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1
Walker, Candace Lynette. "Implementing Inquiry-Based Learning in a General Microbiology Laboratory." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/43973.
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In recent years there has been an increased interest in inquiry-based learning, also known as experiential learning or problem-based learning, as a more appropriate model of teaching science. The purpose of this study was to incorporate inquiry-based learning in a college sophomore-level General Microbiology Laboratory. The goal of this laboratory course is to introduce students to basic techniques and procedures necessary for the study of microorganisms. Laboratory sections were randomly assigned to an experimental group or a control/reference group. The experimental group was taught the concept of serial dilutions using an inquiry-based learning approach whereas the control group was taught using traditional teaching methods. Analysis of the data generated from the students' involvement in the investigation during the fall semester indicated that the experimental group had a slightly greater improvement in their knowledge of serial dilution. The study continued in the spring semester and involved close to 300 students. During the spring semester both the experimental and the control groups had similar attitudes about their learning experience as evaluated by a Lickert Scale survey. However, a statistical analysis of the quiz scores of the students with values within the interquartiles indicated the experimental classes' quiz scores were significantly higher on quiz 2 taken at the midpoint in the study. Thus an inquiry-based learning approach was found to be beneficial to the middle 50% of the class.Master of Science
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Pitt, Sarah Jane. "Managing for quality in clinical microbiology services." Thesis, Liverpool John Moores University, 2001. http://researchonline.ljmu.ac.uk/5526/.
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The technical quality of the work performed in clinical microbiology laboratories is regularly monitored, by external and internal schemes. Among the factors which might affect quality, attitudes of the laboratory staff are rarely considered. In this study, three concepts recognised by occupational psychologists as being important in the work place, Job Satisfaction, Commitment and Climate, were measured among microbiology biomedical scientists (BMSs) in the United Kingdom A self-report questionnaire was developed through preliminary interviews and two pilot studies. The perceptions of Job Satisfaction, Commitment (to both Profession and Organisation) and Climate were measured using established models from the occupational psychology literature. Three scales were devised specifically during this study to assess an individual BMS's perceptions of the standard of their own performance, the attitudes of their colleagues towards their work and the quality within their laboratory. A fourth measure was developed which collated all the ways that technical quality in clinical microbiology laboratories is currently measured in the UK into one scale. A total of 2415 questionnaires were posted to BMSs employed in National Health Service, Public Health Laboratory Service, Privately funded and University laboratories between November 1998 and February 1999. By March 1999,931 replies had been received, a response rate of 39%. BMSs reported lower Job Satisfaction than Medical Laboratory Technologists (the equivalent profession) in the United States. The results supported Meyer and Allen's (1991) three-component model of commitment and showed that BMSs experienced Professional Commitment more strongly than Organisational Commitment. An eight dimension model of Climate was developed, for clinical microbiology staff, from Newman's (1977) Perceived Work Environment scale. BMSs' perceptions of Individual Climate were affected by a number of demographic factors, but the most important was the size of the laboratory. The optimal number of people in a clinical microbiology department for positive Individual Climate was found to be less than 30. Affective Commitment to the Profession was the component of Commitment which most strongly influenced technical quality, through its positive relationship with an individual BMS's performance at work. Through aggregation of Climate scores for selected laboratories, it was shown that Laboratory Climate correlated positively with technical quality. From BMSs' perceptions of their laboratory's quality, a scale to assess `A Climate for Laboratory Quality' was developed. There was a strong positive relationship between `A Climate for Laboratory Quality' and a department's score on the measure of technical quality. Interviews with staff in four clinical microbiology laboratories supported the questionnaire findings with respect to Laboratory Climate. Qualitative data collected from a representative group of users of each of the four microbiology services showed that users' main concern was rapid turnaround time for results. Comments also highlighted the need for more effective communication between laboratory staff their colleagues working directly with patients.
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Boggs, Christine N. "The virtual edge development and evaluation of virtual labs for a general microbiology classroom /." Laramie, Wyo. : [University of Wyoming], 2006. http://proquest.umi.com/pqdweb?did=1260817691&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
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Clarke, Elizabeth Jane. "Understanding and designing sensors in Escherichia coli." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3398874.
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Pirbadian, Sahand. "Bacterial nanowires of Shewanella oneidensis MR-1| electron transport mechanism, composition, and role of multiheme cytochromes." Thesis, University of Southern California, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=3704250.
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In this thesis, we discuss three topics concerning extracellular electron transfer in the Dissimilatory Metal Reducing Bacterium (DMRB) Shewanella oneidensis MR-1. One proposed strategy to accomplish extracellular charge transfer in Shewanella involves forming a conductive pathway to electrodes by incorporating redox components on outer cell membranes and along extracellular appendages known as bacterial nanowires within biofilms. In the first part of this dissertation, to describe extracellular charge transfer in microbial redox chains, we employed a model based on incoherent hopping between sites in the chain and an interfacial treatment of electrochemical interactions with the surrounding electrodes. Based on this model, we calculated the current-voltage (I-V) characteristics and found the results to be in good agreement with I-V measurements across and along individual microbial nanowires produced by the bacterium S. oneidensis MR-1. Based on our analysis, we propose that multistep hopping in redox chains constitutes a viable strategy for extracellular charge transfer in microbial biofilms.
In the second part, we report the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe S. oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by our proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.
In addition, to elucidate the membranous nature of Shewanella nanowires, we imaged these filaments using Transmission Electron Microscopy. The TEM images reported in this thesis also provide the most accurate estimates of bacterial nanowire dimensions to date. Future TEM and cryo-TEM imaging can establish the specific alignment and configuration of outer membrane cytochromes that facilitate electron transport along bacterial nanowires.
In the third part of this thesis, we focus on the molecular conductance of MtrF, the first decaheme outer membrane cytochrome with a solved crystal structure. Decaheme outer membrane cytochromes of Shewanella play a crucial role in all the suggested pathways of extracellular electron transfer. An understanding of the electron transfer properties in MtrF will therefore impact all aspects of extracellular electron transfer research. In this thesis, using purified MtrF, we form monolayers of the protein on atomically flat gold substrates and address the dry monolayer with a Scanning Tunneling Microscope (STM) tip. This technique can be used in the future to examine the conductivity of individual MtrF molecules within the monolayer in the form of I-V curves. This methodology will allow experimental comparison with recently developed simulations of MtrF conductance.
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Joseph, Alton J. "Regulation of S6KL during cell cycle progression." Thesis, California State University, Long Beach, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1527714.
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mTOR (Mammalian Target ofRapamycin), PI3K (Phosphatidylinositol3-kinase) and MEK (Mitogen-activated protein kinase/ERK kinase) have been shown to be potent regulators ofS6Kl at G1 phase of the cell cycle. Research has been concentrated at the Gt phase to elucidate mTOR's role in cell growth and proliferation. Limited information is available on the activity ofmTOR, PI3K and ERKl/2 in cell cycle phases other than G1. Since we have observed that S6Kl is active in phases other than G1 our goal was to ascertain ifmTOR, PI3K or ERKl/2 have a role in regulating S6Kl during these cell cycle phases. Using cell cycle analysis and immunoblot analysis we have determined here that mTORand PI3K could play a role in regulating S6Kl at the G1/S transition iQ. the cell cycle but there is also indications that mTOR and PI3K are potentially involved in regulating S6Kl in the phases post-G1/S of the cell cycle, indicating a complex interaction between the kinases used to regulate S6Kl during the cell cycle. ERKl/2 is demonstrated to have limited involvement in the regulation of S6Kl during the cell cycle.
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Noonan, Jonathan. "Surfaced enhanced Raman spectroscopy (SERS) for the molecular imaging of atherosclerosis." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8939/.
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Cardiovascular diseases are the leading cause of mortality worldwide, with the majority of these deaths being a result of the inflammatory pathology, atherosclerosis. A critical need for multi-parameter molecular imaging has been identified to facilitate improved atherosclerosis diagnosis and the understanding of local inflammatory pathways in humans. Established imaging modalities such as ultrasound and magnetic resonance imaging are being investigated as potential solutions to this clinical problem, however, inherent limitations with these technologies have resulted in the exploration of alternate imaging approaches. This thesis focuses on the development and testing of surface enhanced Raman spectroscopy (SERS), a promising and novel molecular imaging modality, for the molecular imaging of vascular inflammatory biomarkers in vitro, ex vivo and in vivo. SERS detects molecule specific vibrational signals which are enhanced when an analyte is excited with light in close proximity to a noble metal surface. To achieve molecular specificity and surface enhancement, we developed antibody functionalised gold nanoparticles (nanotags) designed to bind to our molecular targets of interest, the adhesion molecules, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and P-selectin, and produce a SERS signal detectable by spectroscopy and/or microscopy based approaches. In vitro, we demonstrate the simultaneous and quantifiable SERS detection of ICAM-1, VCAM-1 and P-selectin on TNFa stimulated human endothelial cells. We subsequently demonstrated the simultaneous SERS detection of ICAM-1, VCAM-1 and P-selectin in freshly isolated atherosclerotic human coronary artery ex vivo. Finally, we explored SERS imaging in a humanised mouse model, demonstrating non-invasive multiplex imaging of adhesion molecules in vivo. In summary, this proof of concept study demonstrates the suitability of SERS and nanotags for the non-invasive molecular imaging of vascular inflammation. We have tested this approach with increasing biological complexity and highlighted SERS as a potential molecular imaging tool for future clinical translation in the context of vascular inflammation, atherosclerosis and cardiovascular disease.
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McArthur, Lisa. "Investigating interactions between rat adult cardiac myocytes and fibroblasts in the heart." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8512/.
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Boulerice, François. "Studies on HIV-1 replication in the monocytoid cell line U-937 : (etude de l'expression du VIH-1 dans la lignee cellulaire monocytaire U-937)." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41051.
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The expression of HIV-1 in cells of monocytic origin was investigated. In order to do this, the cell line U-937 was chosen because it possesses many characteristics of immature monocytes and can be easily infected by HIV-1. We found that long-term chronically-infected cells generate a high amount of defective particles, unable by themselves to successfully infect any target cells. Two main phenotypes were detected, one showing defectiveness in reverse transcriptase activity, and the other one showing inability to express fully mature glycoproteins.We also investigated the biological relevance of these defective particles, particularly in a situation where two cells generating defective virions are fused together by chemical methods. We show evidence that defective virions can complement each other in a hybrid cell and generate fully infectious particles resembling wild-type virus. Such a phenomenon may occur by genetic recombination, as suggested by Southern blot analysis. This finding may prove of clinical relevance since cells of phagocytic lineage can fuse in vivo naturally or by virus-mediated mechanisms.
We next tried to assess the efficacy of the antiviral drug, AZT, in controlling HIV-1 replication in such monocyte-like cells. AZT was found unable to completely abolish HIV-1 replication in vitro but was able to delay in a dose-dependent fashion the expression of the virus. In addition, we also studied the effect of AZT in clonal derivatives of U-937 cells, and found that the inhibitory effect was proportional to the degree of susceptibility of these cells to the virus.
