Biochemistry, Biophysics, and Structural Biology Commons^™

Computational Design Of The Affinity And Specificity Of A Therapeutic T Cell Receptor, Brian G. Pierce, Lance M. Hellman, Moushumi Hossain, Nishant K. Singh, Craig W. Vander Kooi, Zhiping Weng, Brian M. Baker

Molecular and Cellular Biochemistry Faculty Publications

T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. …

Ube3a Role In Synaptic Plasticity And Neurodevelopmental Disorders.The Lessons From Angelman Syndrome., Irina Filonova

USF Tampa Graduate Theses and Dissertations

Angelman Syndrome (AS) is a severe neurodevelopmental disorder that affects 1:12000 newborns. It is characterized by mental retardation, delayed major motor and cognitive milestones, seizures, absence of speech and excessive laughter. The majority of AS cases arise from deletions or mutations of UBE3A gene located on the chromosome 15q11-13. UBE3A codes for E3-ubiquitin ligase that target specific proteins for degradation. To date, a wide variety of Ube3a substrates has been identified. The accumulation of Ube3a-dependent proteins and their effect on the multitude of signal transduction pathways are` considered the main cause of the AS pathology. While the majority of research …

Structural Dynamics Of A Mitochondrial Trna Possessing Weak Thermodynamic Stability, Hari Bhaskaran, Takaaki Taniguchi, Takeo Suzuki, Tsutomu Suzuki, John J. Perona

Chemistry Faculty Publications and Presentations

Folding dynamics are ubiquitously involved in controlling the multivariate functions of RNAs. While the high thermodynamic stabilities of some RNAs favor purely native states at equilibrium, it is unclear whether weakly stable RNAs exist in random, partially folded states or sample well-defined, globally folded conformations. Using a folding assay that precisely tracks the formation of native aminoacylable tRNA, we show that the folding of a weakly stable human mitochondrial (hmt) leucine tRNA is hierarchical with a distinct kinetic folding intermediate. The stabilities of the native and intermediate conformers are separated by only about 1.2 kcal/mol, and the species are readily …

Septin Assemblies Form By Diffusion-Driven Annealing On Membranes, Andrew A. Bridges, Huaiying Zhang, Shalin B. Mehta, Patricia Occhipinti, Tomomi Tani, Amy S. Gladfelter

Dartmouth Scholarship

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make …

Methylseleninic Acid Elevates Redd1 And Inhibits Prostate Cancer Cell Growth Despite Akt Activation And Mtor Dysregulation In Hypoxia, Indu Sinha, Joshua Allen, John T. Pinto, Raghu Sinha

NYMC Faculty Publications

Methylseleninic acid (MSeA) is a monomethylated selenium metabolite theoretically derived from subsequent β-lyase or transamination reactions of dietary Se-methylselenocysteine that has potent antitumor activity by inhibiting cell proliferation of several cancers. Our previous studies showed that MSeA promotes apoptosis in invasive prostate cancer cells in part by downregulating hypoxia-inducible factor HIF-1α. We have now extended these studies to evaluate the impact of MSeA on REDD1 (an mTOR inhibitor) in inducing cell death of invasive prostate cancer cells in hypoxia. In both PTEN+ and PTEN- prostate cancer cells we show that MSeA elevates REDD1 and phosphorylation of AKT along with p70S6K …

The Abcs Of The Ribosome, Kurt Fredrick, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

An ABC protein that binds the ribosomal exit site suggests a new mechanism for direct regulation of translation in response to changing ATP levels in the cell.

