Microbiology Commons^™

Frequency And Outcome Of Graft Versus Host Disease After Stem Cell Transplantation: A Six-Year Experience From A Tertiary Care Center In Pakistan, Natasha Ali, Salman Naseem Adil, Mohammad Usman Shaikh, Nehal Masood

Department of Pathology and Laboratory Medicine

Objective: The objective of this study was to evaluate the frequency and outcome of graft versus host disease after stem cell transplantation for various haematological disorders in Pakistan.
Materials and Methods: Pretransplant workup of the patient and donor was performed. Mobilization was done with G-CSF 300 mu g twice daily for five day. Standard GvHD prophylaxis was done with methotrexate 15mg/m(2) on day +1 followed by 10mg/m(2) on days +3 and +6 and cyclosporine. Grading was done according to the Glucksberg classification.
Results: A total of 153 transplants were done from April 2004 to December 2011. Out of these were …

Evading Innate And Adaptive Immunity During Adenovirus Cell Entry, Shauna Marvin

Dissertations

Adenovirus (Ad), a non-enveloped, dsDNA virus, enters cells via clathrin-mediated endocytosis. For viral genome delivery to the nucleus, Ad must penetrate endosomal membranes to create defects sufficient for the passage of the 90 nm diameter capsid across cell membranes. Recent observations suggest that adenovirus type 5 (Ad5) capsid uncoating occurs at the cell surface upon binding to both the coxsackievirus and adenovirus receptor and α_v integrins. This uncoating event leads to the exposure of the capsid membrane lytic protein VI. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture, we demonstrate that Ad5 membrane rupture occurs …

Exploitation Of The Bacteriophage-Derived Peptidase Chapk, Pierre-Mehdi Hadbi

Theses

This project investigated an anti-staphylococcal bacteriophage-derived peptidoglycan hydrolase enzyme, namely a cysteine-histidine amydohydrolase/peptidase (CHAP_k). The study focused firstly on optimizing the production of recombinant CHAP_k, which was previously cloned in an E. coli expression system, and sets out the resulting optimal Standard Operating Procedures (SOPs) for production, purification and determination of concentration and activity of the resulting protein stock. A typical yield resulting from the investigation was 300pg of CHAP_k from one litre of E.coli culture. The activity of the purified enzyme was typically in the region of 190U nmol ’. Secondly, the project investigated …

Hypoxia Inducible Factor 1 Alpha (Hif-1a): A Major Regulator Of Placental Development, Renee E. Albers

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Hypoxia-inducible factor-1 alpha (HIF-1a) is a critical component of the cellular oxygen-sensing machinery and is essential for placental formation and embryonic survival. In this study, we promoted prolonged expression of HIF-1a, by using a form that is insensitive to oxygen, denoted as CA-HIF-1a. In order to have continual placental specific expression of the CA-HIF-1a, lentiviral infection was performed on embryos at the blastocyst stage of development and transferred into pseudo-pregnant mothers. Placental analysis was performed via in situ hybridization on embryonic day 14.5 (E14.5) to determine the effects of CA-HIF-1a prolonged expression. Data indicate that prolonged activity of CA-HIF-1a restricted …

Antilisterial Characteristics Of Volatile Essential Oils, Leeann L. Slaughter

Theses and Dissertations--Animal and Food Sciences

This study explored the in vitro and in situ antilisterial inhibitory activity of 16 essential oils during indirect exposure: Spanish Basil oil (Ocimum basilicum), Bay oil (Pimenta racemosa), Italian Bergamot oil (Citrus bergamia), Roman Chamomile oil (Anthemis nobilis), Sir Lanka Cinnamon oil (Cinnamomum zeylanicum), Citral, Clove Bud oil (Syzygium aromaticum), Cumin Seed oil (cuminum cyminum), Eucalyptus oil (Eucalyptus globulus), Eugenol, Geranium extract (Pelargonium graveolens), Marjoram oil (Origanum majorana), Neroli extract (Citrus aurantium), Peppermint oil (Mentha piperita L.), …

The Relative Contribution Of Methanotrophs To Microbial Communities And Carbon Cycling In Soil Overlying A Coal-Bed Methane Seep, Christopher T. Mills, Gregpry F. Slater, Robert F. Dias, Stephanie A. Carr, Christopher M. Reddy, Raleigh Schmidt, Kevin W. Mandernack