Finally, we analyzed the susceptibility of U-937 cells by using such clonal derivatives and found that some displayed either high or low susceptibility to infection by HIV-1. The susceptibility which correlated with the amounts of intracellular viral DNA and TNF-alpha mRNA expression was found not to depend on levels of CD4 receptors, a result which suggests that other factors are involved in modulating replication in those cells.
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Sugrue, Daniel Martyn. "Modulation of Natural Killer cell response by human cytomegalovirus." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/42799/.
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The Natural Killer (NK) cell activating receptor DNAM-1 (CD226) is stimulated through recognition of CD112 (nectin-2) and CD155 (nectin-like molecule 5; PVR) on target cells. HCMV UL141 elicits protection from NK-cells by down-regulating CD155 from the cell surface and sequestering it in the ER (Tomasec, 2005). Here, HCMV UL141 was shown to be involved in the down-regulation of CD112. Interestingly, UL141 appeared necessary but not sufficient to modulate CD112 expression. This thesis therefore focused on a hypothesis whereby UL141 was acting with one or more additional HCMV genes to target CD112 for degradation. This project was the first to utilise an entire recombinant adenovirus (RAd) library expressing individual HCMV ORFs (RAd-HCMV-ORF library) to screen for function. The RAd-HCMV-ORF library clearly provided an extremely powerful tool for the screening of HCMV gene function as results were highly repeatable and robust. The co-infection of RAd-UL141 and RAd-US2 resulted in a single, clear, positive hit in the final screening process. This hit was further verified by immunoblot where CD112 appeared to be down-regulated in cells infected with both RAd-UL141 and RAd-US2, compared to controls. While a Hela-US2 cell line which stably expressed US2 also down-regulated CD112 when infected with RAd-UL141. A RCMVΔUS1-11 virus was constructed, which failed to down-regulate CD112 from the cell surface of RCMVΔSU1-11 infected cells. The addition of proteasome inhibitors was able to partially restore CD112 expression in HCMV infected cells (Prod'homme et al., 2010). It therefore appeared that US2 and UL141 act to degrade CD112 via the proteasome during HCMV infection. CD112 downregulation may have the potential to prevent DNAM-1:CD112 interaction between HCMV infected targets and effector cells of the immune system, providing another facet to HCMV’s ability to avoid the human immune response.
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Guo, Jiannan. "PolyA signals located near 5’ of genes are silenced by a general mechanism that prevents premature 3’ end processing." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1297/.
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PolyA signals located at the 3’ end of eukaryotic genes drive the cleavage and polyadenylation reaction to the nascent pre-mRNA. Although these sequences are expected only at the 3’ end of genes, we found that strong polyA signals are also present within the 5’ untranslated regions (UTRs) of many Drosophila melanogaster mRNAs. Although the polyA signals in 5’ UTRs show little activity of triggering 3’ end processing in the endogenous transcripts, they are very active when placed at the 3’ end of reporter genes. We further investigated these unexpected observations and discovered that both these novel polyA signals and standard polyA signals become functionally silent when they are positioned close to transcription start sites in either Drosophila or human cells. This suggests that the transcriptional stage when the polyA signal emerges from the polymerase II (Pol II) transcription complex could determine whether a putative polyA signal is recognized as functional. The data suggest that this mechanism, which probably prevents cryptic polyA signals from causing premature transcription termination, depends on low Ser2 phosphorylation of the C-terminal domain of Pol II and inefficient recruitment of processing factors.
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Woodcock, Stephen Mark. "Modelling the assembly and structure of microbial communities with applications to waste treatment strategies." Thesis, University of Glasgow, 2007. http://theses.gla.ac.uk/4468/.
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The central premise of this thesis is that combining models in theoretical ecology with our newfound ability to observe and measure microbial systems will allow for a suite of laws to be developed which can describe and predict the assembly and structure of microbial communities. This would allow environmental engineers to modify and improve the design and efficiency of wastewater treatment systems. It is demonstrated here that there is significant evidence that microbial community assembly is at least partly a random process. A simple Neutral Community Model (NCM) is shown to replicate much of the variability observed in real systems as diverse as the waste water treatment plants, estuaries and the human lung. This is in contrast to the prevailing view in microbial ecology that community composition is shaped by deterministic processes. Molecular methods in microbial ecology yield very small, sometimes biased, samples from what are ostensibly very large communities. It is demonstrated, using published literature on taxa-area relationships for microorganisms that sampling effects have the capacity to significantly distort the true, underlying ecological patterns. In doing so, a potential reconciliation is offered between some seemingly contradictory published reports on the nature of taxa-area relationships for microorganisms. The effects of sampling are built directly into the NCM, which allows the model to be tested using the data which are typically collected by microbial ecologists. The model is calibrated using the taxa-abundance distribution observed in a small waterborne bacterial community housed in a bark lined tree hole in a beech tree. Using these parameters, unchanged, it is shown that the model can predict the taxa-abundance distributions and taxa-volume relationship observed in 26 other beech tree communities whose sizes span three orders of magnitude. This represents the strongest test, so far, for any biological community, microbial or otherwise, that NCMs provide a useful tool in predicting community composition.
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Wang, Anqi. "Bacterial biofilms and biomineralisation on titanium." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1562/.
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This study investigated bacterial interactions with titanium, and evaluated the use of Serratia biomineralisation to produce a hydroxyapatite (HA) coating on titanium. Adherence of Gram-positive Staphylococcus epidermidis and Streptococcus sanguinis and Gram-negative Serratia sp. NCIMB 40259 and Escherichia coli was compared on commercially pure titanium, Ti6Al4V alloy, pure aluminium and pure vanadium. Grain boundaries, grain orientation and alloy phase structure did not influence adhesion or early proliferation. Adherence of all four strains was equivalent on pure titanium and Ti6Al4V and inhibited on pure aluminium. Serratia biomineralisation was used to introduce a crystalline coating on Al\(_2\)O\(_3\) grit blasted titanium discs and a porous titanium mesh. The porous coating consisted of micro-scale spheres composed of nano-scale calcium deficient HA. Embedded alumina particles and alkali treatment did not noticeably alter precipitation of Serratia HA, nor the structure of the coating in comparison with non-treated substrates. Coatings were retained after sintering at 800\(^\circ\)C in argon, although the original curved plate-like crystals changed to nano-scale β-tricalcium phosphate particles. A phosphorous-rich diffusion zone formed at the coating-titanium interface. This biomineralised coating may have applications for coatings of implants in non load-bearing sites, and other non-clinical applications where a high surface area is the major concern.
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Shaw, Paul B. "Studies of the alkaline degradation of cellulose and the isolation of isosaccharinic acids." Thesis, University of Huddersfield, 2013. http://eprints.hud.ac.uk/id/eprint/19266/.
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Cellulosic materials are expected to form a significant proportion of the waste proposed for disposal in underground repositories being designed for the storage of radioactive waste. Under the alkaline conditions of these facilities, cellulose degrades by a so called „peeling‟ reaction resulting in the production of a complex mixture of products (CDPs), the major components being α- and β isosaccharinic acid (α and β-ISA). A significant amount of research has been performed on ISA as part of the safety assessment for the development of these underground repositories due to the ability of ISA to complex with, and increase the solubility of radioactive isotopes. Until now, the vast majority of this research has involved the readily-available α-ISA, only a limited number of studies have involved β-ISA because no simple procedure is available for its isolation. Therefore, in this project, a method for the synthesis and isolation of β-ISA was developed. Cellulose degradation experiments which were performed to maximise solution concentrations of β-ISA are described in chapter 3. Microcrystalline cellulose was degraded under anaerobic conditions at either RT, 50 °C or 90 °C and comparisons were made between the use of NaOH and Ca(OH)2 as the base catalyst. As expected, the major products of all degradation reactions were α- and β-ISA, in addition, small amounts of free metasaccharinic acid (MSA) was detected in the Ca(OH)2 reactions. The largest solution concentrations of β-ISA were produced when cellulose was degraded at 90 °C using NaOH; after 24 hrs of reaction, solution concentrations of 12.7 g L-1 were achieved, whereas, in the equivalent Ca(OH)2 reaction, after 4 days a maximum concentration of only 5.1 g L-1was produced. For this reason, cellulose was degraded at 90 °C using NaOH to produce degradation solutions to be used in procedures to isolate β ISA. An additional finding was that significant amounts of ISA were being removed from degradation solutions due to absorption on to unreacted cellulose fibres; in the NaOH reaction, absorption was occurring rapidly and the percentage of ISA in both the solution and solid phases were very similar. In the Ca(OH)2 reaction, the absorption was a slow process and the percentage of ISA on the solid phase (61 %) was lower than the percentage of ISA in the solution phase (84 %) suggesting that solid Ca(OH)2 was affecting both the rate at which absorption was occurring and the composition of the absorbed species; this was possibly due to solid Ca(OH)2 physically obstructing the access of ISA to the cellulose fibres and also catalysing the oxidation of some of the ISA into smaller fragmentation products. Methods which were developed to isolate β-ISA are described in chapter 4. Isolation of β-ISA was initially achieved by eluting crude cellulose degradation solutions directly through a column of anion exchange resin. Using an automated system, a large throughput of material was possible resulting in the accumulation of relatively large amounts of β-ISA; after repeating the column 17 times, 1 g of pure β-ISA was isolated. However, using this method, the crude solutions severely fouled the anion exchange resin, concluding that anion exchange was more suited to small scale isolations of β-ISA. A final isolation procedure was developed which involved the elution of mixtures of benzoylated CDPs through normal phase silica columns. It was determined that prior to elution, coloured impurities could be efficiently removed by passing the derivatised mixture through a wide bed of silica. Slow elution of the resulting clean syrup through a large silica column allowed up to 7 g of tribenzoylated β-ISAL to be isolated and following de-benzoylation procedures, 2.6 g of β-ISA was isolated from a single column. The large protecting groups also allowed single crystals of both α- and β-tribenzoate to be produced and the resulting X-ray structures confirmed the absolute configuration of tribenzoylated β-ISAL as being 2R, 4S. Additional NMR analysis of collected fractions allowed several other polyhydroxylated compounds to be identified, also present as their perbenzoylated esters, these being: 3,4-dihydroxybutanoic acid, 2,5-dihydroxypentanoic acid, 2,3-dideoxypentanoic acid and 2,4,5-trihydroxypentanoic acid. The isolation of large amounts of β-ISA allowed several solution phase physical properties of β ISA to be measured and these are reported in chapter 5, including the aqueous pKa (3.61) which was determined using NMR methods. The rate constants for the inter-conversion between ISAH and ISAL were also studied for both α- and β-ISA. In acidic environments, ISAH undergoes an acid catalysed lactonisation to generate isosaccharino-1,4-lactone (ISAL), conversely in basic environments, ISAL undergoes a base catalysed ring-opening to produce ISAH. Using pH-stat autotitration, the second-order rate constants for the lactone hydrolysis reaction were determined, to which values of 25.3 M-1 s-1 for β-ISAL and 97.0 M-1 s-1 for α-ISAL were observed. The acid catalysed lactonisation of ISAH was studied using 1H NMR spectroscopy; the second-order rate constant for the lactonisation of β-ISAH (3.10 x 10-3 M-1 s-1) was larger than the second order rate constant for the lactonisation of α-ISAH (7.04 x 10-4 M-1 s-1).
