Dissertations / Theses on the topic 'Pathogen'
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Panagoda, Gehan Joseph. "Pathogenic features of Candida parapsilosis : an emerging fungal pathogen /." Thesis, Hong Kong : University of Hong Kong, 1998. http://sunzi.lib.hku.hk/hkuto/record.jsp?B20377770.
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Hovhannisyan, Hrant 1992. "Comparative transcriptomics of host-pathogen interactions and hybridization in Candida pathogens." Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/670316.
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Candida pathogenic yeasts represent a global healthcare problem. They comprise phylogenetically diverse species, including newly emerged pathogens. How human-Candida interactions vary across species, and what processes underlie the emergence of novel pathogens are poorly understood. Current thesis addresses these issues using comparative transcriptomics and bioinformatics. We established the global patterns of host-pathogen interactions between human host and the main Candida species, providing novel mechanistic insights into their interplay. We also explored lncRNAs of these pathogens, assessing their implications in infection. Further, we designed and validated a pan-Candida RNA enrichment approach, opening new possibilities for studying host-pathogen interactions in vivo. Then, we assessed the impact of hybridization on transcriptomes of hybrid yeasts, exploring the links between hybridization and virulence emergence. We also developed a new bioinformatics tool facilitating the research in the field. Altogether, results of this thesis expand our knowledge on relevant aspects of human-Candida interactions and yeast evolution.Las levaduras patógenas Candida representan un problema de salud global. Este grupo de levaduras, comprenden especies filogenéticamente diversas, e incluye patógenos emergidos recientemente. La forma en que las interacciones entre humanos y Candida varían de una especie a otra y qué procesos subyacen a la aparición de nuevos patógenos son poco conocidos. La tesis actual aborda estos problemas utilizando una aproximación de transcriptómica comparativa y bioinformática. Establecimos los patrones globales de las interacciones huésped-patógeno entre el huésped humano y las principales especies de Candida, proporcionando nuevas ideas mecanicistas sobre su interacción. También exploramos los lncRNA de estos patógenos, evaluando sus implicaciones en la infección. Además, diseñamos y validamos un enfoque de enriquecimiento de ARN pan-Candida, abriendo nuevas posibilidades para estudiar las interacciones huésped-patógeno in vivo. Luego, evaluamos el impacto de la hibridación en los transcriptomas de levaduras híbridas, explorando los vínculos entre la hibridación y la aparición de virulencia. En su conjunto, los resultados de esta tesis amplían nuestro conocimiento sobre aspectos relevantes de las interacciones humano-Candida y la evolución de las levaduras.
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Gibson, Josie. "In vivo imaging and analysis of host-pathogen interactions of intracellular pathogens." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19047/.
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Cellular and extracellular host-pathogen interactions are important in the progression of infection. Extracellular survival and growth can be significant for pathogen dissemination. Equally, intracellular pathways, such as autophagy, can be employed in host cell defence. Autophagy is a cellular self-degradation process that recycles cellular components through lysosomal degradation. Primarily for regulating starvation and housekeeping pathways, autophagy is also important for degradation of invading intracellular pathogens. Selective autophagy receptors can also target pathogens for autophagic degradation. However, some intracellular pathogens are able to subvert or block host cell autophagy. Cryptococcus neoformans and Staphylococcus aureus represent fungal and bacterial pathogens which can reside either intracellularly or extracellularly. The role of host cell autophagy in C. neoformans and S. aureus infection is unclear. Infection of zebrafish with C. neoformans or S. aureus enabled in vivo imaging of host-pathogen interactions, to examine infection growth dynamics and dissemination, in addition to cellular level imaging of pathogen interactions with host cell autophagy components. Cryptococcal infection can cause cryptococcal meningitis, frequently associated with cerebral infarcts. Analysis of cryptococcal proliferation during infection suggested that formation of intravascular cryptococcal masses precedes invasion of surrounding tissue. Vessel integrity analysis highlighted cryptococcal-mediated vascular damage, potentially providing a route for tissue dissemination. Vasculature damage may explain the origin of cortical infarcts in disease. Autophagy mutant zebrafish were generated to analyse host autophagy in pathogen infection. Characterisation of mutant larvae revealed a clear survival and growth defect, indicating autophagy is required for larval development. Autophagy mutants were subsequently used to analyse the role of autophagy during infection through analysis of pathogenic burden and pathogen association with autophagosome marker LC3-II. LC3II recruitment to pathogens was reduced in autophagy mutant neutrophils. Additionally, selective autophagy receptor p62 was recruited to S. aureus and C. neoformans within neutrophils, highlighting the involvement of host cell autophagy during infection.
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Yan, Ling. "Phagocyte-pathogen interactions." [Lincoln, Neb. : University of Nebraska-Lincoln], 2004. http://www.unl.edu/libr/Dissertations/2004/YanDis.pdf.
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Feldmann, Friederike. "Implication of extraintestinal pathogenic Escherichia coli siderophore receptors in host pathogen interaction." kostenfrei, 2008. http://mediatum2.ub.tum.de/doc/649951/649951.pdf.
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Kärkkäinen, Riikka M. "Production of DNA aptamers with specificity for bacterial food pathogens." Thesis, University of Chester, 2012. http://hdl.handle.net/10034/620695.
