxication …

Effects Of Nicotine Vs Non-Nicotine E-Cigarettes On The Aggression Behavior Of Crayfish, Alex Kimble

Williams Honors College, Honors Research Projects

This study looks at the effects of e-cigarette vapor with and without nicotine on the aggression behavior of crayfish (Procambarus clarkii). The treatment groups were control, nicotine, and non-nicotine. The mass, sex, and condition of their appendages were recorded before random assignment. For the purpose of this study only male crayfish were used as there were not enough female crayfish. The crayfish were then randomly assigned to each group and contained ten crayfish that were subjected to the e-cigarette vapor infused water environment for a twenty-four hour period. The control group did not contain e-cigarette vapor and was exposed to …

Effect Of An Oncogenic Her2 Mutant On Egfr Activity, Paige Kalik

Williams Honors College, Honors Research Projects

Studies have shown that HERs are involved in the progression of human cancers. Researchers have found that HER2 mutations in colorectal cancer are similar to mutations found in breast cancer. Recombinant antibodies are effective therapeutic treatments for cancers. Antibodies can also be used to determine how proteins function under normal and pathophysiological conditions while also identifying interacting partners. In this Honors Project, my aim is to investigate the effect of an oncogenic mutant of HER2. Considering the information above, the question researched in this project was: how does the S310F mutation affect the organization and function of HER2? The specific …

A Characterization Of Key Residues In Class I Viral Fusion Proteins Important For Fusogenic Activity, Hadley E. Neal

Theses and Dissertations--Molecular and Cellular Biochemistry

Viral fusion proteins are critical for viral entry and subsequent infection. Class I fusion proteins are characterized by synthesis as an inactive precursor requiring cleavage by a host cell protease to become fusion competent. Though vaccine and antiviral therapeutic developments often target the fusion protein, questions surrounding cleavage dynamics and protein stability remain. The work presented in this dissertation investigates specific regions of three class I viral fusion proteins in an effort to identify key residues involved in proteolytic processing and membrane fusion.

The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) mediates receptor binding, facilitates fusion …

Characterization Of The Function And Regulation Of The Hmpv Phosphoprotein, Rachel Thompson

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus (NNSV) that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. Despite the initial identification of HMPV in 2001, there are currently no FDA approved antivirals or vaccines available. Therefore, understanding the mechanism of HMPV replication is critical for the identification of novel therapeutic targets. A key feature in the replication cycle of HMPV and other NNSVs is the formation of membrane-less, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). Recent work on NNSV IBs suggests they display characteristics of biomolecular condensates formed …

The Role Of Chromatin Structure In Circular Rna Biogenesis, Function And Disease, Rebekah Eleazer

Theses and Dissertations--Molecular and Cellular Biochemistry

Fundamental to cellular differentiation, development, and physiology is the precise regulation of gene expression. At the center of this control lies chromatin, and disruptions in chromatin biology are key contributors to complex human diseases. By convention, gene expression analysis primarily focuses on transcript abundance, however the splicing of transcripts into distinct isoforms is also of biological relevance. One new form of alternative splicing, backsplicing, is responsible for generating circular RNAs (circRNAs), which are emerging as essential components to gene regulatory networks. However, unlike other forms of alternative splicing, the role that chromatin plays in backsplicing and circRNA formation remains poorly …

Investigating The Roles Of Platelet Bioenergetics In Hemostasis And Thrombosis, Kanakanagavalli Shravani Prakhya

Theses and Dissertations--Molecular and Cellular Biochemistry

Platelets use a series of energy-dependent processes to mediate hemostasis, yet it is unclear which metabolic fuels/pathways are critical for platelet function. Platelets are metabolically flexible, switching between glycolysis and oxidative phosphorylation (OxPhos) depending on oxygen tension and substrate availability. The deletion of GLUT1 and GLUT3 together revealed an essential role in platelet function and hemostasis. To study platelet bioenergetics, we used clot contraction. We created a sensitive and inexpensive method to simultaneously assess clot contraction kinetics to directly compare and define the effects of different treatments or genetic manipulations on platelet function.

Since the pharmacological inhibitors cannot be used …

The Role Of Dynamin Related Protein 1-Mediated Mitochondrial Dynamics In Colorectal Cancer, Sumati Raj Hasani

Theses and Dissertations--Molecular and Cellular Biochemistry

Cancer cells are known for their ability to adapt variable metabolic programs depending on the availability of specific nutrients. Consequently, metabolic reprogramming has been increasingly recognized as a major mechanism that fuels tumorigenesis and disease progression. Here, we have investigated the role of a pro-fission factor, Dynamin-Related Protein 1 (Drp1), in promoting metabolic adaptation in colon cancer.

