Biochemistry, Biophysics, and Structural Biology Commons^™

Proteomic Approaches To Identify Unique And Shared Substrates Among Kinase Family Members, Charles Lincoln Howarth

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease.

Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies …

Deciphering Phosphoprotein Phosphatase Signaling Networks Using Proteomics Approaches, Brooke Brauer

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a highly regulated mechanism of cell signaling control and its deregulation is implicated in disease. The kinases that catalyze the addition of phosphate groups onto their substrate proteins have been well studied, their signaling pathways mapped, and their effects on cell and organismal health observed. Knowledge of the phosphatases that reverse the reaction only recently began to come into focus. Phosphoprotein phosphatases (PPPs), long thought to be housekeeping enzymes, are now known to be exquisitely specific towards their substrates, but the exact nature of phosphatase regulation—both upstream and downstream of the phosphatase—is unclear.

PPPs recognize substrates through …

Probing The Role Of Astrocytes In The Pathology Of Fragile X Syndrome With Human Stem Cells, Baiyan Ren

Theses & Dissertations

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder related to intellectual disability and the most common monogenic cause of autism spectrum disorder. FXS is mainly caused by an expansion of CGG repeats in the 5’-untranslated region of fragile X mental retardation 1 (FMR1) gene, leading to the loss of expression of fragile X mental retardation protein (FMRP). Astrocytes are the most abundant glial cells in the central nervous system (CNS). Loss of FMRP in astrocytes has been found to contribute to structural and functional synaptic deficits in the Fmr1-KO mouse model. The contribution of human astrocytes, however, to the …

Biochemical And Proteomic Approaches To Determine The Impact Level Of Each Step Of The Supply Chain On Tomato Fruit Quality, Robert T. Madden

USF Tampa Graduate Theses and Dissertations

Fresh fruits and vegetables (FFVs) are the most frequently wasted foods because of their perishability and handling requirements. However, there is a lack of information on how much each step of the supply chain impacts FFVs quality, particularly on tomatoes, and what measures need to be taken for an immediate and effective impact on waste reduction. There is also no information on how the supply chain affects the proteome of the tomato and what proteins are differentially regulated by the most impactful steps of the supply chain. The objectives of the work presented on this thesis were to evaluate the …

Composition Of The Survival Motor Neuron (Smn) Complex In Drosophila Melanogaster, A. Gregory Matera, Amanda C. Raimer, Casey A. Schmidt, Jo A. Kelly, Gaith N. Droby, David Baillat, Sara Ten Have, Angus I. Lamond, Eric J. Wagner, Kelsey M. Gray

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated its composition in Drosophila …

Functional Analysis Of The Replication Fork Proteome Identifies Bet Proteins As Pcna Regulators, Sarah R. Wessel, Kareem N. Mohni, Jessica W. Luzwick, Huzefa Dungrawala, David Cortez

Molecular Biosciences Faculty Publications

Identifying proteins that function at replication forks is essential to understanding DNA replication, chromatin assembly, and replication-coupled DNA repair mechanisms. Combining quantitative mass spectrometry in multiple cell types with stringent statistical cutoffs, we generated a high-confidence catalog of 593 proteins that are enriched at replication forks and nascent chromatin. Loss-of-function genetic analyses indicate that 85% yield phenotypes that are consistent with activities in DNA and chromatin replication or already have described functions in these processes. We illustrate the value of this resource by identifying activities of the BET family proteins BRD2, BRD3, and BRD4 in controlling DNA replication. These proteins …

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 …

Mapping Molecular Datasets Back To The Brain Regions They Are Extracted From: Remembering The Native Countries Of Hypothalamic Expatriates And Refugees, Arshad M. Khan, Alice H. Grant, Anais Martinez, Gully Apc Burns, Brendan S. Thatcher, Vishwanath T. Anekonda, Benjamin W. Thompson, Zachary S. Roberts, Daniel H. Moralejo, James E. Blevins

Arshad M. Khan, Ph.D.

The Role Of Phosphohistidine Phosphatase 1 In Ethanol-Induced Liver Injury, Daniel Richard Martin

USF Tampa Graduate Theses and Dissertations

Chronic liver diseases, which includes alcoholic liver disease (ALD), are consistently among the top 15 leading causes of death in the United States. ALD is characterized by progression from a normal liver to fatty liver disease (hepatic steatosis), which can lead to cirrhosis, alcoholic hepatitis, and liver failure. We have identified a novel role of phosphohistidine signaling, mediated through phosphohistidine phosphatase 1 (PHPT1), in the onset of hepatic steatosis. We have identified PHPT1 as a target of selective oxidation following acute ethanol exposure as well as being downregulated following chronic ethanol exposure. We mapped the oxidative modification site and developed …