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Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.
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Ahmad, Sarah. "Identification of pathogen-specific protein-encoding genes from microbial pathogens based on bioinformatic analysis." Thesis, University of Exeter, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425246.
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Almiman, Bandar F. "Molecular genetic and genomic characterization of an emerging mycotoxigenic pathogen Fusarium proliferatum." Thesis, University of Bedfordshire, 2018. http://hdl.handle.net/10547/622835.
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This aim of this research was to elucidate the genotypic diversity of the mycotoxigenic species Fusarium proliferatum associated with diverse hosts and distributed in wide geographic locations to gain new insights into the biology of this emerging pathogen. This study developed a novel molecular genetic marker FG1056. Multilocus typing of F. proliferatum isolates (52) using F. verticillioides (2) and F. oxysporum (3) as references was carried out with FG1056 and a set of known genetic markers (ITS, TEF1, CAL and FUM1). This distinguished up to 10 genetic groups, 2 clusters and 23 haplotypes among the F. proliferatum isolates. FG1056 marker showed the highest number of SNPs (169), informative sites (89) and haplotypes (23) relative to other markers used and was comparable to the multi locus typing. Varying patterns of relationships were observed between isolates represented in the genetic groups and their host and geographic origin. Considerable biological variability was recorded among the F. proliferatum isolates in morphology, growth, sporulation and most notably fumonisin production (up to 140-fold differences) with reference to variable temperature, water activity and duration. De novo genome assemblies with the size ranging from 43.96 - 50 Mb have been developed for four diverse F. proliferatum isolates. In silico analysis led to the identification of 12,980 genes common to all isolates and up to 134 genes potentially unique to an isolate. Using these resources, FUM gene cluster (~45.3 Kb) was identified for the first time in F. proliferatum. Order and orientation of the 16 FUM genes and the complete flanking genes (MSF1 and ZCB1 at 5’; ANK1 and GAT1 at 3’) have been determined. This study has provided new insights into the genetic and biological diversity of F. proliferatum and also developed new genetic and genomic resources, which will serve as a solid platform for further research particularly to understand the regulation of fumonisins production in the laboratory and in the field.
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Berriri, Souha. "Identification of constitutively active forms of Arabidopsis MAP Kinases : brings more evidence on MPK4 function in plant immunity." Thesis, Evry-Val d'Essonne, 2012. http://www.theses.fr/2011EVRY0024.
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La phosphorylation/déphosphorylation des protéines est un mécanisme de signalisation intracellulaire commun. Parmi les kinases végétales, les Mitogen-Activated Protein Kinases (MAPKs) sont impliquées dans de nombreux processus biologiques importants, comme la réponse aux stress biotiques et abiotiques, le développement et la dynamique du cytosquelette. Chez Arabidopsis thaliana et ce malgré de nombreux efforts, les fonctions des kinases impliquées dans les cascades MAPK restent peu inconnues. L'activation des kinases en utilisant des mutations mimant la phosphorylation des sites normalement phosphorylés est une approchequi a fait ses preuves dans le cas de MAP2Ks et a largement contribué à élucider leurs fonctions. Cette stratégie s’est révélée impossible dans le cas des MAPKs, puisque les résidus à muter restent encore à identifier. Pour contourner ce problème, nous avons adapté un crible basé sur la complémentation fonctionnelle d’un mutant MAPK de levure avec des formes aléatoirement mutées de MPK6d’Arabidopsis dans le but d'identifier des mutants présentant une activité constitutive. Nous en avons identifiés plusieurs et avons montré que ces formes constitutivement actives (CA) de MPK6 sont actives sans phosphorylation par les MAP2Ks. Par ailleurs, les mutations des résidus équivalents dans d'autres MAPKs les rendent également hyperactives, ce qui indique que cette stratégie peut être utilisée comme approche générale pour activer les MAPKs afin d’en comprendre les fonctions. L’étude des interactions protéine-protéine et l’analyse des profils dephosphorylation indiquent que les MAPKs CA conservent leur spécificité envers leurs substrats et interacteurs. Comme preuve de concept, nous avons généré des formes actives du MPK4. La MPK4 CA exprimée sous son propre promoteur a parfaitement complémenté le mutant mpk4. La caractérisation des lignées exprimant MPK4 CA confirme le rôle négatif de cette kinase dans les réponses de défense aux pathogènes des plantes que ce soit dans la PTI (PAMP Triggered Immunity) ou dans la ETI (Effector Triggered Immunity). Globalement, ce travail permettra de fournir des informations directes sur les cibles des MAPKs et devrait contribuer à la compréhension globale de la transduction du signal chez les plantesProtein phosphorylations and dephosphorylations are common events occurring duringintracellular signaling processes. Among plant kinases, Mitogen-Activated Protein Kinases (MAPKs) are involved in signaling of many important biological processes, including biotic and abiotic stresses, development and cytoskeleton organization. Despite an abundant literature on MAPKs, the exact roles and direct targets of many Arabidopsis thaliana MAPKs are not clear yet. The activation of kinases using phospho-mimicking mutations of the phosphorylated residues was a successful approach in the case of MAP2Ks, helping to elucidate their functions. This strategy failed in the case of MAPKs since the necessary residues to mutate remain unclear. To bypass this problem, we adapted a screen based on the functional complementation of a MAPK yeast mutant with randomly mutated Arabidopsis MPK6 in order to identify the ones mutants showing constitutive activity. We identified several clones and showed that these constitutively active (CA) of MPK6 candidates are indeed active without phosphorylation by MAP2Ks. Interestingly, mutations of the equivalent residues in other MAPKs triggered constitutive activity as well, indicating that this strategy may be used as a general approach to activate MAPKs and identify their functions. Interaction and phosphorylation assays indicatedthat CA MAPKs retain their substrate and interactor specificity. As proof-of-concept, we generated active versions of MPK4. CA MPK4 expressed under itsown promoter successfully complements mpk4 mutant plants. Characterization of CA MPK4 lines further confirmed the negative role of MPK4 in plant pathogen defense responses and its implication in both PTI (PAMP Triggered Immunity) and ETI (Effector Triggered Immunity). Overall, the work will help to provide direct information on all MAPK targets and should be an important contribution to the overall understanding of signal transduction in plants
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Su, Fan. "Modifications physiologiques induites par Burkholderia phytofirmans chez Arabidopsis thaliana. Applications à la protection contre les stress biotique et abiotique." Thesis, Reims, 2015. http://www.theses.fr/2015REIMS032.
