Microbiology Commons^™

Virus Purification, Detection And Removal, Khrupa Saagar Vijayaragavan

Dissertations, Master's Theses and Master's Reports - Open

The biopharmaceutical industry has a growing demand and an increasing need to improve the current virus purification technologies, especially as more and more vaccines are produced from cell-culture derived virus particles. Downstream purification strategies can be expensive and account for 70% of the overall manufacturing costs. The economic pressure and purification processes can be particularly challenging when the virus to be purified is small, as in our model virus, porcine parvovirus (PPV). Our efforts are focused on designing an easy, economical, scalable and efficient system for virus purification, and we focused on aqueous two-phase systems. Industry acceptable standards for virus …

Novel Cul3 Binding Proteins Function To Remodel E3 Ligase Complexes, Wananit Wimuttisuk, Mark West, Brittney Davidge, Kebing Yu, Arthur Salomon, Jeffrey D. Singer

Biology Faculty Publications and Presentations

Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and …

Sumoylation Affects Ns1'S Ability To Neutralize The Interferon Response, Jeanette Cecilia Gonzalez

Open Access Theses & Dissertations

It is of huge concern the possibility that at some point in the future we may face a new pandemic involving a highly pathogenic virus due to our current preventive and treatment options to fight this viral disease. The main reason for such limitations is the ability of the virus to go over constant antigenic shifts and drifts on its viral surface proteins HA and NA. The constant mutations that affect this virus cause the need of developing new vaccines for each influenza season. Periodically, the World Health Organization (WHO) reports that around 10% of the world population gets infected …

The Role Of Surface Chemistry In The Toxicity Of Manufactured Cerium Dioxide Nanomaterials To Caenorhabditis Elegans, Emily Kay Oostveen

Theses and Dissertations--Plant and Soil Sciences

Manufactured CeO₂ nanomaterials (CeO₂-MNMs) are used for a wide variety of applications including diesel fuel additives and chemical/mechanical planarization media. To test the effects of CeO₂-MNM surface coating charge on to model organism Caenorhabditis elegans, we synthesized 4 nm CeO₂ with cationic (DEAE-), anionic (CM-), and neutral (DEX) coatings. In L3 nematodes exposed for 24 hours, DEAE-CeO₂ induced lethality at lower concentrations than CM- or DEX-CeO₂. Feeding slightly decreased CeO₂ toxicity, regardless of coating. In L2 nematodes exposed for 48 hours with feeding, DEAE-CeO₂ caused lethality at the …

Coronavirus Proteases As Therapeutic Targets: Development Of Biosensors To Detect Inhibition Of Protease Activity And Separation Of The Multiple Functions Of Coronavirus Papain-Like Proteases, Andrew Kilianski

Dissertations

Coronaviruses are important human pathogens and have the potential to severely impact public health on an international scale. The emergence of SARS-CoV and MERS-CoV highlight the need for research to identify antivirals and vaccines against coronaviruses. To develop therapeutics against current and potentially emergent coronaviruses, I utilized two approaches targeting the proteases encoded within all coronaviruses. The papain-like protease and 3C-like protease of coronaviruses are responsible for cleaving viral polyproteins early during infection, and this step is required for viral replication. To quantitatively assess the inhibition by small-molecule compounds on MERS-CoV protease activity, I developed a luciferase-based biosensor to monitor …

Role Of The Pkna And Pknb Kinases In Mycobacterium Tuberculosis, Tripti Anandan

Wayne State University Dissertations

To respond to environmental changes, M. tuberculosis possesses eleven "eukaryotic-type" Ser/Thr protein kinases. The aim of the study described in this dissertation was to identify role of two of these kinases; PknA and PknB that are essential in M. tuberculosis. Two approaches are described to screen for potential in vivo substrates of PknA/PknB. First approach is based on proteomic search by over-expressing PknA/PknB in M. tuberculosis. Proteomic search led to identification of proteasome to be a substrate of PknA and PknB in M. tuberculosis. Furthermore, I demonstrate that the phosphorylation of PrcA and PrcB by PknA regulates processing of Pre-PrcB. …

The Mechanism For Inhibition Of Vibrio Cholerae Virulence Gene Expression By Bile And Its Fatty Acid Components, Sarah Plecha

Wayne State University Dissertations

Vibrio cholerae is responsible for the diarrheal disease cholera by producing two major virulence factors: cholera toxin (CT) and toxin-coregulated pilus (TCP). During infection, V. cholerae downregulates its motility genes, which are actively expressed in the environment, and upregulates its virulence genes, which are inactive in the environment. Virulence gene transcription is regulated by the major transcriptional activator, ToxT. ToxT is regulated, in part, by host signals, which in turn, make ToxT active or inactive in respect to virulence gene expression. Host signals include temperature and pH as well as the chemical molecules bicarbonate and bile, which are positive and …