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Thomas, Elizabeth Baby. "Analysis of protein kinases regulating the Trypanosoma brucei cell cycle." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6229/.
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Trypanosoma brucei spp. are protozoan parasites that cause Human African Trypanosomiasis in humans, and Nagana in cattle. These diseases are mostly fatal if left untreated and there is an urgent need for safe, effective drugs that can be easily administered. T. brucei has a complex cell cycle, the regulation of which appears to be divergent compared to other model eukaryotes. This implies that the regulators of the T. brucei cell cycle could be exploited as a source of novel drug targets. One such cell cycle regulator of interest is T. brucei polo-like kinase (TbPLK), a serine/threonine protein kinase thought to diverge from its canonical functions in eukaryotic mitosis and be mainly involved in the duplication of the parasite’s cytoskeletal structures. This study sought to investigate how the activity and localisation of TbPLK is regulated in procyclic form (PCF) and bloodstream form (BSF) parasites. A second aim of this study was to identify novel protein kinases (PKs) which regulate the T. brucei cell cycle by screening part of a kinome-wide RNA interference (RNAi) library of BSF cell lines, that has recently been established (Jones et al. 2014). The cell lines had already been assessed for proliferation defects upon RNAi induction by using an Alamar blue viability assay. In this study, the cell lines which displayed proliferation defects were further screened for cell cycle defects using growth curves and DAPI staining, to identify as yet uncharacterised protein kinases required for T. brucei cell cycle regulation. 50 PKs had been shown to be required for viability in vitro and were screened for potential cell cycle roles. Of these, 25 were identified as potential cell cycle regulators, 15 of which were detected for the first time. The majority of the hits were deemed to be involved in either just cytokinesis, or cytokinesis in combination with kinetoplast duplication or mitosis, with surprisingly few in G1/S. Knockdown of a number of these putative cell cycle PKs induced cell death signifying their potential as drug targets. Indeed, one of the hits, CLK1, was genetically validated as a potential drug target in a mouse model. The identification of these putative cell cycle kinases has also provided valuable starting points by which the signalling pathways that regulate the cell division cycle of these parasitic organisms can be elucidated.
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Aboklaish, Ali F. "The development of methods to investigate the mechanisms underlying serum resistance of Ureaplasma species." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/70797/.
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The human Ureaplasma species are among the smallest and simplest self-replicating bacteria known to date. These microbes cause infection in humans, particularly in the upper genital tract during pregnancy, leading to several adverse outcomes including preterm birth, chorioamnionitis, and respiratory diseases of neonates. Little is known about the pathogenesis of Ureaplasma and mechanisms by which they avoid recognition and killing by the complement system. In this thesis, some mechanisms underlying serum resistance of Ureaplasma spp. were investigated. This goal was achieved by creating serum-resistant models of serum-sensitive laboratory Ureaplasma strains and developing and using some proteomic and molecular biology methods to study the role of potential factors, which mediate serum resistance and play a role in pathogenesis of Ureaplasma. My original contribution to the knowledge in this work was the development of transposon mutagenesis method that can now be used to study virulence genes of Ureaplasma. This method will also allow genetic manipulation of Ureaplasma for future studies. Monitoring and investigating induced serum-resistant strains using immunoblot analysis and proteomics revealed significant changes in two candidate proteins coincident with serum resistance. The first was the elongation factor Tu protein that found to be immunogenic and had altered pI isoforms. The observed change in this protein was consistent in all serum-resistant strains, which suggests a possible role in mechanism of serum resistance, possibly as a mediator for binding complement regulators, such as factor H and C4BP, at the cell surface of Ureaplasma. The second candidate protein was a novel 41 kDa protein that was uniquely expressed in all induced serum-resistant strains. Expression of this protein in all resistant strains strongly indicates its involvement in mechanism(s) of serum resistance of Ureaplasma. The possible gene that encodes for this 41 kDa protein has putatively been identified as UUR10_0137 in the genome of U. urealyticum serovar 10 (strain ATCC 33699) using the transposon mutagenesis method developed in this study. Although the gene product of UUR10_0137 gene is not known (hypothetical protein), this protein is now identified and proposed to have a role in serum resistance of Ureaplasma. The product of the UUR10_0137 gene could function as a complement regulator or inhibitor that prevents the activation of complement system, protecting Ureaplasma from the complement attack. The contribution of the multiple- banded antigen, MBA, was proven to be unimportant to serum resistance. Sole antigenic variations in this major surface antigen of Ureaplasma did not play any role in mediating serum resistance. Confirmation of a gene that mediates complement resistance would dramatically increase our understanding of Ureaplasma pathogenicity and provide a target for future human studies with preterm birth and Ureaplasma infection.
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Kantawong, Fahsai. "Use of comparative proteomics to study a novel osteogenic nanotopography." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/919/.
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The principal aim of this thesis was to investigate the ability of surface topography in inducing bone cell differentiation for biomedical purposes. In orthopedic research, regeneration of bone defects can be performed in vitro using biomaterials. Third generation biomaterials aim not only to support tissue (first generation) and not only to be ‘bioactive’ (second generation), but to stimulate specific, known and desirable responses at the molecular level. Nanoscale topography offers a possible route to the development of third generation biomaterials. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) is a new method for assessing protein expression strategies and here, a micro-grooved topography was used as a model for protocol optimization. The protocol was successfully developed and proved that 2D DIGE can be used as a powerful tool in the evaluation of biomaterial can direct cell behavior and cell fate. Next, the refined protocol was applied to the evaluation of the novel nanotopographic features; near-square nanopits (120 nm diameter, 100 nm depth with the pitch between the pits was set to an average of 300 nm with a ± 50 nm error). Protein expression profiles indicated that ERK1/2 might play part in cell proliferation and cell differentiation. However, to make a clear conclusion about molecular signalling, the study of sub-cellular proteome is needed in the future work. Additionally, the use of another comparative proteomic technique; dimethyl labelling, implicated the possibility of sub-population differentiation, i.e. the formation of multiple cell types that could be advantageous in tissue engineering of complex organs. Furthermore, the application of fluid-flow bioreactors was shown to enhance the growth rate and possibly increased differentiation of cells cultured on nanotopographical features.
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Kremer, Katrin. "Vesicular trafficking in Toxoplasma gondii." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4753/.
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Toxoplasma gondii is an obligate intracellular protozoan parasite with a worldwide prevalence. Together with the causative agent of malaria (Plasmodium falciparum) and other medically important pathogenic parasites it belongs to the phylum of the Apicomplexa. Besides identifiable eukaryotic organelles, apicomplexan parasites differ from other eukaryotic cells by an extra set of specialised secretory organelles (micronemes, rhoptries and dense granules), that are sequentially secreted during invasion of the host cell. Upon host cell contact the apically located micronemes are the first organelles to be released and contain crucial virulence factors that are secreted. In order to systematically analyse vesicular traffic with a special focus on the secretory pathway of rhoptry and microneme proteins the ddFKBP system was used to perform a systematic analysis of Rab proteins in Toxoplasma gondii. Rab proteins are small GTP- binding proteins that are involved in targeting and fusion of vesicles from a donor to an acceptor membrane. Whereas higher eukaryotes like human cells encode more than 60 different Rab proteins apicomplexan parasites possess only a reduced core set of Rab proteins. Performing co-localisation studies with generated parasite lines expressing ddFKBPmyc-tagged versions of Rab1A, 1B, 2, 4, 5A, 5C, 7, 18 and Rab5B-ddFKBPHA revealed, that all these Rabs localise to the early secretory pathway (Rab1B, 2 and 18), the Golgi (Rab4), or the late secretory pathway (Rab5A, Rab5B, Rab5C and Rab7). No exact localisation could be defined for Rab1A. Rab5A and Rab5C, normally involved in endocytic uptake, were identified as important regulators of traffic to micronemes and rhoptries in Toxoplasma gondii, using an overexpression screen of Rabs and the analysis of trans-dominant mutants of promising candidates. Intriguingly, some microneme proteins could be found to traffic independently on functional Rab5A and Rab5C, indicating the existence of independent transport routes to micronemes, which again indicates that apicomplexans have remodelled Rab5-mediated vesicular traffic into a secretory system that is essential for host cell invasion. By using two-colour super-resolution stimulated emission depletion (STED) microscopy, distinct localisations of independent microneme proteins could be verified. This demonstrated that micronemal organelles are organised in distinct subsets or subcompartments. Given these results, it can be assumed that apicomplexan parasites modify classic regulators of the endocytic system to carry out essential parasite-specific roles in the biogenesis of their unique secretory organelles.
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Dobson, Rachel Pamela. "Analysis of the functions and interactions of RAD51 paralogues in Trypanosoma brucei." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/939/.
Full textAbstract:
Trypanosoma brucei evades host acquired immunity by antigenic variation, involving periodic switches in the variant surface glycoprotein (VSG) coat. DNA recombination is critical in this process and a key component of homologous recombination, RAD51, also plays a role in VSG switching. T. brucei encodes four proteins distantly related to RAD51; termed RAD51 paralogues, named RAD51-3, RAD51-4, RAD51-5 and RAD51-6. Two of these RAD51 paralogues, RAD51-3 and RAD51-5, have been shown to function in homologous recombination, DNA repair and RAD51 re-localization into foci following DNA damage. Surprisingly, however, only RAD51-3 appears to act in VSG switching. To examine the functions of all the RAD51 paralogues in T. brucei, reverse genetics has been used to generate mutants of the two remaining unstudied paralogues, RAD51-4 and RAD51-6. Phenotypic analysis of both mutant cell lines indicates that these factors also play critical roles in RAD51-directed recombination and repair, and both influence VSG switching in the parasite. As homozygous mutant cell lines of RAD51 and the RAD51 paralogues were available, it was possible to comprehensively compare the phenotypes of rad51 -/- with rad51-3 -/-, rad51-4 -/-, rad51-5 -/-, and rad51-6 -/-. From these results it was observed that the phenotypes of the rad51 paralogue mutants are broadly equivalent, with two exceptions. Firstly, as mentioned above, RAD51-5 does not function in VSG switching, and RAD51-4 and RAD51-6 may not have direct roles in VSG switching. Secondly, rad51-4 -/- mutants are less sensitive to the DNA damaging agent phleomycin and a higher percentage of rad51-4 -/- cells form DNA damage-induced RAD51 foci compared with the other homozygous mutant cell lines. These results may imply that RAD51-4 and RAD51-5 have a less central role in RAD51-directed DNA repair or that their functions can be performed by other factors. In addition, the physical interactions of all the RAD51 paralogues were examined. It was found by yeast two-hybrid and co-immunoprecipitation that they form at least two complexes, and probably function in sub-complexes in a similar manner to the Rad51 paralogues of higher eukaryotes. These analyses shed light on the evolution and role of eukaryotic RAD51 paralogues in DNA recombination and repair in general, as well as the contribution that recombination makes to antigenic variation in T. brucei.
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Yahyai, Sadaf. "Optimization of a method for testing ballast water for enterococci and an investigation on the occurrence of antibiotic resistance in vibrio cholerae." Thesis, University of Maryland, College Park, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1552707.