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La PGPR Burkholderia phytofirmans PsJN (Bp) stimule la croissance de diverses plantes et les protège également contre certains stress environnementaux. L’objectif des travaux a été d’approfondir les connaissances sur l’interaction Bp-plante, en se focalisant sur l’aspect physiologique et métabolique des feuilles d’Arabidopsis thaliana. Nous avons également déterminé les mécanismes impliqués dans la réponse des feuilles suite à l’inoculation de cette bactérie lors d’un stress abiotique (froid) ou biotique (Pseudomonas syringae pv. tomato DC3000, Pst).Nos résultats montrent que l’induction de la promotion de croissance d’A. thaliana par Bp pourrait être liée à l’accumulation des teneurs en métabolites primaires (acides aminés, glucides solubles et vitamines) et la variation du niveau des hormones dans les feuilles. La physiologie et le métabolisme des feuilles sont modifiés localement et de façon distale par la colonisation épi- et endophytique de Bp. De plus, les modifications des taux de métabolites sont plus marquées après une interaction plante-bactéries relativement longue. Par ailleurs, l’inoculation de Bp peut réduire les dommages sur l’activité photosynthétique dus au froid par une limitation non-stomatique de la photosynthèse et l’accumulation de pigments photosynthétiques. Enfin, la présence de Bp entraîne localement un retard dans le développement initial de Pst. Cependant, l’inoculation par Bp ne protège pas l’appareil photosynthétique lors d’une attaque par Pst. Ces travaux soulignent donc que le temps de présence et la localisation d’une PGPR dans une plante influencent la physiologie, le métabolisme et la tolérance aux stress de cette même planteEndophytic PGPR Burkholderia phytofirmans PsJN (Bp) promotes growth of various plants and triggers protection against several environmental stresses. To get more insights into the interaction between plant and Bp, we focused on leaf physiological and metabolic aspects of Arabidopsis thaliana. We also determined the mechanisms involved in the defense of leaves after inoculation of the bacteria followed by an abiotic (cold) or a biotic (Pseudomonas syringae pv. tomato DC3000, Pst) stress. Our results show that the induction of growth promotion of A. thaliana by Bp could be related to the accumulation of primary metabolite levels (amino acids, soluble carbohydrates and vitamins) and to the variation of hormone levels in the leaves. Leaf physiology and metabolism are changed locally and distally by Bp epi- and endophytic colonization. In addition, changes in metabolite levels are more pronounced after a relatively long interaction between plant and bacteria.Moreover, Bp inoculation can also reduce cold injury on the photosynthetic activity by a non-stomatal limitation of photosynthesis and accumulation of photosynthetic pigments. Finally, the local presence of Bp causes a delay in the development of Pst, but only in the early stages of the infection. However, the inoculation with Bp does not protect the photosynthetic apparatus during Pst attack.Thus, our results emphasize that the time of presence of a PGPR and his location in the plant could influence the plant physiology and stress tolerance
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Berg, Femke van den. "The evolution of plant defence against pests : pathogen development and pathogen-induced leaf necrosis." Thesis, University of Reading, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421209.
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Nurtay, Anel. "Mathematical modelling of pathogen specialisation." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667178.