Chemistry & Biochemistry Faculty Publications

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH₄ and also a likely source of ancient (i.e. ¹⁴C-dead) carbon to soil microbial communities. Natural abundance ¹³C and ¹⁴C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO₂ and CH₄ were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison …

The Use Of Metagenomic Approaches To Analyze Changes In Microbial Communities, Girish Neelakanta, Hameeda Sultana

Biological Sciences Faculty Publications

Microbes are the most abundant biological entities found in the biosphere. Identification and measurement of microorganisms (including viruses, bacteria, archaea, fungi, and protists) in the biosphere cannot be readily achieved due to limitations in culturing methods. A non-culture based approach, called “metagenomics”, was developed that enabled researchers to comprehensively analyse microbial communities in different ecosystems. In this study, we highlight recent advances in the field of metagenomics for analyzing microbial communities in different ecosystems ranging from oceans to the human microbiome. Developments in several bioinformatics approaches are also discussed in context of microbial metagenomics that include taxonomic systems, sequence databases, …

Thalassiosira Spp. Community Composition Shifts In Response To Chemical And Physical Forcing In The Northeast Pacific Ocean, P. Dreux Chappell, Leann P. Whitney, Traci L. Haddock, Susanne Menden-Deuer, Eric G. Roy, Mark L. Wells, Bethany D. Jenkins

Biological Sciences Faculty Publications

Diatoms are genetically diverse unicellular photosynthetic eukaryotes that are key primary producers in the ocean. Many of the over 100 extant diatom species in the cosmopolitan genus Thalassiosira are difficult to distinguish in mixed populations using light microscopy. Here, we examine shifts in Thalassiosira spp. composition along a coastal to open ocean transect that encountered a 3-month-old Haida eddy in the northeast Pacific Ocean. To quantify shifts in Thalassiosira species composition, we developed a targeted automated ribosomal intergenic spacer analysis (ARISA) method to identify Thalassiosira spp. in environmental samples. As many specific fragment lengths are indicative of individual Thalassiosira spp., …

Expression Of An Epitope Tagged Tarp Effector In Chlamydia Trachomatis, Brenda Nguyen

HIM 1990-2015

Previous studies performed on Chlamydia trachomatis have demonstrated how these obligate intracellular microbes invade host cells through the utilization of secreted effector proteins. One secreted effector called Tarp (translocated actin recruiting protein) is implicated in cytoskeleton rearrangements that promote bacterial entry into the host cell. The focus of our study is to create a plasmid that carries the tarP gene that when transcribed and translated from within Chlamydia trachomatis will generate a c-Myc epitope tagged Tarp. The tag will be used in future studies to track the progression of the protein through the infectious process and will allow us to …

Development Of An Alkaline Phosphatase Reporter System For Use In The Lyme Disease Spirochete Borrelia Burgdorferi, Selina Sutchu

HIM 1990-2015

The use of the periplasmic alkaline phosphatase (PhoA) reporter protein from E. coli has been critical for definition of the topology of transmembrane proteins of multiple bacterial species. This report demonstrates development of a PhoA reporter system in B. burgdorferi. Codon usage of the E. coli phoA in B. burgdorferi was analyzed and an optimized version of the gene was obtained. In order to assess the differential activity of the reporter system, two optimized PhoA-fusion construct using B. burgdorferi proteins were engineered: one using the periplasmic protein OppAIV and one using the cytoplasmic protein PncA. The activity of PhoA requires …

Understanding The Role Of Plasmodium Falciparum Vamp8 Snare Homologue, Katherine Ferreira

HIM 1990-2015

Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia [37]. According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of …

Equine Serum Antibody Responses To Streptococcus Equi And Streptococcus Zooepidemicus, Rafaela De Negri

Theses and Dissertations--Veterinary Science

Streptococcus zooepidemicus (Sz) and Streptococcus equi (Se) share 98% DNA sequence homology, but display different pathogenic properties. Infection by one organism does not cross-protect against the other. To better understand pathogenic differences between these organisms and gain information about which proteins are expressed in horses infected experimentally with Se, intrauterine Sz or naturally with respiratory Sz we compared antibody specificities of convalescent sera using ELISA. These comparisons were based on sets of 8 and 14 immunoreactive recombinant proteins of Se strain CF32 and Sz strain NC78, respectively. Sera from donkeys that were previously naturally affected with strangles and later developed …