Cell Envelope Modifications By Lcp Family Proteins Of Group B Streptococcus And Virulence Singaling By Peptides Of The Salivaricin Locus Of Streptococcus Pyogenes, Hannah Morrison Rowe

Wayne State University Dissertations

ABSTRACT

CELL ENVELOPE MODIFICATIONS BY LCP FAMILY PROTEINS OF GROUP B STREPTOCOCCUS AND VIRULENCE SIGNALING BY PEPTIDES OF THE SALIVARICIN LOCUS OF STREPTOCOCCUS PYOGENES

by

HANNAH MORRISON ROWE

December 2014

Advisor: Dr. Melody Neely

Major: Immunology and Microbiology

Degree: Doctor of Philosophy

Streptococci are important human and animal pathogens. Group B Streptococcus (GBS) is a zoonotic pathogen that is the leading cause of human neonatal sepsis and meningitis. In order to cause systemic disease GBS must produce a polysaccharide capsule. Capsule expression is controlled in part by the protein CpsA. We demonstrate that deletion of the cpsA gene, or expression …

Socs1/Socs3 Expression And Virus Replication Of Denv2 And Hsv-1 In Cytokine-Polarized Subsets Of Raw 264.7 Macrophages, Nagarjuna Reddy Cheemarla

Browse all Theses and Dissertations

Cytokine activation of macrophages leads to "macrophage polarizatio" The M1 subset is polarized by exposure to lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) and M2 polarized cells develop after exposure to interleukin-4 (IL-4) or IL-13. In this study, the RAW 264.7 murine macrophage cell line was used to study the effect herpes simplex virus-Type I (HSV-1) and Dengue virus serotype-2 (DENV2) infections have on cultures of unpolarized (M0) or polarized subsets. The macrophage subsets were characterized using cluster of differentiation markers CD14 and CD86. Uninfected M1 macrophages showed up regulation in expression of the co-stimulatory factor B7.2 (CD86) compared to expression by …

Development Of A Co-Culture System To Mimic The Transfection Of Hsv-1 From Keratinocytes To Neuronal Cells, David A. Dixon

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To more closely mimic the in vivo progression of HSV-1 a novel in vitro method was created. In this study HEL-30 keratinocytes were infected with a 0.1 MOI of HSV-1 and plated unto a membrane with 8μm diameter pores. The membrane insert was then placed into culture wells that contained Neuro-2a cells (N2A). The neurites from the neuronal cells made cell-to-cell contact with the infected HEL-30 cells and the virus was transmitted into the neuronal cells. To determine if infection occurred in the N2A cells the cells were lysed and the lysate incubated with Vero cells to titrate virus plaque …

White Blood Cell Counts, Parasite Prevalence, And Plasma Cortisol Levels Of Dogs In A County Animal Shelter: Changes Over Days And Impact Of A Program Of Repeated Human Interaction, Emily S. Dudley

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Animal shelter housing is highly stressful for a dog, compromising welfare and leading to undesirable behaviors and unknown health consequences. We documented the changes in circulating numbers of white blood cells, plasma cortisol, and fecal parasite shedding of dogs housed for 10 days at a county animal shelter. White blood cell changes were most prominent on Day 10 after arrival to the shelter. Changes included increased total leukocytes, mature neutrophils, and lymphocytes, with less consistent increases in monocytes and neutrophil to lymphocyte ratio (N:L). Fecal parasite shedding was elevated and not affected by day. Cortisol levels of shelter dogs declined …

Identification Of The Sites Of Action Of Inhibitors Of Mammalian Phospholipase D2 (Pld2) And The Role Of Interacting Protein Partners, Ramya Ganesan

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Phospholipase D (PLD) is a key enzyme for the remodeling of phospholipids in the cell membrane. PLD has been implicated in many physiological functions such as chemotaxis and phagocytosis as well as pathological functions such as ischemia/reperfusion and cancer metastasis. Several small molecule inhibitors of PLD have been recently developed to overcome these pathological effects. We have concentrated on 5-Fluoro-2-Indolyl des-Chlorohalopemide (FIPI) which is an indole derivative of halopemide that inhibits the two mammalian isoforms PLD1 and PLD2. We also concentrate on N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT) and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide (NOPT) that are specific inhibitors of the PLD2 isoform. In spite of knowing their …