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L’aparició de nous virus causants de malalties està estretament lligada a l’especialització de subpoblacions virals cap a nous tipus d’amfitrions. La modelització matemàtica proporciona un marc quantitatiu que pot ajudar amb la predicció de processos a llarg termini com pot ser l’especialització. A causa de la naturalesa complexa que presenten les interaccions intra i interespecífiques en els processos evolutius, cal aplicar eines matemàtiques complexes, com ara l’anàlisi de bifurcacions, al estudiar dinàmiques de població. Aquesta tesi desenvolupa una jerarquia de models de població per poder comprendre l’aparició i les dinàmiques d’especialització, i la seva dependència dels paràmetres del sistema. Utilitzant un model per a un virus de tipus salvatge i un virus mutat que competeixen pel mateix amfitrió, es determinen les condicions per a la supervivència únicament de la subpoblació mutant, juntament amb la seva coexistència amb el cep de tipus salvatge. Els diagrames d’estabilitat que representen regions de dinàmiques diferenciades es construeixen en termes de taxa d’infecció, virulència i taxa de mutació; els diagrames s’expliquen en base a les característiques biològiques de les subpoblacions. Per a paràmetres variables, s’observa i es descriu el fenomen d’intersecció i intercanvi d’estabilitat entre diferents solucions sistemàtiques i periòdiques en l’àmbit dels ceps de tipus salvatge i els ceps mutants en competència directa. En el cas de que diversos tipus d’amfitrions estiguin disponibles per a ser disputats per ceps especialitzats i generalistes existeixen regions de biestabilitat, i les probabilitats d’observar cada estat es calculen com funcions de les taxes d’infecció. S’ha trobat un rar atractor caòtic i s’ha analitzat amb l’ús d’exponents de Lyapunov. Això, combinat amb els diagrames d’estabilitat, mostra que la supervivència del cep generalista en un entorn estable és un fet improbable. A més, s’estudia el cas dels diversos ceps N>>1 que competeixen per diferents tipus de cèl·lules amfitriones. En aquest cas s’ha descobert una dependència no monotònica, contraria al que es preveia, del temps d’especialització sobre la mida inicial i la taxa de mutació, com a conseqüència de la realització d’un anàlisi de regressió sobre dades obtingudes numèricament. En general, aquest treball fa contribucions àmplies a la modelització matemàtica i anàlisi de la dinàmica dels patogens i els processos evolutius.La aparición de nuevos virus causantes de enfermedades está estrechamente ligada a la especialización de las subpoblaciones virales hacia nuevos tipos de anfitriones. La modelizaci ón matemática proporciona un marco cuantitativo que puede ayudar a la predicción de procesos a largo plazo como la especialización. Debido a la naturaleza compleja que presentan las interacciones intra e interespecíficas en los procesos evolutivos, aplicar herramientas matemáticas complejas, tales como el análisis de bifurcación, al estudiar dinámicas de población. Esta tesis desarrolla una jerarquía de modelos de población para poder comprender la aparición y las dinámicas de especialización, y su dependencia de los parámetros del sistema. Utilizando un modelo para un virus de tipo salvaje y un virus mutado que compiten por el mismo anfitrión, se determinan las condiciones para la supervivencia únicamente de la subpoblación mutante, junto con su coexistencia con la cepa de tipo salvaje. Los diagramas de estabilidad que representan regiones de dinámicas diferenciadas se construyen en términos de tasa de infección, virulencia y tasa de mutación; los diagramas se explican en base a las características biológicas de las subpoblaciones. Para parámetros variables, se observa y se describe el fenómeno de intersección e intercambio de estabilidad entre diferentes soluciones sistemáticas y periódicas en el ámbito de las cepas de tipo salvaje y las cepas mutantes en competencia directa. En el caso de que varios tipos de anfitriones estén disponibles para ser disputados por cepas especializadas y generalistas existen regiones de biestabilidad, y las probabilidades de observar cada estado se calculan como funciones de las tasas de infección. Se ha encontrado un raro atractor caótico y se ha analizado con el uso de exponentes de Lyapunov. Esto, combinado con los diagramas de estabilidad, muestra que la supervivencia de la cepa generalista en un entorno estable es un hecho improbable. Además, se estudia el caso de los varias cepas N>> 1 que compiten por diferentes tipos de células anfitrionas. En este caso se ha descubierto una dependencia no monotónica, contraria a lo que se preveía, del tiempo de especialización sobre el tamaño inicial y la tasa de mutación, como consecuencia de la realización de un análisis de regresión sobre datos obtenidos numéricamente. En general, este trabajo hace contribuciones amplias a la modelización matemática y el análisis de la dinámica de los patógenos y los procesos evolutivos.
The occurrence of new disease-causing viruses is tightly linked to the specialisation of viral sub-populations towards new host types. Mathematical modelling provides a quantitative framework that can aid with the prediction of long-term processes such as specialisation. Due to the complex nature of intra- and interspecific interactions present in evolutionary processes, elaborate mathematical tools such as bifurcation analysis must be employed while studying population dynamics. In this thesis, a hierarchy of population models is developed to understand the onset and dynamics of specialisation and their dependence on the parameters of the system. Using a model for a wild-type and mutant virus that compete for the same host, conditions for the survival of only the mutant subpopulation, along with its coexistence with the wild-type strain, are determined. Stability diagrams that depict regions of distinct dynamics are constructed in terms of infection rates, virulence and the mutation rate; the diagrams are explained in terms of the biological characteristics of the sub-populations. For varying parameters, the phenomenon of intersection and exchange of stability between different periodic solutions of the system is observed and described in the scope of the competing wild-type and mutant strains. In the case of several types of hosts being available for competing specialist and generalist strains, regions of bistability exist, and the probabilities of observing each state are calculated as functions of the infection rates. A strange chaotic attractor is discovered and analysed with the use of Lyapunov exponents. This, combined with the stability diagrams, shows that the survival of the generalist in a stable environment is an unlikely event. Furthermore, the case of N=1 different strains competing for different types of host cells is studied. For this case, a counterintuitive and non-monotonic dependence of the specialisation time on the burst size and mutation rate is discovered as a result of carrying out a regression analysis on numerically obtained data. Overall, this work makes broad contributions to mathematical modelling and analysis of pathogen dynamics and evolutionary processes.