Full textAbstract:
Several methods of enumerating Enterococci in water are suggested in the literature, notably membrane filtration and mEA plating. To establish optimal growth conditions, including incubation time, (24 and 48 hr) and temperature (35°C and 41°C), samples of 0.1 mL, 1 mL and 10 mL filtered water collected from Lake Artemisia, MD, USA were amended with known concentrations of Enterococcus faecalis (ATCC 29212), filtered using 0.45 µm membrane filters, and incubated on mEA agar under different conditions: 35°C/24h, 35°C/48h, and 41°C/48h, following U. S. Environmental Protection Agency guidelines. Results demonstrated no significant difference among the volume and time of incubations used but a significant difference in the temperatures employed. Being the etiological agent of cholera, V. cholerae is a major public health problem in several developing countries. The prevalence of β-lactamase-producing strains and their isolation from life-threatening infections as well as the environment is alarming and presents a major therapeutic challenge for clinicians. The extended-spectrum β-lactamase profile of a collection of 210 V. cholerae O1 strains isolated from clinical and water samples was investigated. The strains were collected during ongoing epidemiological and ecological cholera surveillance in the provinces of Chhatak and Mathbaria in Bangladesh, between March 2009 and April 2012. Resistance to penicillins, monobactams, carbapenems, second-, third- and fourth- generation cephalosporins were tested by disk diffusion. Genotypic analysis of the resistance determinants was performed by PCR to detect ESBL (blaCTX, blaTEM, blaSHV), carbapenemases (blaIMP, blaSPM, blaVIM, blaBIC, blaNDM, blaKPC, blaAIM, blaSIM, blaDIM, and blaGIM). All strains were sensitive to the 4th-generation beta-lactam cefepime. This is the first report documenting such extensive resistance to monobactams and third-generation cephalosporin in V. cholerae.
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Khan, Mohammad Khalid. "In vitro fermentation of mixtures of indigestible carbohydrates by the human faecal bacteria." Thesis, University of Glasgow, 2000. http://theses.gla.ac.uk/5315/.
Full textAbstract:
Aim of this thesis was to evaluate mixtures of indigestible carbohydrates in vitro to predict their effects on gut function. In this study, I investigate the effect of combining carbohydrates with different fermentative properties and their interactive influences, reflected in the end products from in vitro fermentation. The study focused on the rate of fermentation and fermentability of such mixtures and the SCFA produced to gain an index of the likely site of fermentation in the colon. The main aim of the thesis was to produce a mixture of carbohydrates which would delay but preserve butyrate production from rapidly fermenting carbohydrates such as raftilose. This was achieved in several mixtures but mostly those containing raftilose and ispaghula. In general, mixtures of carbohydrates were fermented more slowly than raftilose alone. Overall, ispaghula was the most effective in slowing the rate of fermentation compared with pectin or gums. Mixing raftilose with ispaghula or guar gum gave the best preservation of n-butyrate and propionate production. The rate of n-butyrate production was less rapid in mixed cultures of three carbohydrates (raftilose, ispaghula and pectin) than cultures of 100mg raftilose but production of n-butyrate was preserved. In summary, ispaghula and raftilose in two-carbohydrate mixtures and ispaghula, pectin and raftilose in three-carbohydrate mixtures delayed the release of butyrate with no loss in butyrate production and may move butyrate further round the colon, at the same time reducing the potential adverse effects of raftilose. Moreover, the addition of pectin (or guar gum) may add the therapeutic effect of delaying nutrient absorption in the small intestine was well. These studies have identified at least two mixtures (raftilose & ispaghula; raftilose, ispaghula & pectin) worthy of study in more detail in man.
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Carmichael, Sarah J. "Signalling molecule production by Escherichia coli and Campylobacter jejuni." Thesis, University of Central Lancashire, 2006. http://clok.uclan.ac.uk/7647/.
Full textAbstract:
Quorum sensing (QS) is a density-dependent gene regulation signalling mechanism utilized by bacteria to enable the simultaneous expression of bacterial phenotypes in a given population. Several QS mechanisms and different classes of signalling molecules, including acyl-homoserine lactones (AHLs), have now been identified in numerous bacterial species. AHL production by clinical and laboratory isolates of Escherichia coli and Campylobacter jejuni has been investigated in this study. Laboratory, blood and urine E.coli isolates were analysed via three reporter strain bioassays for putative AHL production. The initial results indicated that the E.coli blood isolates produced a compound(s) capable of activating one of the bioassays. Subsequent analysis by thin layer chromatography and mass spectroscopy suggested that this active compound may have been a cyclic dipeptide, although the apparent inability to isolate subsequent samples of this active compound has prevented confirmation of this finding. All of the C. jejuni isolates tested induced activity in the Agrobacterium liquid culture bioassay, in a growth-dependant manner, indicating the possible presence of an exogenous AHL. Comparative analysis of the genome sequenced C. jejuni strain, NCTC 11 168-GS and its clinical counterpart, C. jejuni NCTC 11168-0, has indicated that these variants respond differently to changes in the levels of dissolved oxygen and toxic oxygen derivatives and as a consequence produce different levels of the active compound. FIPLC separation and HPLC-mass spectroscopy has indicated that this active compound may be N-hexadecanoylhomoserine lactone, providing preliminary evidence of a previously unidentified AHL-mediated QS mechanism within the Epsilon Prozeobacteria class.
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Grace, Cooper A. "Evolutionary genomics of transposable elements in the Saccharomyces sensu lato complex." Thesis, University of Huddersfield, 2018. http://eprints.hud.ac.uk/id/eprint/34743/.
Full textAbstract:
Transposable elements (TEs) are almost ubiquitous components of eukaryotic genomes that have long been considered solely deleterious or ’junk DNA’. They are split into two main forms, retro-transposons and DNA transposons, depending on the method of replication employed. Hosts have developed strategies for combating TEs including RNAi, methylation and copy number con-trol. TEs have also evolved ways of persisting in the genome in order to survive, such as target site specificity. Two additional ways which may be utilised by TEs, positive selection and horizontal transfer, were investigated here primarily using the budding yeasts in the Saccharomyces sensu lato complex. These species typically contain up to five families of retrotransposons, designatedTy1-5, and multiple subfamilies, all of varying transpositional activity. Discoveries of insertions evolving under positive selection and providing benefits to their hosts have been sporadic and serendipitous findings in a number of organisms. Full genome screenings for such insertions are rarely published, despite the impact TE insertions have upon their hosts. A population genomics approach was performed to address this issue in the genomes of Saccha-romyces cerevisiae and sister species S. paradoxus. Signatures of positive selection acting upon Ty insertions were identified using Tajima’s statistical D test. Neighbouring genes were also analysed to ascertain the true target of selection where hitchhiking linked the two. A subset of LTR-gene pairings were explored using qPCR in order to identify any effects on host gene expression the occupied loci may cause. Two genes displayed significantly increased levels of expression, which may be due to the presence of positive selection candidate LTRs, which in turn may contribute to improving host fitness. This thesis further documents the systematic screening for Ty-like elements of all available genomes of budding yeast and related species. Extensive phylogenetic analyses estimated evolutionary relationships and possible horizontal transfer events of elements between the species. Evidence for in excess of 75 horizontal transfer events was uncovered here, around half of which were successful in propagating in new genomes. The occurrence of horizontal transfer of TEs in the genomes of budding yeast is therefore far more common than previously documented. During screening of genomes, a further potential method of avoiding host defences was uncovered. The divergence of the highly active Ty4 family, which coincided with population isolation of multiple Saccharomyces species into subfamilies, was surprising given previous reports of this family being of particularly low activity. Such events are rarely recorded in eukaryotic genomes, and may also illustrate the compulsive spread of a new subfamily via horizontal transfer. The investigations reported here represent the first genomic screening of Ty insertions in Saccharomyces for signatures of positive selection, and an updated, comprehensive search for evidence of HT between species of budding yeast. Both may act as methods for TE families to persist in the genomes of their hosts, and represent far more than simply ’junk DNA’.
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Abdulla, Othman. "[1,2]-Sigmatropic rearrangement of benzylic ammoniumy lids ; Catalytic sp3-sp3 functionalisation of sulfonamides ; Annulation of arynes in the synthesis of sultams." Thesis, University of Huddersfield, 2018. http://eprints.hud.ac.uk/id/eprint/34758/.
Full textAbstract:
The first chapter in this thesis describes research on the asymmetric [1,2]-sigmatropic rearrangement of benzylic ammonium ylids. Our group previousely developed method showing that DMSO as solvent, and BTPP as base, in the presence of 5Å molecular sieves, dramatically improves the yield of the reaction. Hence, we applied the developed the method using8-(–)-phenylmenthol and (2S)-camphorsultam as chiral auxiliaries. In the second chapter, a new application of Pd-catalysed allylation is reported. This enabled the synthesis of (30)of branched sp3-functionalised sulfonamides, a compound class for which few reported methods exist. By reacting benzyl sulfonamides with allylic acetates in the presence of Pd0 catalysts and a base, at room temperature, direct allylation was efficiently performed, yielding products that are analogues of structural motifs seen in biologically active small molecules. The reaction was performed under mild conditions and could be applied to nanomolar sigma-receptor binders, thus enabling a late-stage functionalisation and efficient expansion of drug-like chemical space. The third chapter described a synthesis of benzosultam without recourse to transition-metal catalysis, or stoichiometric amounts of organometallic building blocks. Iodomethane sulfonylamide adds to benzyne (generated using fluoride sources), and then the formed intermediate undergos an intramolecular cyclisation to afford sultam. Using this method that procceds under simple reaction conditions, (11) benzosultams were synthesised in modest yield.
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Ruanto, Patcharawarin. "Architecture of bacterial promoters : the case of E. coli ogt promoter." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/4480/.
Full textAbstract:
The ogt gene encodes an O\(^6\)-alkylguanine DNA alkyltransferase, which is reported to repair DNA against methylation damage caused by reactive nitrogen species, which are generated during an anaerobic respiration in a presence of nitrate. This study examined transcription activation by NarL at the ogt promoter using biochemical techniques with various semi-synthetic ogt promoters. The ogt promoter has two crucial DNA sites for NarL dimers at positions -78.5 and -45.5 relative to the transcript start site. An interaction between NarL and αCTD was investigated using site-specific mutagenesis. Locations and orientations of NarL and RNA polymerase in the transcript initiation complex were also confirmed in this study. It was found that NarL, at position 45.5, is located on the different DNA helical face from αCTD, which binds to the minor groove immediately upstream the -35 element. This unrecognized promoter architecture allows residue 273 of αCTD to interact with residue 178 of NarL.
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Canestraro, Martina. "Role of MAPK and NF-κB signalling pathways in the regulation of the human GM-CSF gene in normal and leukaemic blood cells." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5720/.