The Search For Catalytic Subunit Of Dna Polymerase Α In Thermomyces Lanuginosus, Chelsea Elyse Meier

Honors Program Theses

In eukaryotes, the enzyme DNA polymerase a (DNAPa) begins the process of DNA replication. Ultimately, this means DNAPa is responsible for all cell proliferation and growth of an organism. The mechanism by which this enzyme operates is interesting due to its implications in medicine; however, that mechanism remains largely elusive. DNAPa, including its catalytic subunit, has been studied in several eukaryotes. Unfortunately, these organisms present issues with the ease of study and with ethics. This research delved into a novel model organism that was potentially devoid of all these issues: the fungus Thermomyces lanuginosus. Though T. lanuginosus was easy to …

Presenting Fake Figures: A Tool To Teach Effective Scientific Figure Design, Verónica A. Segarra, Stephanie Pulford, Susan Walsh

Faculty Publications

No abstract provided.

Bacterial Assemblages And Microbial Enzyme Activity In The Major Tributaries Of The Lower Mississippi River, Justin Millar

Electronic Theses and Dissertations

Spatial variation in the structure and function of bacterial communities is far more complex than previously believed. High throughput sequencing techniques have begun to elucidate the dynamic worldwide patterns of bacterial biogeography; however, there are still many ecosystems for which there is little to no representative data. Currently there are no high throughput-based analyses of bacterial assemblages in large rivers. Rivers form important connections between terrestrial land use and marine biogeochemistry and act as conduits for organisms and allochthonous nutrients. Bacteria dominate carbon cycling and nutrient processing in rivers, and therefore can influence regional and global elemental cycles. This study …

The Characterization Of The Interplay Between Sumoylation And Influenza A Virus, Andres Santos

Open Access Theses & Dissertations

Influenza A virus is a negative sense single stranded RNA virus responsible for multiple devastating pandemics in the past. This virus is capable of entering multiple eukaryotic hosts, sequestering, and using their cellular machinery for its own replicative purposes. This plethora of molecular events is all orchestrated through a shockingly limited repertoire of only 11 viral proteins. All of which are all strictly dependent on cellular processes such as post-transcriptional regulation, protein translation, post-translational modifications, etc. This study is focused on the characterization of the interplay between Influenza A virus and the cellular SUMOylation system. The first part of this …

Metagenome Sequence Analysis Of Filamentous Microbial Communities Obtained From Geochemically Distinct Geothermal Channels Reveals Specialization Of Three Aquificales Lineages, Cristina Takacs-Vesbach, William P. Inskeep, Zackary J. Jay, Markus J. Herrgard, Douglas B. Rusch, Susannah G. Tringe, Mark A. Kozubal, Natsuko Hamamura, Richard E. Macur, Bruce W. Fouke, Anna-Louise Reysenbach, Timothy R. Mcdermott, Ryan Dem. Jennings, Nicolas W. Hengartner, Gary Xie

Biology Faculty Publications and Presentations

The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems world- wide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal “filamentous streamer” communities (∼40 Mbp per site), which targeted three different groups of Aquificales found in Yellow- stone National Park (YNP). Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobac- ulum (Aquificaceae) populations, whereas the circum-neutral pH (6.5–7.8) sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae). Thermocrinis (Aquificaceae) populations were found primarily in the circum-neutral sites with undetectable …

Managing Soil Microbial Communities With Organic Amendments To Promote Soil Aggregate Formation And Plant Health, Shawn T. Lucas

Theses and Dissertations--Plant and Soil Sciences

The effects of managing soil with organic amendments were examined with respect to soil microbial community dynamics, macroaggregate formation, and plant physio-genetic responses. The objective was to examine the possibility of managing soil microbial communities via soil management, such that the microbial community would provide agronomic benefits. In part one of this research, effects of three amendments (hairy vetch residue, manure, compost) on soil chemical and microbial properties were examined relative to formation of large macroaggregates in three different soils. Vetch and manure promoted fungal proliferation (measured via two biomarkers: fatty acid methyl ester 18:2ω6c and ergosterol) and also stimulated …

Type I Interferon Activation Of Natural Killer (Nk) Cells By Cytomegalovirus (Cmv) And Their Interaction With Dendritic (Dc) And Nkt Cells, Howaida A. Oulad Abdelati