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Akhras, Michael S. "Nucleic Acid Based Pathogen Diagnostics." Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4684.
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Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis. Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras. Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering.
QC 20100624
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Ho, Timothy Boon Leong. "Pathogen polymorphisms of mycobacterium tuberculosis." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399538.
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Sealfon, Rachel (Rachel Sima). "Computational investigation of pathogen evolution." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/99858.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 105-118).
Pathogen genomes, especially those of viruses, often change rapidly. Changes in pathogen genomes may have important functional implications, for example by altering adaptation to the host or conferring drug resistance. Accumulated genomic changes, many of which are functionally neutral, also serve as markers that can elucidate transmission dynamics or reveal how long a pathogen has been present in a given environment. Moreover, systematically probing portions of the pathogen genome that are changing more or less rapidly than expected can provide important clues about the function of these regions. In this thesis, I (1) examine changes in the Vibrio cholerae genome shortly after the introduction of the pathogen to Hispaniola to gain insight into genomic change and functional evolution during an epidemic. I then (2) use changes in the Lassa genome to estimate the time that the pathogen has been circulating in Nigeria and in Sierra Leone, and to pinpoint sites that have recurrent, independent mutations that may be markers for lineage-specific selection. I (3) develop a method to identify regions of overlapping function in viral genomes, and apply the approach to a wide range of viral genomes. Finally, I (4) use changes in the genome of Ebola virus to elucidate the virus' origin, evolution, and transmission dynamics at the start of the outbreak in Sierra Leone.
by Rachel Sealfon.
Ph. D.
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Sidhu, Jatinder. "Pathogen regrowth in composted biosolids." Thesis, Sidhu, Jatinder (2000) Pathogen regrowth in composted biosolids. PhD thesis, Murdoch University, 2000. https://researchrepository.murdoch.edu.au/id/eprint/52513/.
Full textAbstract:
Composting is commonly used as an effective means of stabilising wastewater biosolids and reducing pathogen concentrations to very low levels. However, under certain conditions enteric bacteria such as Salmonella and E. coli can regrow in previously composted biosolids and compost based soil amendments. Regrowth of Salmonella in composted biosolids can pose a potential threat to public health.
Pathogen regrowth in composted biosolids is affected by a number of factors, such as moisture content, bio-available nutrients, temperature and indigenous microorganisms. However, assessment of the bio-safety of composted biosolids on the basis of these parameters is very difficult and unreliable. Moreover regrowth of
pathogens in composted biosolids is unpredictable. The main aim of this study was to find out whether regrowth of pathogens in composted biosolids could be prevented or controlled. To accomplish this goal, the effect of composting and storage on survival and regrowth of Salmonella typhimurium was investigated. The role of bio-available nutrients and indigenous microorganisms in suppression of Salmonella regrowth was investigated in detail.
The results of this study suggest that a full-scale windrow composting process carried out in accordance with recommended guidelines (ARMCANZ) is effective in reducing Salmonella concentrations to below detection limit. However, Salmonella regrowth in stored biosolids after 26 weeks, coinciding with a rainfall after a dry spell was observed. This suggests that Salmonella can survive the composting process in low numbers and regrowth can take place in the presence of favourable growth conditions.
A pathogen regrowth potential test using antibiotic-resistant S. typhimurium was developed to evaluate the pathogen regrowth potential of composted biosolids. The regrowth potential test was found to be a very useful tool for laboratory investigation. However, further validation of the pathogen regrowth potential test is required, prior to its routine use for monitoring composted biosolids.
The antagonistic activity of indigenous microorganisms was found to be the most significant factor in suppression of S. typhimuriwn growth in composted biosolids. Rapid growth of seeded S. typhimurium, with a maximum population density of more than 108 MPN g-1, was observed in sterilised biosolids. Conversely, growth of S.typhimurium was suppressed in non-sterilised compost with a maximum population density of less than 103 MPN g-1. The inactivation rate of Salmonella was also found to be significantly greater in non-sterilised compost as compared to sterilised compost. Maximum inhibition of Salmonella growth was observed in biosolids that had been composting for two weeks.
The specific growth rate of Salmonella was found to have a strong negative correlation (-0.85) with the maturity of the compost. However, a decline in bioavailable nutrients was not sufficient to prevent regrowth of Salmonella in composted biosolids stored for two years. The role of bio-available nutrients (age. of compost) was non significant (P<_ 0.05) as compared to the role of indigenous microorganisms in inhibition of Salmonella regrowth.
The antagonistic effect of indigenous microorganisms towards Salmonella declined with the storage of compost. A strong negative correlation (-0.85) between the Salmonella inactivation rate and age of compost was observed. Salmonella inactivation rate was also found to be seven times higher in biosolids composting for two weeks as compared to compost stored for two years. Consequently, it can be concluded that all composted biosolids had a Salmonella regrowth potential. However, the presence of biologically active indigenous microflora significantly reduced this regrowth potential. As a result of a decline in the antagonistic activity of indigenous microflora with storage, a longer Salmonella survival time could be expected in stored compost as compared to freshly composted biosolids. Consequently, long term storage of compost is not recommended as this may lead to an increased pathogen regrowth potential and longer survival time.
