Biochemistry, Biophysics, and Structural Biology Commons^™

Genome-Wide Identification Of Conditionally Essential Genes In Salmonella Typhimurium Using Tn-Seq Method, Anita Khatiwara

Graduate Theses and Dissertations

ABSTRACT

As more whole genome sequences become available, there is an increasing need for high-throughput methods that link genes to phenotypes and facilitate discovery of new gene functions. The objective of this study was to develop a high-throughput method to study gene functions in bacteria and use this method to study gene functions of S. enterica serotype Typhimurium (S. Typhimurium) under various environmental conditions encountered during its life cycle. Chapter I of this dissertation reviews the history and evolution of functional genomics in bacteria with focus on Salmonella, along with the recent techniques available. Chapter II, deals with the development …

Analysis Of The Effects And Current Treatments Of Laminin Deficiency, Joshua Mark Reynolds

Senior Honors Theses

Laminin (LM) is a network of proteins that functions as a connective framework of most cells in the body. It is composed of multiple different subunits and therefore has many different variations. It is a trimeric protein, meaning that it is composed primarily of ⍺, β, and γ chains. The differentiation of these subunits is what gives the different variants their functions. In addition, although LM is the primary molecule in scope, the network of other connective proteins involved in LM-associated diseases will also be covered in lesser detail because molecules like dystrophin, dystroglycan, collagen, and integrin are vital to …

Cloning And Characterization Of The Cell Wall Acting Enzyme Cd1034 From The Pathogen Clostridium Difficile, Zachary Suter

Honors Projects in Science and Technology

The manifestation of multidrug resistance in bacteria over the past several decades has resulted in one of the foremost challenges in the management of infectious diseases. The question arises, “How do we address this growing problem?” One solution to stem the growing rise in antimicrobial resistance is to investigate new targets, while another approach is to re-examine classical antibacterial targets with a fresh perspective. The aim of this paper is to begin the process of antibacterial development for the pathogen Clostridium difficile by characterizing the cell wall acting glucosaminidase CD1034. It is inunderstanding how CD1034 functions biochemically that it can …

Nano- And Micro-Second Electrical Pulsing Of B16-F10 Mouse Melanoma Cells: Plasma Membrane And Sub-Cellular Organelle Changes, Yiling Chen

Theses and Dissertations in Biomedical Sciences

High electric field-treated cells are permeable to molecular dye through either opening of pores in the plasma membrane or other unknown processes which can disturb the membrane in an organized way. However, direct morphological evidence is lacking and responses of intracellular organelles are not clear. We used traditional chemical fixatives and biochemical techniques to capture cell membrane and organelle changes immediately after pulsing with high voltage electric field application. Different pulse durations, nanosecond (ns) and microsecond (µs), and field magnitudes, 60 kV/cm and 1.2 kV/cm, were applied to mouse melanoma B16-F10 cells. Two different ns durations (60 and 300 ns) …

Nonosecond Pulsed Electric Field Induced Changes In Dielectric Properties Of Biological Cells, Jie Zhuang

Electrical & Computer Engineering Theses & Dissertations

Nanosecond pulsed electric field induced biological effects have been a focus of research interests since the new millennium. Promising biomedical applications, e.g. tumor treatment and wound healing, are emerging based on this principle. Although the exact mechanisms behind the nanosecond pulse-cell interactions are not completely understood yet, it is generally believed that charging along the cell membranes (including intracellular membranes) and formation of membrane pores trigger subsequent biological responses, and the number and quality of pores are responsible for the cell fate. The immediate charging response of a biological cell to a nanosecond pulsed electric field exposure relies on the …

Alteration In 5ht1a Receptor Activity From A Prenatal Exposure To Dexamethasone In A Stressed And Non-Stressed Adult Male Rat, Darshan S. Shah