Full textAbstract:
GM-CSF is an important haematopoietic growth factor and immune modulator. Studies on T cells revealed that efficient activation of the human GM-CSF gene is dependent upon the activation of an enhancer located 3 kb upstream of the promoter, inducible by phorbol myristate acetate and calcium ionophore (PMNI) via kinase-and Ca\(^2\)\(^+\) -dependent signalling pathways, respectively. This enhancer is often aberrantly remodelled as a constitutive DNase hypersensitive site (DHS) in acute myeloid leukaemia (AML). To investigate the role of MAPKs in enhancer activity and chromatin remodelling, I used activated T blasts and human leukaemic cell lines as inducible model systems. The combination of MEK and p38 MAPK inhibitors reduced PMNI-induced GM-CSF gene expression and the DHS at the enhancer. This was associated with a reduction in DNA-binding activity for the MAPK-inducible AP-1 and in the phosphorylation of MSK1, which in turn stimulates NF-κB transcriptional activity by phosphorylating p65 at Ser276. The combination of MEK and p38 inhibitors also reduced the PMNI-mediated recruitment of AP-1, MSK1 and NF-κB at the enhancer. These data demonstrate a cross-talk between the MAPK and NF-κB signalling pathways in regulating GM-CSF gene transcription and therefore represent potential targets for the treatment of AML cases where aberrant chromatin remodelling occurs.
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Kuravi, Sahithi Jyothsna. "The role of extra and intra vascular cells and their molecules in modulating glomerular inflammation during health and disease." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3363/.
Full textAbstract:
Endothelium forms an interface between vasculature and cellular sub endothelial environment responding to the changes in the immediate environment in health and disease. During vasculitic glomerulonephritis (VGN), autoantibodies raised against neutrophil serine proteases (SP) activate the neutrophils resulting in the release of neutrophilic products onto the endothelial surface changing its ability to support neutrophil recruitment and leading to its damage. We have hypothesized that endothelial functions are modulated by intra and extracellular environments of the vasculature and that the glomerular epithelial cells (podocytes) regulate glomerular endothelial cell (GEnC) functions. This work investigated the effects of SP (Proteinase-3 (PR3) and human neutrophil elastase (HNE)) in mediating neutrophil recruitment on EnC prior to the development of injury in a concentration and time dependent fashion. Most of the effects were inhibitable by the presence of α1-antitrypsin (α1-AT). Further, using an in vitro static co-culture system, this work demonstrates an endogenous mechanism by which podocytes modulate endothelial response to inflammatory stimuli in the glomerulus under healthy and disease conditions. Overall, these studies elucidate that GEnC presents an adhesive phenotype attracting neutrophils under the influence of inflammatory cytokines. During disease conditions as in VGN, exposure of endothelium to SPs results in a pre-activation stage leading to damage with high and/or prolonged exposures. Podocytes modulate glomerular endothelial responses to cytokine stimulation in health and disease. Thus, endothelial functions are tightly regulated by interacting cells and their molecules and could be an important phenomenon in the disease process of VGN where neutrophil recruitment plays a central pathogenic role.
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Balasiny, Basema Kasem. "Regulation and sources of nitric oxide in Escherichia coli." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6748/.
Full textAbstract:
The enteric bacterium \(Escherichia\) \(coli\) is exposed to nitric oxide (NO) in its oxygen-limited environment. Various transcription factors regulate gene expression to provide protection against nitrosative stress, but their respective roles remain controversial. Key questions answered in this thesis were whether S-nitrosylated OxyR directly regulates the expression of NO-regulated genes; whether NsrR is required for the synthesis of an important protective system; and whether FNR is a physiologically relevant sensor of environmental NO. Transcription from the NO-activated \(hcp\) promoter was almost totally dependent upon a functional FNR protein, which is inactivated by oxidative stress, but unaffected by deletion of the \(oxyR\) gene. This indicated that the effects of an \(oxyR\) mutation on resistance to nitrosative stress are indirectly due to inactivation of FNR rather than to direct activation of \(hcp\) transcription by S-nitrosylated OxyR. The NO-sensitive repressor, NsrR, is not essential for protection against nitrosative stress. Nitric oxide produced during nitrite reduction had no effect on the ability of FNR to activate or repress gene expression, which is primarily due to NO sensing by NsrR, not by FNR. Some NO accumulates in the \(E. coli\) cytoplasm even in mutants that lack known sources of NO. The source of most of this residual NO is the NsrR-regulated protein, YtfE. Contrary to the proposal by others that YtfE repairs iron-sulfur centres by replacing iron atoms released during nitrosative stress, it is proposed that YtfE releases NO from nitrosylated iron-sulfur proteins, and that Hcp reduces this NO to the less toxic N2O.
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Robinson, Ashley. "The development and application of a transposon insertion sequencing methodology in Escherichia coli BW25113." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7389/.
Full textAbstract:
Escherichia coli is one of the most studied model organisms in biology. Even with decades of research, there are a substantial number of genes with an as yet unknown function. Previously, to determine the link between gene function and phenotype took significant experimental effort. However, newer methods are capable of providing large amounts of biological data in short timeframes. One such method, transposon insertion sequencing, is a powerful research tool, which couples transposon mutagenesis and next generation sequencing to identify genes that have important or essential functions. Here, three transposon insertion sequencing methods were compared. The techniques were adapted from previously published literature. Based on a number of metrics one technique was shown to be superior for data generation. This method was chosen for application in further transposon-insertion sequencing experiments. Subsequently, the optimised method was used to assess which genes were essential for the viability of the model organism E. coli K12. The results of this work were compared with the literature and other databases of gene essentiality. A high degree of concordance was observed between our datasets and those generated previously through other methods. Indeed, the method described here was shown to have several benefits over previously used approaches. Finally, genes involved with maintenance of the outer membrane were identified by using markers for membrane permeability in tandem with the chosen method. In keeping with previous literature multiple genes involved with many aspects of the cell envelope were reported. Many of the reported genes were shown to be involved with metabolic processes related to the biogenesis and maintenance of the cell envelope.
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Al-Jassar, Caezar. "Biophysical characterization of the plakin family." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/4406/.
Full textAbstract:
The desmosome is an inter-cellular complex required for strong cell-cell adhesion in skin and heart tissue. The plakin family of proteins is important to the desmosome as their main function is to act as linkers between the cell surface and cytoskeleton. The structures, dynamics, impact of heart disease causing mutations and interactions of these component domains have remained largely unknown. In order to better understand this, biophysical techniques were used to shed light into the specific effects of pathological mutations using complementary techniques including nuclear magnetic resonance spectroscopy, small angle X-ray scattering and X-ray crystallography. Novel insights included the modular organization and flexibility of serial spectrin repeat constructs in the N-terminal plakin domain was established. Biophysical characterization of a variety of plakin family C-terminal tail constructs was successfully conducted in addition to understanding the molecular consequences of disease causing point mutations linked to Arryhthmogenic Right Ventricular Cardiomyopathy. Together this provides a better understanding of the specific roles of the plakin family’s N- and C- termini in linking the desmosomal machinery on the cell membrane to the intermediate filaments.
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Rajendran, Ranjith. "Adaptive resistance mechanisms of Aspergillus fumigatus biofilms." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4333/.
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Biofilm formation is one of several significant virulence factors associated with life threatening pulmonary infections in immunocompromised individuals caused by Aspergillus fumigatus. Previous studies have demonstrated phase dependant antifungal activity against A. fumigatus biofilms. Antifungal resistance associated with fungal biofilms is a complex multifactorial phenomenon, and it remains unclear specifically how this manifests itself in A. fumigatus. This study therefore aimed to investigate adaptive resistance mechanisms in A. fumigatus biofilms. Different phases of A. fumigatus biofilms were grown for 8, 12, 24 and 48h in polystyrene plates in RPMI media. Functional efflux pump activity was subsequently assessed using an Ala-Nap fluorescent uptake assay. Extracellular material was extracted from each phase and the level of extracellular DNA (eDNA) was quantifiedusing a microplate fluorescence assay. The minimum inhibitory concentrations (MIC) of different classes of antifungals were assessed in the presence and absence of different inhibitors using a checkerboard assay, or with a fixed concentration, by the broth microdilution method to assess synergism, antagonism, or otherwise. The presence of eDNA and phenotypic changes in biofilm caused by antifungal agents and inhibitors were assessed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) techniques. The resultant biofilm biomass for different experiments was evaluated using a crystal violet assay. SYBR green qRT-PCR was used to assess the expression of different genes implicated in biofilm resistance (AfuMDR 1-4, ChiA-E, HSP90 and Fks1) over the period of multicellular development, using a diffusion chamber in a murine model and a Galleria mellonella infection model. The results from this study demonstrated phase dependant expression of efflux pumps in A. fumigatus biofilm populations, which actively contributes to azole resistance. Moreover, voriconazole treatment induced efflux pump expression in both in vitro and in vivo models.These data suggest that A. fumigatus efflux pump proteins, which evolved to become integral to their natural physiological function, have inadvertently induced resistance to azole drugs, albeit in the early phases of biofilm development. Assessment of A. fumigatus biofilm extracellular matrix (ECM), associated with maturing biofilms, showed that eDNA is an important architectural component of the biofilm, helping to maintain its stability. The antifungal sensitivity of different phases of A. fumigatus growth decreased significantly in the presence of DNase, indicating that decreased susceptibility to antifungals in the A. fumigatus is mediated in part by eDNA.Its release was shown to correlate withchitinase activity, a marker of autolysis, suggestive that autolysis was associated with eDNA release. It was hypothesised that heat shock protein 90 (HSP90) was involved in this autolytic pathway. Therefore, when HSP90 was pharmacologically inhibited this led to a decrease in matrix eDNA level, providing a compelling mechanism through which HSP90 might regulate biofilm antifungal resistance. To test whether these mechanisms of adaptive resistance had any bearing clinically, a G. mellonella model was developed. It was shown that each of the key genes were expressed during infection, both in control and antifungal treated larvae. This validates the potential use of this insect model for resistance and virulence studies. Overall, this study establishes several novel adaptive resistance mechanisms regulating biofilm drug resistance in A. fumigatus biofilms. Moreover, it highlights the potential to target these mechanisms as a therapeutic strategy for managing and improving clinical outcomes in these hard-to-treat infections.
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Evans, Stephanie Kaye. "Studies on the response to, and recovery from, rapamycin in Saccharomyces cerevisiae." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6168/.
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The Target of Rapamycin Complex 1 (TORC1) is a key and conserved regulator of cell growth and proliferation. The xenobiotic compound rapamycin is a potent inhibitor of TORC1 in yeast. The EGO complex, a non-essential activator of TORC1 is required for recovery of cells following rapamycin treatment. Why? Here, we find that rapamycin is in fact only a partial inhibitor of yeast TORC1; wild-type cells are able to maintain slow proliferation in the presence of high concentrations of the drug (i.e. concentrations multiple times the minimum inhibitory concentration). We find that this residual, rapamycin-insensitive, proliferation is dependent on the EGO complex and on TORC1 activity. We show that the ability of cells to maintain slow proliferation in the presence of rapamycin dictates their ability to recover. We find that rapamycin is not actively detoxified in yeast; instead, rapamycin is cleared by dilution-by-proliferation. The cell-associated intracellular pool of rapamycin is stable, decreasing only very slowly following washout of the drug and only diminishing at the rate of cell proliferation. The rapamycin-insensitive growth rate also persists long after rapamycin washout, indeed, until cells recover from the drug. The rapamycin-insensitive growth rate is not only able to quantitatively account for the observed kinetics of recovery from the drug in wild-type cultures, but also explains the severity of the ego- recovery defect. We contributed to a large-scale genetic screen seeking mutants that, like ego- mutants, fail to recover from rapamycin treatment. We find that loss of any one of 10 proteins identified results in a rapamycin recovery defect and a slow rapamycin-insensitive growth rate. Our data propose important or novel roles of the core HOPS/CORVET complex, threonine biosynthesis, Vps15p, Vsp34p, Ccr4p and Dhh1p activities in modulating the activity or efficiency of TORC1. Overall our results reveal that rapamycin is only a partial inhibitor of yeast TORC1, that persistence of the drug within the cell limits recovery and that rapamycin is not actively detoxified in yeast. Instead, recovery occurs due to dilution-by-proliferation and distribution of the drug among an increasing number of progeny cells. We also identify a set of potentially novel regulators of TORC1 activity.