The dilution-plate procedure adopted in this study showed that bacterial concentrations in compost declined by two log10 during storage for two years, whereas population of actinomycetes and fungi increased during the same period. Indigenous bacteria and actinomycetes isolated from composted biosolids of different maturity were screened for their role in the suppression of Salmonella regrowth. Some of the indigenous bacteria were found to suppress Salmonella growth by one to two log10 when Salmonella was seeded into stationary phase culture of indigenous bacteria. None of the isolated indigenous microorganisms produced secondary metabolites active against Salmonella.
Somatic Salmonella (SS) phages were found to survive in composted biosolids for up to two years. Growth of Salmonella was suppressed by one to two log10 in the presence of SS phages. It is possible that Salmonella growth suppression in compost is due to an intense competition for limited nutrients in the presence of biologically active indigenous microorganisms, with some anti-Salmonella activity from SS phages.
The results of this study suggest that prevention of pathogen regrowth in composted biosolids is difficult due to the availability of nutrients and limited control over environmental factors which influence the antagonistic activity of indigenous microorganisms. However, regrowth of pathogens in composted biosolids can be prevented if a biologically active population of indigenous microorganisms is maintained. It is possible that by preventing rapid drying of compost during maturation biological activity of indigenous microorganisms can be maintained. Covering of compost piles during maturation can preserve moisture and the effect of preserving moisture on the antagonistic activity of indigenous microorganisms should be investigated. Research should be carried out further to identify the indigenous microorganisms which suppress Salmonella regrowth. Additional research work should also be carried out to determine the mechanism of growth suppression. Resolving this issue could provide a better understanding of the antagonistic effect of indigenous microflora towards pathogenic bacteria in composted biosolids.
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le, Roux Marie Cecilia. "Mycoplasma genitalium,passenger or pathogen?" Thesis, University of Limpopo (Medunsa), 2010. http://hdl.handle.net/10386/251.
Full textAbstract:
Thesis (D Phil. (Microbiology))--2010.Mycoplasma genitalium is the smallest existing self-replicating prokaryote, lacks a cell wall and has a genome consisting of only 580 kilo base pairs. It has characteristic pear/flask-like morphology with a terminal tip organelle used for attachment. Many researchers, mainly in developed countries, have investigated the role the organism plays in the aetiology of male urethritis and the majority of studies show an association between M. genitalium and male urethritis. In this study, the modified Koch’s postulates were applied to answer the question whether M. genitalium is a true pathogen, or merely a passenger, invading already inflamed or damaged cells. A total of 300 urine specimens were collected from adult males with symptoms and/or signs of urethritis and 75 from asymptomatic men. In the first study, three molecular assays; viz, a commercial conventional PCR test, a real-time PCR (q- PCR) test and a transcription mediated amplification (TMA) assay were evaluated for the detection of M. genitalium. The comparison between the assays was based on the extended gold standard concept, where a specimen was deemed positive when any two nucleic acid amplification tests were positive. In the second study, the specimens were tested for four common urethral pathogens (N. gonorrhoeae, C. trachomatis, T. vaginalis and M. genitalium) using TMA assays. Finally, the bacterial loads for M. genitalium were determined using the q-PCR assay. v All three assays tested were highly specific (98-99%) for the detection of M. genitalium. However, where q-PCR and TMA demonstrated high sensitivities (96% and 100%), the sensitivity of the conventional PCR assay was low (78%). One or more pathogens were detected in a total of 129 (43%) men with urethritis. M. genitalium was the most frequently detected pathogen in men with urethritis (129; 43%), and significantly more (p= 0.04) than in asymptomatic men (7; 9.0%). There is a strong association with M. genitalium bacterial load and clinical urethritis. Patients with urethral discharge had significantly higher M. genitalium concentrations than those with only burning on micturition (p<0.001), and the bacterial concentrations in men with symptoms and/or signs of urethritis were significantly higher than that in asymptomatic men (p=0.02). As the number of organisms increased, the severity of the symptoms increased; an indication of the role that the organism plays in disease progression. In conclusion, by applying the modified Koch postulates, it was shown that Mycoplasma genitalium is by no means a passenger, but rather an important cause of adult male urethritis that should be taken into account when making diagnosis and when designing treatment strategies.
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Thomas, Graham. "The host-pathogen interface : characterising putative secreted proteins of the honeybee pathogen Nosema ceranae (Microsporidia )." Thesis, University of Exeter, 2015. http://hdl.handle.net/10871/21445.