Honors Theses

Synthetic glucocorticoids (GC) are used as a clinical therapeutic to stimulate lung development in fetuses that present the risk of preterm delivery. Previous studies have shown that a prenatal exposure to Dexamethasone (DEX) causes a disturbance in normal GC mediation of neuritic outgrowth, cell signaling, and serotonergic systems. Our hypothesis is that a prenatal exposure to DEX during the third trimester of pregnancy alters 5HT1A receptor function. Pregnant dams were injected daily with 150μg/ml/kg of DEX from gestation day 14 through 19. Control dams were treated with and equal volume of saline. Swim stress followed by elevated plus maze testing …

The Dietary Isoprenoid Perillyl Alcohol Inhibits Telomerase Activity In Prostate Cancer Cells, Tabetha Sundin

Theses and Dissertations in Biomedical Sciences

This is the first evidence that a plant-derived compound–perillyl alcohol regulates telomerase activity via the mammalian target of rapamycin (mTOR) pathway in prostate cancer cells. Telomerase–the enzyme responsible for immortalizing cells through telomeric repeats addition–is de-repressed early in an aspiring cancer cell. We hypothesized that perillyl alcohol regulates hTERT (human telomerase reverse transcriptase) at the translational and post-translational levels via its effects on the mTOR pathway. A rapid suppression of telomerase activity was detected in prostate cancer cell lines (PC-3 and DU145) in response to biologically-relevant concentrations and short incubations of perillyl alcohol or the mTOR inhibitor—rapamycin.

Western blot analysis …

Single-Molecule Analysis Of The Microtubule Cross-Linking Protein Map65-1 Reveals A Molecular Mechanism For Contact-Angle-Dependent Microtubule Bundling, Amanda Tulin, Sheri Mcclerklin, Yue Huang, Ram Dixit

Biology Faculty Research

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also …

The Role Of Tumor Suppressors, Ship And Rb, In Immune Suppressive Cells, Michelle Marie Collazo Ruiz

USF Tampa Graduate Theses and Dissertations

Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) have been extensively studied in the past 30-40 years. Their potent suppressive capacity shown in several pathological and clinical settings, such as cancer and transplantation, has made it evident that better understanding their development and function is critical.

Specifically, Tregs play a pivotal role in preventing autoimmunity, graft-versus-host disease (GvHD), and organ graft rejection. We previously demonstrated that germline or induced SH2 domain-containing inositol 5-phosphatase (SHIP) deficiency in the host abrogates GvHD. Here we show that SHIP-deficiency promotes an increase of FoxP3+ cells in both the CD4+CD25+ and the CD4+CD25- T …

[Accepted Article Manuscript Version (Postprint)] Plant Peroxisomes: Biogenesis And Function, Jianping Hu, Alison Baker, Bonnie Bartel, Nicole Linka, Robert Mullen, Sigrun Reumann, Bethany Zolman

Biology Department Faculty Works

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of …

Bacterial Host Interactions In Cystic Fibrosis, Máire Callaghan, Siobhan Mcclean

Articles

Chronic infection is a hallmark of cystic fibrosis (CF) and the main contributor to morbidity. Microbial infection in CF is complex, due to the number of different species that colonise the CF lung. Their colonisation is facilitated by a host response that is impaired or compromised by highly viscous mucous, zones of hypoxia and the lack of the cystic fibrosis transmembrane regulator (CFTR). Successful dominant CF pathogens combine an effective arsenal to establish infection and counter-attack the host response, together with an ability to adapt readily to an unfavourable environment. Hypermutability is common among CF pathogens facilitating adaptation and as …

Estradiol Modulation Of Calcium Dynamics In Pituitary Mmq Lactotroph Cells, Monika Pauckova

Electronic Theses and Dissertations

Pituitary lactotrophs are excitable cells that exhibit spontaneous, calcium influx triggering prolactin (PRL) secretion to stimulate lactation. Lactotrophs express estrogen receptors (ER) and are a well established estrogen-responsive cell system. 17B-estradiol (E2) is known to directly affect lactotrophs by increasing PRL transcription and biosynthesis, intracellular Ca2+ concentration ([Ca2+]i) and PRL secretion. This study demonstrates that the MMQ clonal cell line, isolated from the 7315a rat pituitary tumor, is a model lactotroph cell line that is E2-responsive. Spontaneous and evoked Ca2+ transients were especially sensitive to L-type channel block, but not affected by block of omega-conotoxin-GVIA-sensitive CaV channels or TTX-sensitive voltage-gated …

