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Desorption Electrospray Ionization Mass Spectrometry Imaging: Instrumentation, Optimization And Capabilities, Manan Dhunna

Theses and Dissertations

Desorption Electrospray Ionization Mass spectrometry Imaging (DESI-MSI) is an area of great interest and a promising tool in the field of chemical imaging. It is a powerful, label-free technique, which can determine, map and visualize different molecular compounds on a sample surface. The amount of information acquired in a single DESI-MSI experiment is enormous compared to other techniques, as it can simultaneously detect different compounds with their spatial distribution on the surface. The experiment can be used to produce two-dimensional and three-dimensional images. Chapter 2 focuses on the design and optimization of the setup for performing DESI-MS imaging on various …

Facile Synthesis And Characterization Of A Thermally Stable Silica-Doped Alumina With Tunable Surface Area, Porosity, And Acidity, Maryam Khosravi Mardkhe

Theses and Dissertations

Mesoporous γ-Al2O3 is one of the most widely used catalyst supports for commercial catalytic applications. The performance of a catalyst strongly depends on the combination of textural, chemical and physical properties of the support. Pore size is essential since each catalytic system requires a unique pore size for optimal catalyst loading, diffusion and selectivity. In addition, high surface area and large pore volume usually result in higher catalyst loading, which increases the number of catalytic reaction sites and decreases reaction time. Therefore, determination of surface area and porosity of porous supports is critical for the successful design and optimization of …

An Optogenetic Gene Expression System With Rapid Activation And Deactivation Kinetics, Laura B. Motta-Mena, Anna Reade, Michael J. Mallory, Spencer Glantz, Orion D. Weiner, Kristen W. Lynch, Kevin H. Gardner

Advanced Science Research Center

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range, or slow activation/deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach utilizes an engineered version of EL222, a bacterial Light-Oxygen-Voltage (LOV) protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (< 10 s) and deactivation kinetics (< 50 s), and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

Structural Insights Into The Interaction Between A Potent Anti-Inflammatory Protein, Viral Cc Chemokine Inhibitor (Vcci), And The Human Cc Chemokine, Eotaxin-1, Nai-Wei Kuo, Yong-Guang Gao, Megan Schill, Nancy Isern, Cynthia Dupureur, Patricia Liwang

Chemistry & Biochemistry Faculty Works

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N …

Structural Insights Into The Interaction Between A Potent Anti-Inflammatory Protein, Viral Cc Chemokine Inhibitor (Vcci), And The Human Cc Chemokine, Eotaxin-1*, Nai-Wei Kuo, Yong-Guang Gao, Megan S. Schill, Nancy Isern, Cynthia M. Dupureur, Patricia J. Liwang

Educator Preparation & Leadership Faculty Works

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β …

Systems Thinking With Biology Models, Joseph Dauer

Discipline-Based Education Research Group Speaker Series

The recent AAAS call to improve undergraduate biology education suggested university instruction should focus on teaching core concepts like matter and energy, evolution, and systems and core competences like quantitative reasoning, modeling and integrating disciplines. My research has focused on how undergraduate biology students organize their knowledge of biological systems and how they reason about the myriad interactions and potential outcomes inherent to these systems. I will report ongoing research into students’ model construction during an introductory biology course and during clinical interviews 2 years after the course. My colleagues and I have found students’ models change dramatically in both …

Structures Of The Puta Peripheral Membrane Flavoenzyme Reveal A Dynamic Substrate-Channeling Tunnel And The Quinone-Binding Site, Harkewal Singh, Benjamin W. Arentson, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004- residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal …

Evaluation Of Novel Oral Vaccine Candidates And Validation Of A Caprine Model Of Johne’S Disease, Murray E. Hines Ii, Sue E. Turnquist, Marcia R.S. Ilha, Sreekumari Rajeev, Arthur L. Jones, Lisa Whittington, John P. Bannantine, Raul G. Barletta, Yrjö T. Gröhn, Robab Katani, Adel M. Talaat, Lingling Li, Vivek Kapur

School of Veterinary and Biomedical Sciences: Faculty Publications

Johne’s disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn’s disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne’s Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne’s disease go at challenge model. …

Molecular Pap Biomarkers For Cervical Cancer Risk Assessment In Thinprep® Clinical Specimens, Jennifer Taylor

Graduate School of Biomedical Sciences Theses and Dissertations

Approximately 500,000 patients are diagnosed with cervical cancer worldwide each year, thus making it the second most common cause of cancer-related deaths in women. Persistent high-risk human papillomavirus (HR-HPV) infections induce cervical dysplasia by increasing the expression of two viral oncogenes, E6 and E7. Utilization of the Papanicolaous smear test (Pap test) has contributed to a decrease in morbidity and mortality of cervical cancer; however, subjective analysis of cell morphology with a limited amount of cells has led to a substantial rate of false positive and false negative diagnoses. A more objective and sensitive diagnostic tool for the detection of …