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In our current study the roles of natural killer (NK) cells in regulation of the acute phase of murine cytomegalovirus (MCMV) infection were demonstrated. NK cells utilize perforin and gamma interferon to kill MCMV infected cells. Activation of NK cells is controlled by the balance between inhibitory and activation receptors and modulated by cytokines produced from the infected cells. Type I interferons are produced mainly by plasmacytoid dendritic cells (PDCs). Type I interferons have a substantial role in enhancing NK cells cytotoxic activity and NK cells proliferation and maintain cell survival through STAT mediated signaling pathway. IL-12 produced by dendritic …

Characterization Of Brer Interaction With The Bile Response Promoters Breab And Brer In Vibrio Cholerae, Francisca A. Cerda-Maira, Gabriela Kovacikova, Brooke A. Jude, Karen Skorupski, Ronald Taylor

Dartmouth Scholarship

The Vibrio cholerae BreR protein is a transcriptional repressor of the breAB efflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol. 190:7441-7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at the breAB promoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in the breAB promoter. However, only one of …

Inhibition Of The Host Translation Shutoff Response By Herpes Simplex Virus 1 Triggers Nuclear Envelope-Derived Autophagy, Kerstin Radtke, Luc English, Christiane Rondeau, David Leib

Dartmouth Scholarship

Macroautophagy is a cellular pathway that degrades intracellular pathogens and contributes to antigen presentation. Herpes simplex virus 1 (HSV-1) infection triggers both macroautophagy and an additional form of autophagy that uses the nuclear envelope as a source of membrane. The present study constitutes the first in-depth analysis of nuclear envelope-derived autophagy (NEDA). We established LC3a as a marker that allowed us to distinguish between NEDA and macroautophagy in both immunofluorescence and flow cytometry. NEDA was observed in many different cell types, indicating that it is a general response to HSV-1 infection. This autophagic pathway is known to depend on the …

Mitochondrial Dna Instability In Cells Lacking Aconitase Correlates With Iron Citrate Toxicity, Zhengchang Liu, Fei Xiao, Zhejun Dong, Tammy Pracheil, Muhammad Farooq

Biological Sciences Faculty Publications

Aconitase, the second enzyme of the tricarboxylic acid cycle encoded by ACO1 in the budding yeast Saccharomyces cerevisiae, catalyzes the conversion of citrate to isocitrate. aco1Δ results in mitochondrial DNA (mtDNA) instability. It has been proposed that Aco1 binds to mtDNA and mediates its maintenance. Here we propose an alternative mechanism to account for mtDNA loss in aco1Δ mutant cells. We found that aco1Δ activated the RTG pathway, resulting in increased expression of genes encoding citrate synthase. By deleting RTG1, RTG3, or genes encoding citrate synthase, mtDNA instability was prevented in aco1Δ mutant cells. Increased activity of citrate synthase leads …

Human Cytomegalovirus Us17 Locus Fine-Tunes Innate And Intrinsic Immune Responses, Stephen James Gurczynski

Wayne State University Dissertations

HCMV employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves "capture" of a host gene and then its successive duplication and divergence, forming a gene family, many of which have immunomodulatory activities. The HCMV US12 family consists of ten tandemly arranged sequence-related genes in the unique short region of the HCMV genome (US12-US21). Each gene encodes a protein possessing seven predicted transmembrane domains, and patches of sequence similarity with cellular GPCRs and the bax inhibitor-1 family of anti-apoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray …

Critical Events In Human Metapneumovirus Infection: From Entry To Egress, Brent A. Hackett

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a respiratory pathogen in Paramyxovirus family that demonstrates extremely high morbidity in the population, with most individuals having been infected by the age of five. Despite the prevalence of this negative-sense RNA virus in the population for decades, it was only identified in 2001. As such, there is currently no specific treatment for HMPV and the potentially severe consequences of infection for elderly and immunocompromised individuals and particularly infants make development of antivirals targeting HMPV of high significance. HMPV constitutes a quarter of all respiratory hospitalizations among infants, placing it second only to RSV, in addition …

Amp-Activated Protein Kinase Knockdown In Labyrinthine Trophoblast Cells Results In Altered Morphology And Function, Erica Ashton Kayleigh Carey