Full textAbstract:
Microsporidia are obligate intracellular eukaryotic parasites related to fungi, possessing greatly reduced genomic and cellular components. The microsporidian Nosema ceranae threatens honeybee (Apis mellifera) populations. Nosemosis has a complex epidemiology affected by host, pathogen and environmental factors. Although a draft of the N. ceranae genome has been published, the molecular basis underpinning pathogenicity is not known. The lack of established culturing techniques and a tractable genetic system necessitates use of model systems for both host and parasite such as Saccharomyces cerevisiae. I hypothesise effectors essential to disease progression exist amongst N. ceranae secretome genes. In this study I have started characterising these genes using a combination of established and novel techniques for studying microsporidia proteins including bioinformatics, heterologous expression in S. cerevisiae, and the genome-wide analysis platform of Synthetic Genetic Arrays. This effort has yielded new insights into N. ceranae secreted proteins which lack similarity to known sequences. I identified N. ceranae protein NcS77 as a candidate effector implicated in targeting host nuclear pores. NcS50 and NcS85 co-localise with ERG6 a marker for lipid droplets (an organelle known to be targeted by another obligate intracellular pathogen Chlamydia trachomatis) when expressed in S. cerevisiae. N. ceranae polar tube proteins (PTP) induce filament formation when expressed in S. cerevisiae and PTP2 co-localises with the cell wall. Interestingly this phenotype is replicated by another secreted protein which may infer a common function. Together these data contribute to knowledge on N. ceranae pathology bringing us closer to understanding the disease and ultimately lead the way to mitigation.
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VieBrock, Lauren. "ORIENTIA TSUTSUGAMUSHI ANKYRIN-REPEAT PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4023.
Full textAbstract:
Abstract
ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM
By Lauren VieBrock, B.S.
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University
Virginia Commonwealth University, 2015
Director: Jason A. Carlyon, Ph.D.
Professor
Microbiology and Immunology
Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These
results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology.
We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.
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Hennecke, Berthold Rembertus. "Host-pathogen interactions between the fungal pathogen Phloeospora mimosae-pigrae and Mimosa pigra, giant sensitive plant /." [St. Lucia, Qld.], 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17081.pdf.
Full text
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Filippini, Chiara. "Multiple pathogen detection using nanoplasmonic sensing." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amslaurea.unibo.it/7344/.
Full textAbstract:
Opportunistic diseases caused by Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) is an omnipresent global challenge. In order to manage these epidemics, we need to have low cost and easily deployable platforms at the point-of-care in high congestions regions like airports and public transit systems. In this dissertation we present our findings in using Localized Surface Plasmon Resonance (LSPR)-based detection of pathogens and other clinically relevant applications using microfluidic platforms at the point-of-care setting in resource constrained environment. The work presented here adopts the novel technique of LSPR to multiplex a lab-on-a-chip device capable of quantitatively detecting various types of intact viruses and its various subtypes, based on the principle of a change in wavelength occurring when metal nano-particle surface is modified with a specific surface chemistry allowing the binding of a desired pathogen to a specific antibody. We demonstrate the ability to detect and quantify subtype A, B, C, D, E, G and panel HIV with a specificity of down to 100 copies/mL using both whole blood sample and HIV-patient blood sample discarded from clinics. These results were compared against the gold standard Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). This microfluidic device has a total evaluation time for the assays of about 70 minutes, where 60 minutes is needed for the capture and 10 minutes for data acquisition and processing. This LOC platform eliminates the need for any sample preparation before processing. This platform is highly multiplexable as the same surface chemistry can be adapted to capture and detect several other pathogens like dengue virus, E. coli, M. Tuberculosis, etc.
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Petzold, Katja. "NMR studies of host-pathogen interactions." Doctoral thesis, Umeå universitet, Medicinsk kemi och biofysik, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-25710.
Full textAbstract:
This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.
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Ringqvist, Emma. "Host-Pathogen Responses during Giardia infections." Doctoral thesis, Uppsala universitet, Mikrobiologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-108980.
Full textAbstract:
Giardia lamblia is a eukaryotic parasite of the upper small intestine of humans and animals. The infecting trophozoite cells do not invade the epithelium lining of the intestine, but attach to the brush border surface in the intestinal lumen. The giardiasis disease in humans is highly variable. Prior to this study, the molecular mechanisms involved in establishment of infection or cause of disease were largely uncharacterized. In this thesis, the molecular relationship between Giardia and the human host is described. The interaction of the parasite with human epithelial cells was investigated in vitro. Changes in the transcriptome and proteome of the parasite and the host cells, and changes in the micro-environment of the infection have been identified using microarray technology, and 1- and 2-Dimensional SDS-PAGE protein mapping together with mass spectrometry identification. The first large-scale description of cellular activities within host epithelial cells during Giardia infection is included in this thesis (Paper I). We identified a unique activation of the host immune response and induction of apoptosis upon infection by Giardia. Four important virulence factors of the parasite, directly linked to the success of Giardia infection, were characterized and are presented in Papers II and III. The parasite was shown to have immune-modulating capacities, and to release proteins during host-interaction that facilitate the establishment of infection. Additional putative virulence factors were found among Giardia genes transcriptionally up-regulated during early infection (Paper IV). In summary, this thesis provides important insights into the molecular mechanisms of the host-parasite interaction.
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McDonald, Allison. "Pathogen-induced inflammation in immunocompromised conditions." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/47546.