Trans Fatty Acids – A Risk Factor For Cardiovascular Disease, Mohammad Perwaiz Iqbal

Department of Biological & Biomedical Sciences

Trans fatty acids (TFA) are produced either by hydrogenation of unsaturated oils or by biohydrogenation in the stomach of ruminant animals. Vanaspati ghee and margarine have high contents of TFA. A number of studies have shown an association of TFA consumption and increased risk of cardiovascular disease (CVD). This increased risk is because TFA increase the ratio of LDL cholesterol to HDL cholesterol. Food and Agriculture Organization of the United Nations and World Health Organization have come up with the recommendation that the contents of TFA in human dietary fat should be reduced to less than 4%. There is high …

On Chip Preconcentration And Labeling Of Protein Biomarkers Using Monolithic Columns, Device Fabrication, Optimization, And Automation, Rui Yang

Theses and Dissertations

Detection of disease specific biomarkers is of great importance in diagnosis and treatment of diseases. Modern bioanalytical techniques, such as liquid chromatography with mass spectrometry (LC-MS), have the ability to identify biomarkers, but their cost and scalability are two main drawbacks. Enzyme-linked immunosorbent assay (ELISA) is another potential tool, but it works best for proteins, rather than peptide biomarkers. Recently, microfluidics has emerged as a promising technique due to its small fluid volume consumption, rapidness, low fabrication cost, portability and versatility. Therefore, it shows prominent potential in the analysis of disease specific biomarkers. In this thesis, microfluidic systems that integrate …

Computational Insights Into The Oxygen Evolving Complex Of Photosystem Ιι, Muhamed Amin

Dissertations, Theses, and Capstone Projects

The Oxygen Evolving Complex (OEC) of Photosystem II (PSII) is a unique Mn4O5Ca2+ cluster that catalyzes the photoactivated water splitting reaction. The OEC is a model system for bio-inspired artificial systems to use solar energy to pull electrons from water to produce fuel. The OEC goes through a cycle of 5 S states storing 4 holes, via electron transfer to P680+, the primary electron donor in PSII to generate a high valence S4 state that oxidizes water. The key questions are what controls the order of oxidation and deprotonation of the OEC complex and how does the PSII protein modulate …

Conformational Features Of The Human U2-U6 Snrna Complex, Ravichandra Bachu

Dissertations, Theses, and Capstone Projects

The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several …

Polymerase Alpha Components Associate With Telomeres To Mediate Overhang Processing, Raffaella Diotti

Dissertations, Theses, and Capstone Projects

Telomeres consist of TTAGGG repeats, which end with a 3' G-overhang and are bound by a six-protein complex, known as Shelterin. In humans, telomeres shorten at each cell division, unless telomerase is expressed and able to add telomeric repeats to the 3' G-overhang. However, for effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. In this study, I focused on the Polα/primase complex, in particular the subunits p68 (POLA2, the regulatory subunit) and p180 (Polα, the catalytic subunit), and their potential roles at telomeres. I was able to detect p180, p68 and OBFC1, a …

Mechanism Of Phospho-Alpha-Tubulin-Driven Motility In Human Breast Epithelial Cells, Shatarupa De

Dissertations, Theses, and Capstone Projects

Protein kinase C (PKC), an enzyme important in signaling pathways that give rise to various cell phenotypes, has been closely associated with metastatic phenotypes of breast cancer. Tissues from patients of varying degrees of tumorgenicity have shown an elevated expression of the PKCα isoform as well as other diacylglycerol (DAG)-sensitive isoforms. Moreover, independent studies with highly invasive breast cell lines, such as MDA-MB-231 cells, have also shown an elevated level of PKCα. Therefore, PKCα has long been a therapeutic target for breast cancer. Among its known substrates (recently identified by our laboratory), α6-tubulin will, upon phosphorylation by PKC, enhance migration …

Iron Regulates Mrna Translation Initiation Through Rna Iron Responsive Element (Ire), Jia Ma