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Venzi, Marcello. "5-HT2A/2C receptor modulation of absence seizures and characterization of the GHB-model." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/70589/.
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Absence seizures (ASs) are non-convulsive epileptic events which are common in pediatric and juvenile epilepsies. They consist of EEG generalized spike-and-wave-discharges (SWDs) accompanied by an impairment of consciousness and are expressed within the thalamocortical network. This thesis initially focused on investigating the modulation of ASs by two serotonin receptors (5-HTRs), 5-HT2A and 5-HT2C, in a polygenic (i.e. Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and a pharmacological (i.e. γ-hydroxybutyrate, GHB) model of ASs. It was found that, in GAERS, pharmacological activation of 5-HT2A/CRs blocked ASs, whereas 5-HT2AR antagonists increased seizure length. However, experiments on the GHB-model revealed that GHB induced not only ASs but also a period of sedation/hypnosis, a behavioural state that had been neglected in the literature. Thus, the rest of this thesis was devoted to further characterizing the GHB-model. The main result was that GHB-elicited ASs can be distinguished at the level of both EEG and behaviour. In vivo characterization of thalamic firing during GHB-elicited ASs and hypnosis via silicon probes in freely moving animals revealed that both states were accompanied by a decrease in firing rate. In particular, contrary to what was predicted by in vitro and in vivo experiments under neurolept anaesthesia, T-type Ca2+ channel-dependent burst firing in thalamic neurons was found in <10% of spike-and-wave complexes of SWDs. The prevalent activity of nucleus reticularis thalami neurons during ASs was either silence or tonic firing. Indeed, thalamic application of the potent T-type channel antagonist, TTAP-2, by reverse microdialysis did not affect GHB-elicited ASs. Finally, the development of an algorithm to classify GHB-elicited ASs demonstrated that the spectral properties of SWDs can be used to discriminate hypnosis and SWDs. Moreover, spectral coherence can be used in different experimental models of ASs to characterize SWDs according to their waveform regularity.
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Sahl, Yaser. "Exploiting C. elegans to investigate the key combinatorial toxicology associated with the marine environment in the proximity of Jeddah City in the Red Sea." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/71989/.
Full textAbstract:
The interface between urban combinations and associated industry with fragile ecosystems delivering significant ecosystem services denotes one of the critical frontiers for ecological genomic investigation. The major issue when evaluating diffuse pollution generated at this interface revolves around the possible interactions between mixtures of contaminates that individually remain below trigger level but together may result in significant environmental impact. To determine whether mixture effects need to be considered, it is essential to define geochemical parameters by performing a survey for major classes of contaminates and to evaluate their penetrance into the food chain. The coastal marine environment of the Saudi Red Sea is subject to direct and indirect influences of major populations and industrial facilities found along the coast such as those discovered in proximity to Jeddah City in Saudi Arabia. Sampling of both sediment and sea water was performed at contrasting sites representative of near-shore with off-shore locations. Possible food-chain transference of any contaminates was evaluated by sampling fish (L.nebulosus) and plankton at the off-shore sites. Biomarkers are mostly useful in the evaluation of progressive diseases that apparent their symptoms long after exposure to the initiating factor. In such cases, traditional early warning symptoms of developing disease may be lacking. Thus, detection of earlier events can provide a valuable timely warning of risk. It is important to identify and address the growing environmental problems being faced by the community and address it before it takes the shape of an epidemic. To assess toxicity of single and paired metals to the nematode C. elegans, toxicity tests were designed to first determine the impact of single metal exposure Copper, Zinc and Aluminium and then nematodes were exposed to paired combinations. Exposures with paired metals showed a variety of interactions which ranged from antagonistic to synergistic effects.
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Alzahrani, Ahmed. "Communities of Arbuscular Mycorrhizal Fungi in salt marsh habitats : diversity, structure, and ecosystem function." Thesis, University of Essex, 2017. http://repository.essex.ac.uk/20196/.
Full textAbstract:
The relationship between ecosystem functioning and the biodiversity of microorganisms has been a central focus of recent ecological studies, and fungi are considered to play a key role in this relationship. For example, the Mycorrhizae’s association with two-thirds of terrestrial plants makes them one of the most common forms of symbiosis on Earth. Although Arbuscular Mycorrhizal Fungi (AMF) have been shown to be important in nutrient cycling, soil stability and enhancing plant growth by increasing root mycelia, much of the information we have on them is restricted to a small sample of woodland and grassland habitats. Consequently, the mechanisms regulating the diversity and community structure of fungi remain poorly studied across many habitats. This is particularly true for salt marshes, which are key conservation priority habitats in many countries, including the UK. Using the latest sequencing technologies, this work examined the community ecology of fungi across six different salt marsh habitats over two locations in Essex and Lancashire, UK and related this to local environmental variables and sediment nutrient statuses. In addition, by linking with other datasets from the same sites, this study also examined the role of biotic factors that are likely to influence the relationship of rhizosphere fungal communities with each other. Although a range of local abiotic factors significantly influenced the composition of fungal communities within each salt marsh, at larger scales, the compositions of these fungal communities were significantly affected by the pattern of the biotic factors that also differed over seasons. Indeed, the ability of these variables to predict fungal richness and abundances differed greatly between sites, suggesting that drivers of fungal II community structure may be site-specific to some extent. Indeed, fungal richness in relation to abiotic or biotic factors explained considerably different amounts of variation in each site, and generalised poorly to other sites. Therefore, it is possible now to argue against deriving general environment-diversity relationships without empirical validation in multiple sites. Finally, functionally similar fungi ponded similarly to the environmental gradients, suggesting a possible disconnect between functional redundancy and resilience in microbial communities.
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Suleman, Louise. "The role of bacterial proteases in the development of chronic wounds." Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2045659/.
Full textAbstract:
A large number of bacteria are able to secrete extracellular proteases that play vital roles in nutrient acquisition and biofilm formation but are also important biochemical mediators in bacterial virulence, facilitating host invasion. Microorganisms within chronic wounds have been hypothesised to form biofilms, which result in perpetuated inflammation and delayed wound closure. Whilst it is thought that exaggerated secretion of host-derived proteases within chronic wounds prevents successful wound closure, the contribution of bacterial proteases secreted from planktonic microorganisms and biofilms in chronic wound pathology has yet to be elucidated. It is therefore the primary research aim of this thesis is to assess the proteolytic activity of Pseudomonas aeruginosa and Staphylococcus aureus isolated from equine and human chronic wounds and determine whether there is a difference in activity between planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM). The next aim will be to identify these bacterial-derived proteases using zymography and mass spectrometry, and determine whether these proteases reduce wound closure in in vitro scratch wound models. More specifically, the effect of P. aeruginosa and S. aureus PCM and BCM from equine and human clinical isolates, and also purified proteases of these preparations, on the in vitro wound closure of equine normal fibroblasts (NFs), equine chronic wound granulation tissue fibroblasts (GTFs) and human dermal fibroblasts (HDFs), shall be investigated. In these wound models, zymography will be utilised to determine the release of host-derived matrix metalloproteases (MMPs). In this thesis I have identified, for the first time, high protease activity in equine chronic wound-derived P. aeruginosa PCM and BCM, by which 52kDa and 42kDa proteases were detected. P. aeruginosa PCM and BCM were shown to significantly reduce the wound closure of NFs (P < 0.0001) and GTFs (P < 0.0001). Furthermore, the soluble products of P. aeruginosa biofilms, but not planktonic P. aeruginosa, elicited the release of metalloproteases from equine NFs and GTFs. In human studies, high protease activity specific to P. aeruginosa BCM but not PCM (P < 0.0001) was determined. P. aeruginosa BCM significantly reduced the wound closure of HDFs (P <0.0001) and cell viability (P < 0.0001), and further induced the release of metalloproteases from HDFs. P. aeruginosa in biofilm form secreted 62kDa and 52kDa proteases, however, through the partial purification of these proteases, it was determined that these proteases did not play a role in the reduction of wound closure in HDFs. The P. aeruginosa-derived partially purified proteases did however reduce HDF cell viability (P < 0.05). The results in this thesis support the theory that bacterial-derived proteases may contribute to the emphasised proteolytic environment of human chronic wounds. Furthermore, the presence of bacterial biofilms within chronic wounds may induce the release of host-derived proteases. However the role of these specific P. aeruginosa-derived bacterial proteases in chronic wound pathology remains undetermined.
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Whitelaw, Jamie Adam. "The dynamic nature and functions of actin in Toxoplasma gondii." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8072/.
Full textAbstract:
Toxoplasma gondii is an obligate intracellular pathogen. Due to its experimental tractability it has acted as an excellent model system to understand the fundamental principles of pathogenic mechanisms within the group Apicomplexa, including Plasmodium spp. the causative agent of malaria. Work on T. gondii has provided the foundation to understanding how apicomplexan parasites power motility and invasion, which centres around the parasites gliding machinery. This movement depends on the parasite's acto-myosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system. Deletions of core components of the gliding machinery, such as parasite actin or subunits of the glideosome indicate that the parasites remain motile and invasive, albeit at significantly reduced efficiencies. These findings could be explained by different possibilities, such as functional redundancies or compensatory mechanisms for multiple components of the glideosome. Toxoplasma only encodes a single copy of ACT1, therefore redundancies for ACT1 are unlikely. Much of the research in to the role(s) of TgACT1 focuses on motility and invasion. Interestingly, while the conditional act1 KO shows a deficiency in gliding and invasion, severe defects affecting parasite survival were observed during intracellular replication and egress. The amount of actin remaining in the act1 KO parasites was disputed which led to alternate conclusions about actins role in the parasites. Therefore, this study provides a much more detailed characterisation of the conditional act1 KO and when the phenotypes are observed in relation to actin levels within the parasite. Furthermore, the study provides evidence of an alternative model for motility that is independent of the parasites acto-myosin system. Several studies assert that the polymerisation kinetics of TgACT1 is unusual, allowing the formation of only short, unstable actin filaments. However, to date, it has not been possible to study actin in vivo, therefore its physiological role has remained unclear. In order to investigate this, parasites expressing a chromobody that specifically binds to F-actin were generated and characterised. Importantly, TgACT1 forms a vast network during the intracellular life-stages that is important for parasite replication and egress. Moreover, these filaments allow vesicle exchange and produce F-actin connections between parasites in neighbouring vacuoles. This study also demonstrates that the formation of F-actin depends on a critical concentration of G-actin, implying a polymerisation mechanism akin to all other actins. This work is important for understanding the mechanisms used by Toxoplasma to move and invade with regards to the functions of the acto-myosin system. Moreover, it highlights a novel role of actin that is required to control the organisation of the parasitophorous vacuole during division. The role of actin during the lifecycle may have wider implications to other apicomplexan species, such as Plasmodium spp. and also much further in the field of parasitology where F-actin information is scarce.