Mass-Spectrometry Based Proteomics Of Age-Related Changes In Murine Microglia, Antwoine Flowers

USF Tampa Graduate Theses and Dissertations

The last century has seen a steady increase in the extension of the average lifespan. This has concomitantly produced higher incidences of age-related chronic degenerative diseases like Alzheimer’s and Parkinson’s diseases. Age is the single greatest risk factor for the development of not just these degenerative conditions but cancer as well. The aged niche undergoes a number of maladaptive changes that allow underlying conditions to present and progress. Exactly which changes, contribute to the progression of which disease is currently an area of intense study. However, these answers often present therapeutic targets for disease prevention. Age is characterized by a …

Comparative Proteomic Analysis Of Extracellular Vesicles From Prostate Cancer-Derived Cell Lines, Gloria Polanco

Open Access Theses & Dissertations

Prostate cancer (PCa) is the leading non-cutaneous malignancy and the second deadliest among American men. PCa mortality rates among African American men are much higher than any other ethnic group, and the same is true for men of African ancestry world-wide. There is also a lack of reliable diagnostic markers and effective treatment options. Extracellular vesicles (EVs) have been observed to play an important role in cancer processes such as promotion of tumor growth and metastasis. They are also a promising source of diagnostic markers. This study addresses these problems by studying the proteome of EVs derived from PCa cells …

The Role Of Srsf3 In Control Of Alternative Splicing Of Cpeb2 In Triple Negative Breast Cancer, Brian P. Griffin

Theses and Dissertations

In the presented study, we identified that SRSF3 controls the alternative splicing of CPEB2 and consequently promotes a metastatic phenotype in triple negative breast cancer (TNBC). TNBC causes thousands of deaths annually, frequently due to a lack of effective treatments and a high rate of metastasis in patients. Alternative splicing has been found to be dysregulated in numerous cancers, while splicing factors such as SRSF3 are variably expressed. In this study we performed a siRNA panel to screen potential splicing factors, then used specific siRNA to study the effect of its knockdown on cellular function. These results showed that SRSF3 …

Proteomic And Biochemical Studies Of Estrogen-Mediated Signaling And Novel Estrogen Receptor-Interacting Proteins In Breast Cancer Cells, Zhenqi Zhou

Graduate Theses and Dissertations

Estrogen plays essential roles in the growth, development, and homeostasis of a number of tissues, and can also be linked to the growth of breast cancer. The biological activities of estrogen are mediated by estrogen receptors (ERs) ERá and ERâ, and also orphan G-protein-coupled receptor 30 (GPR30). In order to identify novel proteins that are involved in ER-mediated actions of estrogen, we used mass spectrometry-based quantitative proteomic methods to systematically profile global protein expression in responses to E2 (17â-estradiol) stimulation in human breast cancer cell, and identify and characterize cellular novel proteins that are associated with ERs in breast cancer …

Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham

Doctoral Dissertations

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as …

Mass Spectrometry-Based Proteomics Analysis Of The Matrix Microenvironment In Pluripotent Stem Cell Culture, Christopher Hughes

Electronic Thesis and Dissertation Repository

The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.

One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable …

Integrative Bayesian Analysis Of High-Dimensional Multi-Platform Genomics Data, Wenting Wang, Veerabhadran Baladandayuthapani, Jeffrey S. Morris, Bradley M. Broom, Ganiraju C. Manyam, Kim-Anh Do

Jeffrey S. Morris

Motivation: Analyzing data from multi-platform genomics experiments combined with patients’ clinical outcomes helps us understand the complex biological processes that characterize a disease, as well as how these processes relate to the development of the disease. Current integration approaches that treat the data are limited in that they do not consider the fundamental biological relationships that exist among the data from platforms.

Statistical Model: We propose an integrative Bayesian analysis of genomics data (iBAG) framework for identifying important genes/biomarkers that are associated with clinical outcome. This framework uses a hierarchical modeling technique to combine the data obtained from multiple platforms …

Functional Analysis Of Chromodomain Helicase Dna Binding Protein 2(Chd2) Mediated Genomic Stability, Sangeetha Rajagopalan

Doctoral Dissertations

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. …

Prostate Cancer Biomarker Identification: A Comparative Study, Wenjuan Jiang

Computational Modeling & Simulation Engineering Theses & Dissertations

With the current development of proteomics techniques, the discovery of potential molecular biomarkers for early detection of prostate cancer has been greatly improved. In this thesis, we implemented five classifiers including the support vector machine (SVM), Bayesian Classifier, Decision Tree, Random Forest and the Multilayer perceptron (MLP) to test their effectiveness in prostate cancer biomarker identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) tissue imaging data. The classifiers were utilized to discriminate cancer mass spectra from normal ones for the data collected at the Eastern Virginia Medical School (EVMS). There are 94 7 spectra from normal tissues and 27 from …