Our studies have shown that fatty acid (FAs) uptake alters cellular metabolic pathways in colon cancer cells to favor fatty acid oxidation through the activation of Drp1. Uptake of FAs induces mitochondrial fragmentation by promoting ERK-dependent phosphorylation of Drp1 at the S616 site, …

The Development And Characterization Of Nanobodies Specific To Protein Tyrosine Phosphatase 4a3 (Ptp4a3/Prl-3) To Dissect And Target Its Role In Cancer., Caroline Smith

Theses and Dissertations--Molecular and Cellular Biochemistry

Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, …

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

The Effect Of Pomegranate Consumption On The Gut Microbiome, Brant Bandow

Browse all Theses and Dissertations

Pomegranate is known to have antioxidant and prebiotic qualities that have been shown to promote the growth of beneficial bacteria while reducing inflammation in the gut. Inflammation in the gut is an issue that results in many health problems including obesity and colon cancer. In this study, an experimental group received a daily pomegranate supplement for three weeks where a control group did not receive any supplement. After sequencing gDNA isolated from fecal samples from both before and after the trial period there was a significant difference between the two groups. The largest amount of variability is attributed to the …

A Novel Role Of Lipin1 In The Regulation Of Expression And Function Of Nnos, Ayat Azzam

Browse all Theses and Dissertations

Duchenne muscular dystrophy (DMD) is a severe and progressive muscular dystrophy that develops in the skeletal muscles because of mutations in the dystrophin gene. Dystrophin stabilizes sarcolemma and assembles neuronal nitric oxide synthase (nNOS) into the dystrophin-associated protein complex on the sarcolemma. The absence of dystrophin triggers the delocalization of nNOS and contributes to the misregulation of muscle development, blood flow, muscle fatigue, and inflammation. Lipin1 was reduced in the skeletal muscles of patients with DMD and the mdx mouse model of DMD. In this study, we explored the role of lipin1 in the regulation of nNOS expression and function. …

Specialized Metabolism In Retina, Retinal Pigmented Epithelium, And Testis, Siyan Zhu

Graduate Theses, Dissertations, and Problem Reports (ETD)

The retina and its neighboring retinal pigmented epithelium (RPE) are high energy-demanding and metabolically active tissues with specialized and complementary metabolism. They are metabolically interdependent and impact each other’s viability. Interestingly, many of the metabolic features in the retina and RPE are shared with the testis. For example, testis is also energy costly due to continuous sperm differentiation and it has similar metabolic inter-dependence between different testis cells. Both the retina and testis are vulnerable to mitochondrial metabolic impairments.

We conducted three research projects to understand 1) the nutrient utilization and communication in retina and RPE; 2) The profiling of …

Lipid Binding Properties Of Huntingtin As A Novel Therapeutic Target, Chathuranga Siriwardhana

Graduate Theses, Dissertations, and Problem Reports (ETD)

As protein aggregation is the defining hallmark of all amyloid diseases, a common therapeutic strategy is to develop molecules that inhibit aggregation. However, this approach has yielded limited success. Many amyloid proteins directly interact with lipid membranes. These interactions promote distinct aggregation pathways and often result in membrane damage leading to toxicity. As a result, directly targeting the ability of amyloids to bind lipid membranes represents a novel therapeutic strategy. As a proof of principle, the interaction between lipid membranes and mutant huntingtin protein (htt) aggregates was used to test this strategy. Mutant htt containing an expanded polygulatmine (polyQ) domain …

Exogenous Factors That Impact Huntingtin Aggregation, Adam Skeens

Graduate Theses, Dissertations, and Problem Reports (ETD)

While expansion of a polyglutamine (polyQ) domain is the immediate cause of huntingtin (htt) aggregation associated with Huntington’s Disease (HD), other cellular factors modify aggregation. These include interactions with cellular membranes, protein biding partners, molecular crowding, and proteinaceous seeds. Here, two important factors are biophysically characterized: 1) the interaction of htt with endomembranes and 2) proteinaceous seeds obtained from a variety of htt-derived peptides. In the first project, the aggregation of htt at bilayer interfaces and in the presence of divalent cations was investigated. A major cellular factor implicated in altered htt aggregation is the binding of lipids. Furthermore, the …

Investigation Of Early Complex Formation Of Huntingtin Protein With And Without Lipids, Alyssa R. Stonebraker

Graduate Theses, Dissertations, and Problem Reports (ETD)

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by the expansion of the polyglutamine (polyQ) domain of the huntingtin protein (htt). The expansion of the polyQ domain beyond a threshold of approximately 35 repeats triggers complex toxic aggregation mechanisms and results in altered interactions between htt and lipid membranes. Many factors modulate these processes. One such modulator includes sequences flanking the polyQ domain, most notably the first 17 amino acids at the N-terminus of the protein (Nt17), and environmental factors including the presence of membranous structures. Nt17 has the propensity to form an amphipathic a-helix in the presence of …