Dissertations, Theses, and Capstone Projects

The non-coding IRE-RNA structure, a 30 nt stem loop structure, regulates synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA binding with iron regulatory protein (IRP) proteins inhibits ribosome accessing mRNA. Increasing iron concentration decreases IRP binding with IRE-RNA. Previous models of IRE-mRNA translation regulation concentrate on Fe-S binding to IRP and IRP degradation after release from IRE-RNA. These models lack information on the details of decreasing IRE-RNA/IRP protein binding with iron concentration elevation. This research shows 1. Eukaryotic initiation factor 4F (eIF4F) binds to IRE-RNA with high affinity and works as a positive control element …

The Sex Of The Cell Dictates Its Response, Carlos Ganesh Penaloza

Dissertations, Theses, and Capstone Projects

Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of …

Lipid Dependence In Ras-Driven Tumors, Darin Salloum

Dissertations, Theses, and Capstone Projects

Over past decade, metabolic alterations in cancer cells have received a substantial amount of interest. It had been established that cancer cells undergo a significant amount of metabolic alterations, and some of these alterations are similar to those in normal highly proliferative cells. However, it is becoming more apparent that many of the metabolic alterations are specific to particular oncogenic signaling pathways. Although altered metabolic machinery makes cancer cells more efficient at promoting growth when nutrients are supplied at the sufficient amounts, the dependency of cancer cells on particular metabolic reprogramming deems cancer cells susceptible to disruptions within metabolic network. …

Metabolic Checkpoints In Cancer Cell Cycle, Mahesh Saqcena

Dissertations, Theses, and Capstone Projects

Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from …

The Chromatin Architectural Proteins Hmgd1 And H1 Bind Reciprocally And Have Opposite Effects On Chromatin Structure And Gene Regulation, Narasimharao Nalabothula, Graham Mcvicker, John Maiorano, Rebecca Martin, Jonathan K. Pritchard, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins.

RESULTS: Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1 in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. We find …

An Experimental Investigation Into The Mechanisms Of Bacterial Evolution, Zhenmao Wan

Dissertations, Theses, and Capstone Projects

This thesis studies the two fundamental mechanisms of bacterial evolution — horizontal gene transfer and spontaneous mutation, in the bacterium Escherichia coli through novel experimental assays and mathematical simulations. First, I will develop a growth assay utilizing the quantitative polymerase chain reaction (qPCR) to provide real-time enumeration of genetic marker abundance within bacterial populations. Second, I will focus on horizontal gene transfer in E. coli occurring through a process called conjugation. By fitting the qPCR data to a resource limited, logistic growth model, I will obtain estimated values of several key parameters governing the dynamics of DNA transfer through conjugation …

Mechanisms Of Deadenylation Regulation Under Different Cellular Conditions, Xiaokan Zhang

Dissertations, Theses, and Capstone Projects

Control of gene expression by regulating mRNA stability after DNA damage has the potential to contribute to the cells rapid response to stress. The main focus of this dissertation is to elucidate the role(s) of nuclear PARN deadenylase in controlling mRNA stability, hence gene expression, of factors in the p53 signaling pathway during the DNA damage response (DDR). Understanding the mechanisms of these regulatory pathways will provide new insights on how the control of gene expression upon DNA damage decides cellular fate, offering new opportunities for therapeutics. In Chapter II, I presented evidence that PARN along with the cleavage factor …

Roles Of Igh Intronic Enhancer Eμ In Clonal Selection At The Pre-B To Immature B Cell Transition And In The Elimination Of Autoreactive B Cells, Cheng Peng

Dissertations, Theses, and Capstone Projects

The immunoglobulin heavy chain locus (Igh ) intronic enhancer, Eμ, enhances transcription of recombined Igh genes. We have previously shown that in mice with an Eμ-deficient Igh allele (V _H δ^a ), Igμ is expressed at half of the wild-type levels in pre-B cells. We also described an Eμ-dependent "check-point", operating at the pre-B to immature B cell transition, for heavy chain allelic exclusion. We now show that deletion of Eμ results in a smaller immature B cell compartment, and the pre-BCR/BCR signaling is diminished in pre-B cells as a result of the reduced Igμ levels, making it …