Comparison Between Flow Cytometry And Bead Method In Counting Cd4 And Cd8 T Lymphocytes In Mouse Spleen Cells Suspension, Abdulrahman Abdalla Allabidi

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The flow cytometry is gold standard method to count CD4, CD8 human T lymphocytes, and calculate CD8 to CD4 ratio in HIV patients peripheral blood to stage them and monitor their response to treatment. In this study, a comparison was made by counting CD4and CD8 mouse spleen T lymphocytes by flow cytometry, and a method involving antibody coated latex beads. The bead method yielded results comparable to those obtained by flow cytometry. These results indicate that antibody coated beads are suitable to determine CD4 and CD8 + T lymphocytes counts in situation where flow cytometry is not readily available.

Effect Of Herpes Simplex Virus-1 On Macrophage Cd Marker Expression, Hind Obaid Albeshri

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Macrophages are specialized phagocytic cells derived from the peripheral blood mononuclear cells. Macrophages can assume M1 or M2 cellular states upon activation with specific cytokines. Upon activation, macrophages produce either pro- or anti- inflammatory cytokines and thereby perform their functions of antigen engulfment and debris clearance. Macrophages have several cell surface cluster differentiation (CD) markers including CD80, CD163 and CD200R that differentiate M1 macrophages from M2. Downstream signaling and cytokine production varies based on the interaction between the specific CD marker and antigen.

The present study was aimed at analyzing and comparing the effects of HSV-1 infection on un-polarized and …

Elucidating The Effects Of Tcdd On The Polymorphic Human Hs1,2 Enhancer, Abdullah Freiwan

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin known to inhibit immunoglobulin (Ig) gene expression in various animal studies. We have identified the mouse 3'Ig heavy chain regulatory region (3'IgH RR) as a sensitive transcriptional target of TCDD, which may mediate the inhibitory effect of TCDD on Ig expression. Interestingly, the human hs1,2 enhancer is polymorphic and has been associated with a number of autoimmune diseases.

Suggesting a species difference, TCDD inhibited mouse hs1,2 enhancer activation and activated basal human hs1,2 (hs-hs1,2) enhancer activity in the mouse B-cell line. The objective of this study was to elucidate the effects of TCDD …

Ovarian Sex Cord Stromal Tumours In Children And Young Girls - A More Than Two Decade Clinicopathological Experience In A Developing Country, Pakistan, Saroona Haroon, Romana Idrees, Aleena Zia, Aisha Memon, Saira Fatima, Naila Kayani

Department of Pathology and Laboratory Medicine

Background: Ovarian sex-cord stromal tumours (SCST) are rare, and relatively infrequent in children. These have to be distinguished from more common germ cell tumors in children and also from benign epithelial neoplasms.
Objectives: The purpose of our study was to report the clinical and pathological findings in young patients with these tumours in our population.
Material and Methods: The present observational cross-sectional study included all subjects SCST, in Aga Khan University Hospital Histopathology Laboratory, Karachi, Pakistan, from January 1992 till July 2013.
Results: Of the total of 513 SCSTs presented during the study period, 39 fulfilled inclusion criteria and were …

Adeno-Associated Virus (Aav) Transduction Of Primary Human Cd4+T Lymphocytes, Sahar Hussein Kamel

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Development of latent HIV-infected CD4+ T lymphocytes is the major cause of HIV treatment failure. Adeno-associated virus (AAV) is an attractive vector for anti-HIV gene therapy due to its lack of pathogenicity, low immunogenicity, and persistent transgene expression. However, a major limitation for AAV gene transfer is the cell-specific tropism of each serotype. Only AAV serotypes 2 and 5 have been investigated in hematopoietic cells, both of these serotypes have shown low transduction efficiency. Since each of the currently described AAV serotypes demonstrate distinct tissue tropism, I hypothesized that other serotypes such as AAV1, 4, 8, or 9 may demonstrate …

Expression Of The Alpha, Beta, And Gamma Subunits Of The Interleukin-2 Receptor By Human Vascular Smooth Muscle Cells, Sultan Mohammed Alhayyani

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Interleukin 2 (IL-2) is a member of the cytokine family and contributes to the proliferation, survival, and death of lymphocytes [1]. The interleukin-2 receptor (IL-2) is a tripartite receptor commonly expressed on the surfaces of many lymphoid cells and is composed of three non-covalently associated subunits, alpha (CD25), beta (CD122), and gamma (CD132) [2]. Our laboratory has previously described IL-2 receptor Beta expression by vasculature smooth muscle cells (VSMC) in mice and humans [3]. The current work expands our previous observations by assessing the expression of the alpha and gamma subunits of the IL-2R by VSMCs. Analysis of IL-2R expression …