Investigating The Role Of Nudt7-Mediated Peroxisomal Coa Degradation In The Regulation Of Hepatic Coa Levels And Lipid Metabolism, Schuyler Dan Adams Vickers

Graduate Theses, Dissertations, and Problem Reports (ETD)

Coenzyme A (CoA) is a crucial cofactor required to support many essential metabolic processes, including fatty acid oxidation and synthesis, the TCA cycle, and bile acid synthesis. In order to support the proper functioning of these pathways, CoA levels within the cell must be properly regulated. The regulation of CoA levels is achieved through a balance between CoA synthesis and CoA degradation. While the regulation of CoA synthesis has been extensively characterized, the degradation of CoA has received less attention. As such, the contribution of CoA degradation to the regulation of metabolism is poorly understood. NUDT7 is a CoA-specific Nudix …

Elucidating Mechanisms Of Proteasome Modulation By Small Molecules To Treat Neurodegenerative Diseases And Cancer, Janelle Joo Yee Chuah

Graduate Theses, Dissertations, and Problem Reports (ETD)

Maintaining protein homeostasis, or proteostasis, is crucial for cellular function, and protein degradation through the proteasome plays a pivotal role in this process. Dysregulation of the proteasome can lead to proteostasis collapse and has been linked to the development of neurodegenerative diseases and cancers. Neurodegenerative diseases often exhibit proteasome impairment, whereas proteasome upregulation is frequently observed in cancer. Although the use of proteasome inhibitors as a pharmaceutical approach has shown success in certain cancers, it has yet to demonstrate efficacy in neurodegenerative diseases due to the absence of drug-like small molecules capable of effectively reversing proteasome impairment and/or activating the …

Heat Shock Protein 90 (Hsp90) System In Health And Disease., Daniella Munezero

Graduate Theses, Dissertations, and Problem Reports (ETD)

Cells rely on heat shock proteins (HSP) to facilitate and regulate the folding of the substrate proteins into their native state, and degradation if misfolding cannot be prevented. HSP90, a member of the HSP family, is a potential target for treatment of cancer and neurodegenerative diseases. Unfortunately, several clinical trials for cancer treatment have been discontinued due to cell toxicity accompanying HSP90 inhibition. HSP90 has four distinct but structurally similar paralogs. HSP90 inhibitors target all the paralogs despite increasing proof of functional differences among the paralogs. Understanding the in vivo function of HSP90 and the role played by each paralog …

Application Of Computational Biophysics Techniques To Characterize Cell Membrane-Associated Events, Kyle Billings

Graduate Theses, Dissertations, and Problem Reports (ETD)

Cell membranes are crowded environments which can modulate protein structure-function relationships through interaction with lipids, other proteins, carbohydrate structures and so on. This work focuses the impact of the membrane environment on two varieties of peptides: Microbial rhodopsin proteins, and cyclic peptides.

Life on Earth is dependent on the ability of plants and microbes to harness sunlight for energy production. Their ability to transform light into carbohydrates requires tailor-made machinery, and for a wealth of microorganisms, microbial rhodopsin proteins (MR) are critical for maintaining the concentration gradients used to produce the energy molecule Adenosine triphosphate (ATP). The central retinal molecule …

Cup Is Essential For Oskar Mrna Translational Repression During Early Drosophila Oogenesis, Livia V. Bayer, Samantha Milano, Stephen K. Formel, Harpreet Kaur, Rishi Ravichandran, Juan A. Cambeiro, Lizaveta Slinko, Irina E. Catrina, Diana P. Bratu

Publications and Research

Study of the timing and location for mRNA translation across model systems has begun to shed light on molecular events fundamental to such processes as intercellular communication, morphogenesis, and body pattern formation. In D. melanogaster, the posterior mRNA determinant, oskar, is transcribed maternally but translated only when properly localized at the oocyte’s posterior cortex. Two effector proteins, Bruno1 and Cup, mediate steps of oskar mRNA regulation. The current model in the field identifies Bruno1 as necessary for Cup’s recruitment to oskar mRNA and indispensable for oskar’s translational repression. We now report that this Bruno1-Cup interaction leads …

Tcga Expression Analyses Of 10 Carcinoma Types Reveal Clinically Significant Racial Differences, Brian Lei, Xinyin Jiang, Anjana Saxena

Publications and Research

Epidemiological studies reveal disparities in cancer incidence and outcome rates between racial groups in the United States. In our study, we investigated molecular differences between racial groups in 10 carcinoma types. We used publicly available data from The Cancer Genome Atlas to identify patterns of differential gene expression in tumor samples obtained from 4112 White, Black/African American, and Asian patients. We identified race-dependent expression of numerous genes whose mRNA transcript levels were significantly correlated with patients’ survival. Only a small subset of these genes was differentially expressed in multiple carcinomas, including genes involved in cell cycle progression such as CCNB1 …