Low Doses Of Colony-Stimulating Factors Lead To Resolution Of Neutropenia In Cancer Patients Through Increased Levels Of Dihydrofolate Reductase, Mohammad Perwaiz Iqbal, Ikram Ali Burney

Department of Biological & Biomedical Sciences

Low doses of granulocyte- colony stimulating factor (G-CSF) and granulocyte macrophage- colony stimulating factor (GM-CSF) have been shown to be beneficial in reducing duration of systemic antibiotic therapy and in-patient hospitalization by decreasing the period of neutropenia in cancer patients undergoing chemotherapy. Since the underlying mechanism is unclear, the aim of this study was to investigate whether the administration of G-CSF and GM-CSF in two different doses (low dose and standard dose) would result into resolution of neutropenia with concomitant increase in multiple forms of dihydrofolate reductase (DHFR, a pivotal enzyme in the pathway of de novo DNA synthesis). Thirty …

A Lipid-Rich Gestational Diet Predisposes Offspring To Nonalcoholic Fatty Liver Disease: A Potential Sequence Of Events, Alexandria N. Hughes, Julia Thom Oxford

Biomedical Research Institute Publications and Presentations

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. It affects 20%–30% of the US population, and it is increasing worldwide. Recently, the role of lipid-rich maternal gestational nutrition in spurring the development of NAFLD among offspring has been indicated. Fetal predisposition to NAFLD involves numerous physiological reroutings that are initiated by increased delivery of nonesterified fatty acids to the fetal liver. Hampered ß-oxidation, uncontrolled oxidative stress, increased triacylglycerol synthesis, and the endoplasmic reticulum unfolded protein response are all implicated in sculpting a hepatic phenotype with a propensity to develop NAFLD in the postnatal state. This review …

Survivin: Regulation By Yy1 And Role In Pancreatic Cancer Combination Therapy, Nicholas R. Galloway

Loma Linda University Electronic Theses, Dissertations & Projects

Despite significant clinical and basic science advancements, cancer remains a devastating disease that affects people of all ages, races, and background. Survivin, the fourth most common transcript found in cancer cells, is a protein that is thought to be involved in the enhanced proliferation, survival, and metastasis of cancer cells. Therefore understanding how this gene is regulated is potentially of vital importance to improving cancer management and therapy. Our work has identified a novel transcriptional regulator of survivin called Yin Yang 1 (YY1). YY1 is a transcription factor that has been observed to activate some gene promoters and repress others, …

Use Of Synthetic Isoprenoids To Target Protein Prenylation And Rho Gtpases In Breast Cancer Invasion, Min Chen, Teresa Knifley, Thangaiah Subramanian, H. Peter Spielmann, Kathleen L. O'Connor

Molecular and Cellular Biochemistry Faculty Publications

Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called …

Global Analysis Of Lysine Acetylation Suggests The Involvement Of Protein Acetylation In Diverse Biological Processes In Rice (Oryza Sativa)., Babi Ramesh Reddy Nallamilli, Mariola J. Edelmann, Xiaoxian Zhong, Feng Tan, Hana Mujahid, Jian Zhang, Bindu Nanduri, Zhaohua Peng

CALS Publications

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa). We identified 60 lysine acetylated sites on 44 proteins of diverse …

Single Molecule Fret Reveals Pore Size And Opening Mechanism Of A Mechano-Sensitive Ion Channel, Yong Wang, Yanxin Liu, Hannah A. Deberg, Takeshi Nomura, Melinda Tonks Hoffman, Paul R. Rohde, Klaus Schulten, Boris Martinac, Paul R. Selvin

Physics Faculty Publications and Presentations

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a ‘virtual sorting’ single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches …

Immature Myeloid Cells Promote Tumor Formation Via Non-Suppressive Mechanism, Myrna Lillian Ortiz

USF Tampa Graduate Theses and Dissertations

ABSTRACT

Although there is ample evidence linking chronic inflammation with cancer, the cellular mechanisms involved in early events leading to tumor development remain unclear. Myeloid cells are an intricate part of inflammation. They consist of mature cells represented by macrophages, dendritic cells and granulocytes and a population of Immature Myeloid Cells (IMC), which in healthy individuals are cells in transition to mature cells. There is a substantial expansion of IMC in cancer and many other pathological conditions which is associated with pathologic activation of these cells. As a result, these cells acquire the ability to suppress immune responses and are …

Genome-Wide Rnai Ionomics Screen Reveals New Genes And Regulation Of Human Trace Element Metabolism, Mikalai I. Malinouski, Nesrin M. Hasan, Yan Zhang, Javier Seravalli, Jie Lin, Andrei Avanesov, Svetlana Lutsenko, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Trace elements are essential for human metabolism and dysregulation of their homoeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires …

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 …

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 …

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 …

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. …