A Prospective Study Of Periodontal Disease And Risk Of Gastric And Duodenal Ulcer In Male Health Professionals, Matthew R. Boylan, Hamed Khalili, Edward S. Huang, Dominique S. Michaud, Jacques Izard, Kaumudi J. Joshipura, Andrew T. Chan

Department of Food Science and Technology: Faculty Publications

Periodontal disease is characterized by microbial shifts within the oral cavity and chronic inflammation of the gingival tissue, and can lead to tooth loss. Several studies support an association between periodontal disease and risk of chronic systemic conditions, including vascular disease, chronic obstructive pulmonary disease, diabetes mellitus, and cancer, including gastric adenocarcinoma. These associations may be mediated by systemic inflammation, the immune response to periodontal infection, or direct invasion by pathogens. Previous studies of periodontal disease and risk of peptic ulcer have been limited by cross-sectional design, diagnoses that were not confirmed by medical record review, and a lack of …

Cell- And Virus-Mediated Regulation Of The Barrier-To-Autointegration Factor’S Phosphorylation State Controls Its Dna Binding, Dimerization, Subcellular Localization, And Antipoxviral Activity, Augusta Jamin, April Wicklund, Matthew S. Wiebe

Nebraska Center for Virology: Faculty Publications

Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF’s ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF’s DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable …

Characterization Of A Dual-Tropic Human Immunodeficiency Virus (Hiv-1) Strain Derived From The Prototypical X4 Isolate Hxbc2, Shi-Hua Xiang, Beatriz Pacheco, Dane Bowder, Wen Yuan, Joseph Sodroski

Nebraska Center for Virology: Faculty Publications

Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed …

Live Siv Vaccine Correlate Of Protection: Immune Complex-Inhibitory Fc Receptor Interactions That Reduce Target Cell Availability, Anthony J. Smith, Stephen W. Wietgrefe, Liang Shang, Cavan S. Reilly, Peter J. Southern, Katherine E. Perkey, Lijie Duan, Heinz Kohler, Sybille Muller, James Robinson, John V. Carlis, Qingsheng Li, R. Paul Johnson, Ashley T. Haase

Nebraska Center for Virology: Faculty Publications

Principles to guide design of an effective vaccine against HIV are greatly needed, particularly to

protect women in the pandemic’s epicentre in Africa. We have been seeking these principles by

identifying correlates of the robust protection associated with SIV_mac239Δnef vaccination in the

SIV-rhesus macaque animal model of HIV-1 transmission to women. We have identified one

correlate of SIV_mac239Δnef protection against vaginal challenge as a resident mucosal system for

SIV-gp41 trimer antibody production and neonatal Fc receptor (FcRn)-mediated concentration of

these antibodies on the path of virus entry to inhibit establishment of infected founder populations

at the …

Review Of Epidemiology And Transmission Of Kaposi’S Sarcoma-Associated Herpesvirus, Veenu Minhas, Charles Wood

Nebraska Center for Virology: Faculty Publications

This review summarizes the current knowledge pertaining to Kaposi sarcoma-associated herpesvirus (KSHV) epidemiology and transmission. Since the identification of KSHV twenty years ago, it is now known to be associated with Kaposi’s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman’s disease. Many studies have been conducted to understand its epidemiology and pathogenesis and their results clearly show that the worldwide distribution of KSHV is uneven. Some geographical areas, such as sub-Saharan Africa, the Mediterranean region and the Xinjiang region of China, are endemic areas, but Western Europe and United States have a low prevalence in the general population. This makes …

Seroprevalence Of Human Herpesvirus 8 And Hepatitis C Virus Among Drug Users In Shanghai, China, Tiejun Zhang, Ying Liu, Yuyan Zhang, Jun Wang, Veenu Minhas, Charles Wood, Na He

Nebraska Center for Virology: Faculty Publications

To elucidate and compare the seroprevalence of human herpesvirus 8 (HHV8) and hepatitis C virus (HCV) among Chinese drug users, a cross-sectional study of 441 participants, was conducted in Shanghai, China, from 2012 through 2013. Seventy-seven (17.5%) participants were found to be positive for HHV8 antibodies, while 271 (61.5%) participants were positive for HCV. No significant association between HHV8 seropositivity and drug use characteristics, sexual behaviors, HCV, or syphilis was observed. In contrast, a statistically significant association between HCV seropositivity and injected drug history (OR, 2.18, 95% CI 1.41–3.37) was detected, whereas no statistically significant association between HCV seropositivity and …