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Vaikkinen, Heli Johanna. "Finding a gene for virulence in Trypanosoma brucei." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/8010/.
Full textAbstract:
Trypanosoma brucei is the protozoan parasite that causes sleeping sickness in humans and Nagana in cattle in sub-Saharan Africa. The genome of this diploid parasite has been sequenced and T. brucei has been shown to follow a Mendelian genetic system. This has made the use of classical genetic approaches to finding genes that confer phenotypic traits possible in T. brucei. A genetic map has already been assembled for the non-human infective sub-species of T. b. brucei and this has been used successfully for quantitative trait analysis to identify genetic loci that contribute to parasite specific traits. Variation in virulence between different species, sub-species and strains of trypanosomes is well reported in the literature but the genetic basis underlying these differences has not been extensively studied. However, it is clear that trypanosome virulence is influenced by both host and parasite genetic factors. One study examining the genetic basis of difference in virulence between T. brucei parasite strains was conducted by Morrison et al. (2009). This study found T. brucei genetic loci that were associated with different virulence phenotypes: anaemia, splenomegaly, hepatomegaly and reticulocytosis. The work detailed in this thesis is based on one of the Quantitative Trait Loci (QTLs) identified in the 2009 study, the QTL on chromosome 3 that is associated with enlargement of the host spleen (splenomegaly). This thesis describes the generation of new restriction fragment length polymorphism markers and adaptation of SNP sequencing for use in fine mapping the T. brucei virulence associated QTL, specifically the locus associated with splenomegaly (at that stage covering approximately 300 MB with 383 genes). These new markers were used in combination with existing markers to narrow down the splenomegaly QTL identified by Morrison et al. (2009) to a region 50 kb and 52 genes and from these identified a candidate gene. This thesis then goes on to describe the design and application of a novel allele replacement approach to studying the contribution of different alleles of the identified candidate to the splenomegaly phenotype. This approach has not been used before to examine T. brucei genes. Constructs were designed and produced to enable allele replacement of the candidate gene, and a genotyping validation pipeline established to enable evaluation of construct integration. Alleles of the candidate gene were replaced in two different T. brucei strains with a different allele of the same gene. Multiple clones of four different strains of allele replacement (AR) parasites were generated, two in two different progeny clone backgrounds (77 and 86). For each wild-type (WT) clone, a specific allele of the candidate gene was replaced with either a different allele of the same gene or, as a transfection control, with the same allele to rule out any contribution of the transfection process to the splenomegaly phenotype. This resulted in a panel of successfully transfected clones that are a platform for robustly testing the influence of the candidate gene alleles on the splenomegaly phenotype. These AR clones were then used in an in vivo experiment to assess any changes to the splenomegaly phenotype caused by the introduction of a different allele into the different genetic backgrounds. The resulting phenotypic measurements were unexpected and the implications of those results is discussed in the final discussion.
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Sanders, Jon G. "Disentangling the Coevolutionary Histories of Animal Gut Microbiomes." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17463127.
Full textAbstract:
Animals associate with microbes in complex interactions with profound fitness consequences. These interactions play an enormous role in the evolution of both partners, and recent advances in sequencing technology have allowed for unprecedented insight into the diversity and distribution of these associations. However, our understanding of the processes generating those patterns remains in its infancy. Here, I explore variation in microbiomes across two animal lineages—ants and mammals—to tease apart the role of these process in the evolution of gut microbiota. First, I explore patterns of phylogenetic correlation in gut microbiota of herbivorous Cephalotes ants and hominid apes. By examining the sensitivity of phylogenetic correlation to analytical parameters, I show that these outwardly similar patterns are likely to be the result of very different processes in each host lineage. Next, I examine in more depth the interacting effects of diet and phylogeny on the structure of baleen whale microbiomes. Whales consume a diet that differs dramatically from that of their closest extant relatives, the herbivorous artiodactyls. I use a combination of marker gene and shotgun metagenomic sequencing to show that a phylogentically conserved host trait, the multichambered gut, leads to functional and taxonomic similarities of whale gut microbiomes to those of their herbivorous ancestors via the fermentation of animal polysaccharides in the exoskeletons of their prey. Finally, I return to ants to examine how major shifts in the nature of gut microbial association correspond to host ecology. Using measures of absolute bacterial abundance, rather than diversity, I test the hypothesis that evolution of symbiosis with microbes has facilitated ants’ dominance of tropical rainforest canopies. Surprisingly, I find differences in the abundance of gut bacteria in different ant lineages that span many orders of magnitude, suggesting that evolutionary transitions in the functional role of symbiosis in this animal lineage correspond not only to changes in the diversity of these associations, but to changes in kind. The results of these studies help to clarify the roles of history and selection in structuring animal gut microbiota, hinting that the interaction of these factors may fundamentally differ between animal lineages.Biology, Organismic and Evolutionary
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Kath, James Evon. "DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718716.
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Translesion synthesis (TLS) alleviates replication stalling at DNA lesions. Bypass of lesions by specialized translesion DNA polymerases involves exchange with high-fidelity replicative polymerases. As a consequence of their lesion bypass activity, TLS polymerases are mutagenic, requiring careful regulation of polymerase selection. In this dissertation, I describe a single-molecule reconstitution of polymerase exchange and lesion bypass. Using Escherichia coli polymerases as a model system, I have determined that the dimeric processivity clamp can simultaneously bind a replicative polymerase and a translesion polymerase, facilitating rapid exchange during synthesis and lesion bypass. Overlapping sets of polymerase-clamp interactions additionally allow the TLS polymerase Polymerase IV to displace the replicative polymerase Polymerase III. I finally describe the observation of single Polymerase IV molecules in living cells and initial efforts to determine their localization and dynamics during TLS.Biophysics
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Chung, Hattie. "Genome evolution in structured systems." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493565.
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The evolution of a genome is shaped by spatial interactions at multiple scales. At the angstrom level, structural constraints on both RNA molecules and proteins contribute to the evolution of a gene sequence. Such optimized genes are weaved together in a particular order, out of a near-infinite number of combinations, to result in a genome. The fate of a genome is intricately linked to the evolutionary fate of its host organism; in turn, the fate of an organism is governed by where it resides in space. In this dissertation, I investigate how structure shapes the evolution of a gene, genome content, and pathogen populations residing in a diseased human lung. Chapter 1 provides a brief historical overview of population genetics in structured environments. I motivate why it is important to determine the migration rate of new alleles. Chapter 2 investigates how pathogens evolve within the structure of the cystic fibrosis lung. I find that migration rate and mutation rate are on similar timescales. Selection, rather than spatial isolation, maintains diversity within a pathogen population. Chapter 3 presents a new method to probe how codon choice is optimized throughout a gene. I find that codon choice is dictated by preference for particular RNA secondary structures, rather than intrinsic properties of a codon. Chapter 4 describes an ongoing study of how rapidly P. aeruginosa populations evolve in short-term infections. Preliminary results show that gene duplication events can sweep through a population in just 11 days. Chapter 5 introduces ideas for future directions. I pose questions regarding how pathogens evolve molecular mimicry that can trigger autoimmune disease in the human host, and how cancer-inducing inflammation might be detected from mutational signatures in the microbiome.Systems Biology
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Frenkel, Evgeni Mikhailovich. "Competition and Coexistence in Yeast Experimental Evolution." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493568.
Full textAbstract:
Natural selection gives rise to biodiversity by purging the less-fit among variants that are too similar (a principle known as character displacement), but to predict how fit or different an organism needs to be to survive is hard. In the simplest theoretical case, the probability whether one lineage versus another survives depends only on their relative fitness and random fluctuations. In more complex scenarios, this probability may depend on the fitness of all the other lineages in the population, mutations that these and other lineages acquire before the outcome of competition is decided, and additional ecological interactions. These complexities evolve readily in laboratory microbial populations, suggesting that they are the norm in Nature, and have been extensively studied theoretically. This thesis provides one of the few empirical examples in which the evolution and mechanism of some of these complexities have been characterized and modeled sufficiently to make basic predictions, such as whether a mutation will fix or go extinct, which competing lineages may or may not coexist, and how do these processes relate? This work was carried out in an established system for experimental evolution, populations of asexual budding yeast (S. cerevisiae) in microtiter plates.
Chapter 2 demonstrates an experimental design and modeling approach to infer the distribution of fitness effects of beneficial mutations from the population-dynamics of genetic markers. The inferred distribution accurately predicts fixation probabilities of lineages and adaptation rates of populations. Chapter 3 describes a new example of spontaneously-evolved coexistence between types competing for the same resources, including the physical mechanism, genetic basis and a mathematical model of the coexistence. The conclusion provides additional analyses to connect the findings from these two chapters and discusses their implications for microbial evolution more generally and directions for future work.Biophysics
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Winter, Sandra. "Investigating the steam sterilization of dental handpieces." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7057/.
Full textAbstract:
Dental handpieces are used in a wide variety of dental treatment and oral surgery. During patient treatment handpieces becom contaminated with patient material. Due to the design and function of the dental handpieces, internal contamination of handpiece components frequently occurs during use, raising the risk of iatrogenic infection. Dental handpiece lumens represent a challenge for both cleaning and steam sterilization due to limited access. Manufacturers of handpieces and benchtop sterilizers as well as international standards and several guidelines recommend use of a vacuum steam sterilization process for lumen devices; however, non-vacuum is used in many UK dental practices. Therefore the aim of this thesis was to investigate if benchtop steam sterilization processes commonly used in dental practice are appropriate for sterilizing dental handpieces. Critical variables affecting the outcome of steam sterilization, such as pre-cleaning and lubrication were assessed. In order to investigate the above stated aim, four research questions were formulated: 1- Investigating steam penetration into dental handpieces and lumens in general (chapter 4), which was approached using thermometric measurements, chemical and biological indicators were used in different handpiece types (high-speed turbines, slow-speed motors, surgical handpieces) and process challenge devices using non-vacuum and vacuum sterilization cycles in a laboratory setting (chapter 4) and in general dental practices (chapter 6). 2- Investigating the effect of pre-cleaning dental handpieces, contaminated with different test soils from the standards or clinical contamination after patient treatment using a washer-disinfector or a handpiece cleaner-lubricator, which was assessed using the o-phtalaldehyde and G-box method (chapter 7). 3- Investigating the effect of handpiece lubricating oil on microbial inactivation by altering different parameters during a steam sterilization process using a BIER/CIER vessel in St. Paul (MN, US) (chapter 5). 4- Investigating the effect of different humidity levels on chemical and biological indicators using a BIER/CIER vessel in Neuss (Germany) (chapters 3). Thermometric measurements as well as assessment of chemical and biological indicators suggest that not all handpiece types can successfully be sterilized in all non-vacuum benchtop sterilizers. Especially the surgical handpiece appears to be difficult to sterilize. All non-vacuum sterilizers in general dental practice failed to sterilize handpieces. The comparison of the cleaning efficacy of a washer-disinfector and a handpiece cleaner-lubricator showed that a washer-disinfector is more efficient in cleaning the outside of a handpiece. Handpiece lubrication oil appears to impair steam penetration into handpiece lumens. Pre-conditioning in high humidity (90% RH) causes chemical indicators to perform a colour change and indicate successful sterilization quicker than ones pre-conditioned in low humidity (14% RH), which suggests that it is moisture rather than saturated steam that causes chemical indicators to indicate pass conditions. Non-vacuum sterilization benchtop sterilizers are not adequate for sterilizing dental handpieces. A vacuum process is highly recommended in the interest of patient and staff safety. Chemical and biological indicators are not necessarily reliable and results should be interpreted with care.