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The placenta is a transient organ that develops upon initiation of pregnancy and is essential for fetal development and survival. The rodent placenta consists of three layers with predominantly analogous cell types in the human placenta. The labyrinth layer, which lies closest to the fetus, facilitates nutrient and waste exchange between mother and baby. Abnormalities of the placenta may occur as a result of cellular stress and have been associated with the pregnancy-associated disorders, intrauterine growth restriction and placental insufficiency. AMP-activated protein kinase (AMPK) is a master sensor of cellular stress and changes in AMPK expression or activation could be …

The Effect Of Hsv-1 Infection On Differentiated And Polarized U937 Cells, Allolo Dreiwish Aldreiwish

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Herpes simplex virus type 1 (HSV-1) challenges the host immune system through several mechanisms (Frey, et al., 2009). In vitro, U937 cells (human macrophage-like precursor cell line) are not susceptible to HSV-1 infection when they are not differentiating (Lopez-Guerrero and Alonso, 1997). Differentiation of these cells resistance can abrogates their resistance to HSV-1 (Tenney and Morahan, 1991). In this study, we examined the effect of HSV-1 infection on differentiated and polarized U937 cells. U937 cells are differentiated to M0 cells.

Then, M0 cells are polarized to distinct phenotypes, M1 or M2. M1 are proinflammatory macrophages while M2 are anti-inflammatory cells. …

Establishment Of A Quiscence Herpes Simplex Type 1 Infection In L929 Fibroblasts And Neuro-2a Cells By A Nucleoside Analogue Acyclovir, Noura Shaklawoon

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Herpes simplex type 1 (HSV-1) quiescent infection was established in L929 cells, murine fibroblasts, and in Neuro-2A (N2A) cells, mouse neuroblastoma, by treating them with the nucleoside analogue acyclovir (ACV) 24 hours before infection. Subsequent release of virus from the non-productive state was accomplished by treating the cells with the histone deacetylase inhibitor trichostatin A (TSA). Treatment of both L929 and N2A cell lines with ACV 24 hours before infection induced protection from HSV-1 cytopathic effect. A quiescent state was confirmed by absence of virus plaques when supernatant fluids from ACV-treated HSV-1 infected cultures were titered on Vero cell monolayers. …

N-Glycosylation, Localization And Trafficking Of Endogenous Nkcc1 In Cos7 Cells, Richa Singh

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The Na+K+2Cl^- co-transporter-1 (NKCC1) effects the electroneutral movement of Na+, K+ and 2Cl^- ions across plasma membranes. NKCC1 is considered a widely distributed, highly N-glycosylated ion transporter involved in the regulation of the intracellular chloride concentration ([Cl^-]i), cell volume and salt secretion. NKCC1 locates in the plasma membrane of all cells studied so far, particularly on the basolateral side of most polarized cells. However, the majority of immunoreactive NKCC1 distributes in intracellular compartments of unknown nature whereas a small proportion of the transporter appears to be located in the plasma membrane. Here, we identified and characterized …

Novel Approaches For The Eradication Of Hiv Latently Infected Cells, Sally Al Ali

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The development of a suitable experimental cell model to study HIV latency in primary cells could have a massive effect on the current approaches to eradicate virus in latently infected cells. The main proposal of this paper is to develop an in vitro HIV cell model that represents HIV latency in vivo, then to create a more effective viral vector in order to target HIV reservoirs. For this goal, a directed evolution method is suggested to be used in order to mutate the AAV cap gene to generate a recombinant AAV vector that is capable of infecting primary resting CD4+ …

Conversion Of The U937 Monocyte Into "Macrophage-Like" Populations Exhibiting M1 Or M2 Characteristics, Bradley M. Sharp

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The U937 cell line is an oncogenic human monocyte cell line. These monocytes have the potential of differentiating into either macrophages or dendritic cells (Lawrence et al., 2011). This differentiation pattern depends on the characteristics of the tissue microenvironment (Kigerl et al., 2009). PMA (Phorbol 12-Myristate 13-Acetate) is a phorbol ester capable of transforming monocytic cells toward the macrophage pathway. Upon treatment with PMA, U937 cells under-go a series of morphological and functional changes. Traditionally monocytic cell lines are used as a model of macrophage function, because current human macrophage cell lines require a T-cell conditioned growth medium and contact …