Vaccines Within Vaccines: The Use Of Adenovirus Types 4 And 7 As Influenza Vaccine Vectors, Eric A. Weaver

Nebraska Center for Virology: Faculty Publications

adenovirus Types 4 and 7 (ad4 and ad7) are associated with acute respiratory distress (aRD). In order to prevent wide- spread ad-associated aRD (ad-aRD) the United states military immunizes new recruits using a safe and effective lyophi- lized wildtype ad4 and ad7 delivered orally in an enteric-coated capsule. We cloned ad4 and ad7 and modified them to express either a GFP-Luciferase (GFPLuc) fusion gene or a centralized influenza H1 hemagglutinin (Ha1-con). BaLB/c mice were injected with GFPLuc expressing viruses intramuscularly (i.m.) and intranasally (i.n.). ad4 induced significantly higher luciferase expression levels as compared with ad7 by both routes. ad7 transduction …

Effect Of Prey Richness On A Consumer’S Intrinsic Growth Rate, Brian J. Darby, Michael A. Herman

School of Biological Sciences: Faculty Publications

The intrinsic growth rate of nonselective microbivores increases asymptotically with increasing prey biomass, but we do not know how intrinsic growth rate is affected by prey richness. The objective of this experiment was to determine the effect of prey richness on the growth kinetics of nematode predators while grazing on mixed bacterial lawns. We found that the intrinsic growth rate of Caenorhabditis elegans in laboratory culture increased asymptotically with prey richness. The mechanism of this pattern was primarily due to the best available prey species in the mixture: the intrinsic growth rate of the consumer feeding on a mixture of …

Construction Of A Live-Attenuated Hiv-1 Vaccine Through Genetic Code Expansion, Nanxi Wang, Yue Li, Wei Niu, Ming Sun, Ronald Cerny, Qingsheng Li, Jiantao Guo

Qingsheng Li Publications

A safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) is urgently needed to combat the worldwide AIDS pandemic, but still remains elusive. The fact that uncontrolled replication of an attenuated vaccine can lead to regaining of its virulence creates safety concerns precluding many vaccines from clinical application. We introduce a novel approach to control HIV-1 replication, which entails the manipulation of essential HIV-1 protein biosynthesis through unnatural amino acid (UAA*)-mediated suppression of genome-encoded blank codon. We successfully demonstrate that HIV-1 replication can be precisely turned on and off in vitro.

Includes supporting information.

A Glutathione-Independent Glyoxalase Of The Dj-1 Superfamily Plays An Important Role In Managing Metabolically Generated Methylglyoxal In Candida Albicans, Sahar Hasim, Nur Ahmad Hussin, Fadhel Alomar, Keshore R. Bidasee, Kenneth W. Nickerson, Mark A. Wilson

Kenneth Nickerson Papers

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to D-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys¹³⁶- His¹³⁷-Glu¹⁶⁸. Purified Glx3p has an in vitro methylglyoxalase activity (K_m = 5.5 mM andk_cat = 7.8 s^-1) that is significantly greater than that of …

Bacteria In Bioethanol Fermentations, Qing Li

Theses and Dissertations--Plant and Soil Sciences

To gain a better understanding of contaminating bacteria in bioethanol industry, we profiled the bacterial community structure in corn-based bioethanol fermentations and evaluated its correlation to environmental variables. Twenty-three batches of corn-mash sample were collected from six bioethanol facilities. The V4 region of the collective bacterial 16S rRNA genes was analyzed by Illumina Miseq sequencing to investigate the bacterial community structure. Non-metric multidimensional scaling (NMDS) ordination plots were constructed to visualize bacterial community structure groupings among different samples, as well as the effects of multiple environmental variables on community structure variation. Our results suggest that bacterial community structure is facility-specific, …

Sporulation In Bacteria: Beyond The Standard Model, Elizabeth Hutchison

Biology

Endospore formation follows a complex, highly regulated developmental pathway that occurs in a broad range of Firmicutes. Although Bacillus subtilis has served as a powerful model system to study the morphological, biochemical, and genetic determinants of sporulation, fundamental aspects of the program remain mysterious for other genera. For example, it is entirely unknown how most lineages within the Firmicutes regulate entry into sporulation. Additionally, little is known about how the sporulation pathway has evolved novel spore forms and reproductive schemes. Here, we describe endospore and internal offspring development in diverse Firmicutes and outline progress in characterizing these programs. Moreover, …