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Buencamino, Raphael Hector Domingo. "Novel roles of actin binding proteins in Listeria monocytogenes actin-based motility revealed within a cellular context." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3297798.
Full text
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Inverarity, Donald James. "Genomic diversity in naturally transformable Streptococcus pneumoniae." Thesis, Connect to e-thesis, 2009. http://theses.gla.ac.uk/901/.
Full textAbstract:
Thesis (Ph.D.) - University of Glasgow, 2009.Ph.D. thesis submitted to Faculty of Biomedical and Life Sciences, Division of Infection and Immunity, University of Glasgow, 2009. Includes bibliographical references. Print version also available.
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Groban, Eli S. "The study and engineering of cellular signaling pathways." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3339232.
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Thesis (Ph.D.)--University of California, San Francisco, 2008.Source: Dissertation Abstracts International, Volume: 69-12, Section: B, page: 7352. Advisers: Matthew P. Jacobson; Christopher A. Voigt.
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Timling, Ina. "Peeking through a frosty window| Molecular insights into the communities of Arctic soil fungi." Thesis, University of Alaska Fairbanks, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3607060.
Full textAbstract:
Fungi are thought to be one of the most diverse groups of organisms in the Arctic. They drive mineral and energy cycles and influence the occurrence of other organisms as mutualists (mycorrhizae, endophytes, lichens), decomposers and pathogens. Nevertheless, information on fungal biodiversity and distribution patterns in relation to environments across the Arctic is still sparse. Molecular methods were used to examine the diversity and community structures of ectomycorrhizal fungi (EMF) associated with two principal arctic host plants, Salix arctica and Dryas integrifolia, as well as total soil fungal communities of adjacent disturbed and undisturbed areas of patterned-ground features across the five bioclimatic subzones (A-E) of the North American Arctic. Key findings include the following: (1) More diverse fungal communities had been observed than previously known. These communities encompass nearly all fungal phyla and included all fungal guilds. However, a few species-rich fungal families dominated these fungal communities. (2) Surprisingly, species richness did not decline with latitude. (3) The most abundant fungal taxa were widely distributed in and beyond the Arctic. Yet root (EMF) and soil fungal communities showed niche preferences in regard to bioclimatic subzones. Furthermore, disturbed and undisturbed patterned ground features harbored different soil fungal communities with the exception of the coldest subzone A. In contrast, EMF community composition was not affected by host plant identity. (4) Fungal communities in the warmest subzone E were distinct from the other arctic subzones and the majority of taxa matched fungi from the boreal forest. (5) Key drivers of fungal community and guild composition along the bioclimatic gradient included regional climate, pH as well as vegetation composition and productivity across the subzones. At the local scale of patterned-ground features, fungal communities were correlated with vegetation composition and microclimate. With a warming climate, I would expect an enhanced colonization of patterned-ground features by vascular plants that would then affect fungal community structure not only at the species level, but also at the level of fungal guilds. In particular I would expect increases in fungi that are symbiotic with plants and a northward shift of both plant and fungal taxa.
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Estrella, Sanchez Maria del Rocio 1962. "Biodegradation, sorption and transport of 2,4-D under saturated and unsaturated soil conditions." Thesis, The University of Arizona, 1992. http://hdl.handle.net/10150/278212.
Full textAbstract:
Researchers have traditionally viewed sorption, degradation and transport as separate processes and only recently have these processes viewed as coupled. 2,4-D was chosen as a model system to study the interaction between these processes. A series of laboratory batch and column experiments with a sandy loam soil were conducted to determine the relative contributions of sorption and degradation to transport of 2,4-D under both saturated and unsaturated conditions. The sorption contribution to 2,4-D transport was not significant under saturated (Kd = 0.249 mg/g) nor unsaturated conditions (Kd = 0.566 mg/g). Degradation however, was very significant, specially under unsaturated conditions where the estimated first order biodegradation rate (μ) constant was 4.39 d-1. Rate constants under the saturated transport experiment were restricted by oxygen limitations. There was an order of magnitude difference between μ of batch and column experiments which were attributed to differences in aeration and mixing conditions.
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Slieman, Tony Adel. "DNA repair and photochemistry in Bacillus subtilis spores." Diss., The University of Arizona, 2000. http://hdl.handle.net/10150/284204.
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Bacterial endospores are 10 to 20 times more resistant to ultraviolet radiation than their vegetative counterparts, due to two interlocking mechanisms: the DNA photochemistry in spores, and the presence of two DNA repair systems. Spore DNA is closely associated with small acid soluble spore proteins (SASP) which change the conformation of DNA from the B form to an A-like form. When spores are subjected to UV radiation, SASP-bound DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally referred to as spore photoproduct (SP). Spores possess two DNA repair pathways that repair SP, the general nucleotide excision repair (NER) pathway encoded by the uvr genes and the SP-specific SP lyase repair system encoded by the splB gene. Most of the information regarding spore UV resistance has traditionally been obtained using commercial UV lamps that emit predominantly 254-nm UV (UV-C). In contrast, solar UV radiation that reaches the Earth's surface spans 290 to 400-nm wavelengths, the so-called UV-B and UV-A portions of the UV spectrum, whereas the UV-C portion of solar UV is mainly filtered by the stratospheric ozone layer. Ten percent of bacterial spore dry mass consists of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA). DPA has been implicated in triggering germination in germination-deficient mutant B. subtilis spores. DPA has also been shown to photosensitize spore DNA to UV radiation. In this dissertation the SP lyase repair system, spore DNA damage cause by environmental UV, and the role of DPA in the survival of spores to UV radiation were investigated. SplB protein containing an N-terminal 10-Histidine tag [(10His) SplB] was over-expressed and purified from Escherichia coli. The purified (10 His) SplB was used for characterizing the binding of the enzyme to its substrate, SP. A 35-bp oligonucleotide (oligo) was constructed with a single pair of adjacent thymidines on one strand. The oligo was irradiated with 254-nm UV under conditions to produce either SP or cyclobutane pyrimidine dimers (Py<>Py). By DNase I protection, (10His) SplB was shown to bind specifically to the SP-containing oligo and not to the oligo containing Py<>Py or the unirradiated oligo. (10His) SplB bound to the oligo containing SP exhibited a 9-bp DNase I footprint with two hypersensitive sites within the footprint. Bacillus subtilis spores were exposed to UV-C, UV-B, solar UV-A and full spectrum sunlight. Chromosomal DNA was then extracted and probed for the presence of damage using a combination of enzymatic and electrophoretic treatments. Spores were shown to accumulate Py<>Py, single stranded breaks and double stranded breaks in addition to SP. No apurinic/apyrimidinic sites were detected under any irradiation conditions used. Mutant spores that make DPA (DPA⁻) or that were DPA-deficient (DPA⁻) were exposed to UV-C, UV-B, solar UV-A, and full spectrum sunlight as a dried-film or in suspension. When irradiated as a dried-film DPA⁻ spores were the most sensitive followed by the DPA⁻ spores and wild-type spores under all irradiation conditions except for solar UV-A where the DPA⁻ spores were the most resistant. On the other hand, DPA⁻ spores irradiated with UV-C in suspension were 8 times more resistant in comparison to the same spore irradiated as a dried-film.
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Bennett, Louise Agnes. "BMP-7 : role and regulation in osteoarthritis." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8171/.
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Osteoarthritis (OA) is a disease characterised by changes in the structure and function of articular joints, leading to pain and loss of mobility. Bone Morphogenetic protein 7 (BMP-7), a member of the transforming growth factor β superfamily, has been shown to promote anabolic events within articular cartilage, and confer protection from OA associated destruction in a number of animal models. It has been shown that a disease-associated loss of BMP-7 in OA may contribute to the joint destruction. The mechanism associated with the loss of BMP-7 has yet to be fully elucidated. Recently, small non-coding RNAs (microRNAs) that participate in post-transcriptional gene regulation, have been identified as a potential dysregulated mechanisms in OA. It was therefore hypothesised that disease-associated alterations in these microRNAs could lead to subsequent changes in the expression of BMP-7 and its signalling family. The aims of this thesis were to investigate the expression of BMP-7 and other associated BMP signalling molecules and identify any microRNAs that may regulate these transcripts. Furthermore, the study aimed to elucidate the molecular mechanisms by which BMP-7 is able to confer protection in cartilage. The studies presented in this thesis show that both articular chondrocytes and the synovial membrane can express very low levels of BMP-7 transcript in a subset of patients. In juxtaposition, protein can be clearly detected in both articular chondrocytes and synovial membrane. Interrogation of the BMP-7 signalling family transcripts revealed that all members are detectable in OA cartilage. This expression was independent of the eroded nature of the cartilage. Evaluation of the circulating microRNAs that were predicted to target the BMP-7 pathway revealed that several miRNAs (including miR24-3p) were altered in the plasma of OA patients. Interestingly, miR24-3p was able to target BMP receptors ALK2 and BMPR1B. Moreover, there was a significant negative correlation between the expression of miR24-3p and the ALK2 receptor in OA patients. Thus suggesting there is a role for this miRNA in the negative regulation of BMP-7 signalling in OA cartilage. To complement the work evaluating the endogenous signalling pathway, studies were also undertaken to investigate the impact of exogenous BMP-7 stimulation on chondrocytes. BMP-7 was able to promote its own transcriptional expression in a patient specific manner and induce expression of IL-1β in all of the donors investigated. In addition to the induction of IL-1β, BMP-7 was also able to up-regulated the IL-1β antagonist, IL-1Ra. Taken together this data suggests a role for BMP-7 in the regulation of the inflammatory mediator IL-1β. Finally, BMP-7 was able to up-regulate several pro-inflammatory cytokines and chemokines in both primary OA chondrocytes and in vitro differentiated macrophages. In summation, the work presented in this thesis suggests that BMP-7 may be contributing to the promotion of inflammation and subsequent repair as part of the cartilage homeostatic mechanisms. Further to this, miR24 has been highlighted as a regulator of cartilage homeostasis via the direct targeting of ALK2. Changes in the expression of this miRNA over the course of OA disease progression may be involved in driving disease pathogenesis. Therefore understanding the targets for this disease-associated miRNA may help in the development of disease modifying therapies.