Cell Wall Antibiotics Provoke Accumulation Of Anchored Mcherry In The Cross Wall Of Staphylococcus Aureus, Wenqi Yu, Friedrich Götz

Molecular Biosciences Faculty Publications

A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the −YSIRK …

Discovery Of Marinopyrrole A (Maritoclax) As A Selective Mcl-1 Antagonist That Overcomes Abt-737 Resistance By Binding To And Targeting Mcl-1 For Proteasomal Degradation*, Kenichiro Doi, Rongshi Li, Shen-Shu Sung, Hongwei Wu, Yan Liu, Wanda Manieri, Gowdahalli Krishnegowda, Andy Awwad, Alden Dewey, Xin Liu, Shantu Amin, Chunwei Cheng, Yong Qin, Ernst Schonbrunn, Gary W. Daughdrill, Thomas P. Loughran Jr., Said M. Sebti, Hong-Gang Wang

Molecular Biosciences Faculty Publications

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified …

An Ims/Atp Assay For The Detection Of Mycobacterium Tuberculosis In Urine, Dawn M. Hunter, Daniel V. Lim

Molecular Biosciences Faculty Publications

Background. Although sputum smears are the gold standard for diagnosis of tuberculosis, sensitivity in HIV/TB coinfection cases is low, indicating a need for alternative methods. Urine is being increasingly evaluated. Materials and Methods. A novel method for detecting Mycobacterium tuberculosis (MTB) in synthetic urine using a combined IMS/ATP assay was evaluated. Preliminary work established standard ATP conditions and the sensitivity and specificity of the MTB antibody. Eighty-four blinded samples in four replicate assays were evaluated for the presence of MTB using labeled immunomagnetic beads for capture. Beads were separated, washed, and resuspended in broth and added to a …

Identification Of New Cell Size Control Genes In S. Cerevisiae, Huzefa Dungrawala, Hui Hua, Jill Wright, Lesley Abraham, Thivakorn Kasemsri, Anthony Mcdowell, Jessica Stilwell, Brandt L. Schneider

Molecular Biosciences Faculty Publications

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened …

The Two-Component Signal Transduction System Arlrs Regulates Staphylococcus Epidermidis Biofilm Formation In An Ica-Dependent Manner, Yang Wu, Jiaxue Wang, Tao Xu, Jingran Liu, Wenqi Yu, Qiang Lou, Tao Zhu, Nianan He, Haijing Ben, Jian Hu, Friedrich Götz, Di Qu

Molecular Biosciences Faculty Publications

Due to its ability to form biofilms on medical devices, Staphylococcus epidermidis has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating S. epidermidis biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using S. epidermidis strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms in vitro. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. …

Wipi-1 Positive Autophagosome-Like Vesicles Entrap Pathogenic Staphylococcus Aureus For Lysosomal Degradation, Mario Mauthe, Wenqi Yu, Oleg Krut, Martin Krönke, Friedrich Götz, Horst Robenek, Tassula Proikas-Cezanne

Molecular Biosciences Faculty Publications

Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently …

Inhibition Of Voltage-Gated Na+ Current By Nanosecond Pulsed Electric Field (Nspef) Is Not Mediated By Na+ Influx Or Ca²+ Signaling, Vasyl Nesin, Andrei G. Pakhomov

Bioelectrics Publications

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na⁺ current (I_Na) resulted from (i) reduction of the transmembrane Na⁺ gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca²⁺ -dependent mechanism. We found that a single 300?ns electric pulse at 1.65.3?kV/cm triggered sustained Na⁺ influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a …

Electric Field Exposure Triggers And Guides Formation Of Pseudopod-Like Blebs In U937 Monocytes, Mikhail A. Rassokhin, Andrei G. Pakhomov

Bioelectrics Publications

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to "regular," round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb …

Oxidative Effects Of Nanosecond Pulsed Electric Field Exposure In Cells And Cell-Free Media, Olga N. Pakhomova, Vera A. Khorokhorina, Angela M. Bowman, Raminta Rodaitė-Riševičienė, Gintautas Saulis, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′, 7′-dichlorodihydrofluoresein (H₂DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H₂DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat …

Why Do We Collect?, Allison Marsh

Section 2: Imaging the Microscopic

No abstract provided.

Spotlight On Usc: A.C. Moore Herbarium, Allison Marsh

Section 2: Imaging the Microscopic

No abstract provided.

Imaging And Aesthetics, Allison Marsh

Section 2: Imaging the Microscopic

No abstract provided.

Conclusion Panel, Allison Marsh

Section 6: Conclusion

No abstract provided.

Protein Folding By 'Levels Of Separation': A Hypothesis, Lesley H. Greene, Terri M. Grant

Chemistry & Biochemistry Faculty Publications

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long-range interactions between the transition and native states. Long-range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of ‘degrees of separation’, that we term ‘levels of separation’. It is based on the integration of network science and biochemistry. (C) 2012 Federation of European Biochemical Societies.

Arachidonic Acid Signaling In Invasive And Non-Invasive Breast Cancer Cells, Debarshi Roy

Open Access Theses & Dissertations

Breast cancer is the second largest cause of cancer-related deaths in women all over the world. Epidemiological studies suggest that the consumption of high-fat diets can promote the incidence of breast cancers in both developed and developing countries. In particular, the lipid-rich diet contains arachidonic acid (AA, a C20:4 polyunsaturated fatty acid), which has been shown to be associated with tumor formation in breast tissues. Nevertheless, the actual mechanism by which AA induces the metastatic transformation and malignancy is not well understood. The goal of my dissertation, therefore, is to identify the molecules and unravel the pathways that participate in …

The Role Of Cad,Flash And Fam129b In Cancer Cell Survial And Apoptosis, Song Chen

Wayne State University Dissertations

Apoptosis is a normal process in the human body. However, apoptosis is desregulated in cancer cells. Most cancer cells gain resistance to apoptosis, leading to uncontrolled proliferation. In this dissertation, we identified three proteins, associated with apoptosis pathway. 1) CAD, a large multifunctional complex that is invariably elevated in tumor cells, 2) FLASH, a large protein with multiple growth related functions and 3) FAM129B. We demonstrate that CAD could interact with FLASH by using yeast two hybrid, co-immunopreciptation and fluorescence microscopy. In addition, functional analysis using siRNA technology further indicated that CAD could co-operate with FLASH and play roles in …

Identifying Sm22 As A Key Player In Arterial Diseases, Jianbin Shen

Wayne State University Dissertations

Background : Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and osteochondrogenesis are present. However, the role of this downregulation in arterial pathogenesis is unknown. Hypothesis : Downregulation of SM22 may actively contribute to arterial pathogenesis. Methods : Five Sm22 knockout (Sm22^-/-) mice and their wild type littermates were subjected to carotid artery denudation, an artery injury model. Analyses were conducted on carotid arteries 2 weeks after injury. Primary VSMCs were isolated from mouse aortas and investigated individually at passage 2 to 4. Sm22 knockdown was …

Identification Of Cellular Functions Of Cardiolipin As Physiological Modifiers Of Barth Syndrome, Amit Shridhar Joshi

Wayne State University Dissertations

Cardiolipin (CL) is an anionic phospholipid synthesized in the mitochondrial inner membrane. Perturbation of CL metabolism leads to Barth syndrome (BTHS), a life threatening genetic disorder. I utilized genetic, biochemical and cell biological approaches in yeast to elucidate the cellular functions of CL. Understanding the functions of CL is expected to shed light on the pathology and possible treatments for BTHS.

BTHS is caused by mutations in TAZ1, which encodes a CL remodeling enzyme called tafazzin. BTHS patients exhibit a wide range of clinical presentations, indicating that physiological modifiers influence the BTHS phenotype. A targeted synthetic lethality screen was performed …