Full textAbstract:
Primary immunodeficiencies arise from genetic anomalies and can cause aberrant inflammation, tissue destruction, and uncontrolled infections. Defects in immunity often first present as cases of recurrent or severe infections with unusual pathogens. Patients with chronic granulomatous disease (CGD) have mutations in the NADPH oxidase that prevent the formation of reactive oxygen species (ROS) critical for effective immunity. We investigated the phagocyte response to Burkholderia cenocepacia, which causes severe lung infections in patients with CGD. B. cenocepacia survived within ROS-deficient neutrophils and induced rapid cell death that was not observed by infection with other bacteria, including other CGD pathogens. Increased cell death was associated with enhanced caspase-3 activation and phosphatidylserine surface exposure compared to healthy neutrophils, both hallmarks of apoptotic programmed cell death. Caspase activation was required to induce increased neutrophil death in the absence of ROS. B. cenocepacia-induced apoptotic neutrophils were pro-inflammatory to macrophages, especially in the absence of ROS, inducing the secretion of IL-6, IL-8, and TNF-α but not the anti-inflammatory cytokines IL-10 or TGF-β from human macrophages. Surprisingly, macrophages enhanced the growth of B. cenocepacia by providing a nutrient-rich intracellular replication niche that allowed the bacteria to escape from normal neutrophil killing.
We also report here two sisters with an underlying immunodeficiency who presented with fatal neurodegeneration triggered by infections. Whole exome sequencing identified mutations in perforin1, involved in the granule-mediated cytotoxicity pathway for eliminating infected host cells. Perforin1 mutations are associated with familial hemophagocytic lymphohistiocytosis (FHL). In contrast to FHL, these girls did not have hematopathology or elevated cytokine overproduction. However, 3 years after disease onset, the proband had markedly deficient cytokine production, including IL-1β production following stimulation with a panel of bacterial ligands. These observations extend the spectrum of disease associated with perforin mutations to immune-mediated neurodegeneration triggered by infection.
Studies that investigate how opportunistic pathogens cause disease aim to decrease morbidity and mortality in immunocompromised patients and also provide the opportunity to better understand the molecular mechanisms that allow healthy individuals to combat disease.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Rofe, A. P. "Pathogen manipulation of intracellular membrane traffic." Thesis, University of York, 2015. http://etheses.whiterose.ac.uk/12024/.
Full textAbstract:
The use of in vitro assays to study membrane fusion events has been instrumental in discovering the molecular requirements of membrane fusion. The processes involved in phagosome-lysosome fusion are not clear. Some intracellular pathogens, such as Rhodococcus equi, are able to prevent phagosome-lysosome fusion. Thus, this study aimed to develop an in vitro phagosome-lysosome fusion assay to better understand the fusion of these two organelles and to understand the molecular mechanisms behind R. equi pathogenesis. Transfer of biotin-conjugated horseradish peroxidase (biotin-HRP) from lysosomes to streptavidin-bead containing phagosomes (sBCPs) was used to measure organelle fusion. A protocol for the purification of lysosomes from J774.2 mouse macrophages was developed using superparamagnetic iron oxide nanoparticles (SPIONs). Lysosomes were enriched with LAMP1 and devoid of EEA1. Biotin-HRP from lysosomes could be detected and was able to bind to sBCPs in a binding assay. A protocol for the purification of sBCPs from J774.2 cells was also developed. Phagosomes were enriched with LAMP1 and contained Rab5, Rab7 and EEA1. Fusion between organelles, in cytosol and in the presence of an ATP regenerating system, was not detected and may have been disrupted by factors present in pig brain cytosol used to support the vesicle fusion. R. equi virulence depends a 90 Kb plasmid harbouring several virulence-associated proteins (Vaps). Only one of these, VapA, is essential for virulence but R. equi may have additional virulence factors. To identify these potential additional R. equi genes, a R. equi gene library was expressed in yeast which were then screened for membrane trafficking defects. The screen failed to yield any potential R. equi virulence factors that disrupted membrane trafficking in yeast. In a targeted approach, recombinant VapA when fed to cells, induced swelling of mammalian cell lysosomes and late endosomes. This activity was conferred by the C-terminal core of VapA. VapA induced LAMP1 expression suggesting lysosome dysfunction. The effects of VapA were not seen with other R. equi Vap proteins. This thesis presents the framework for further developing an in vitro phagosome-lysosome fusion assay. Secondly, this work builds on our understanding of how R. equi uses VapA to survive intracellularly.
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Otten, Lucienne. "Pathogen detection based on carbohydrate adhesion." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/77814/.
Full textAbstract:
The rapid detection of pathogenic organisms to ensure appropriate administration of treatment remains a global healthcare challenge. This is becoming increasingly difficult, as identification of the organism alone is no longer enough, with the rise of drug resistance amongst many pathogens it is becoming increasingly important that both the pathogen and drug resistance are identified. Currently, rapid identification can be achieved through a variety of techniques. However, many of these techniques are expensive, require extensive sample preparation, or highly trained personnel to run with results often not rapidly available. This leaves health care professionals to make point-of-care treatment decisions based on symptoms without any indication of drug resistance. The use of carbohydrate microarrays for pathogen detection has been identified as both a method for detection but also as a basis for identifying new drug targets. This exploits the initial protein-carbohydrate interaction that many pathogens utilise in the initial stages of infection. However, the use of microarrays is also challenging, as highly sensitive identification of pathogens often requires expensive or synthetically challenging oligosaccharides or coupling with a highly sensitive detection method thus limiting its point of care application. Herein we describe the coupling of a facile surface chemistry for glycan addition with a powerful statistical algorithm to improve the sensitivity of a cheap monosaccharide functionalised surface without using expensive detection methodologies. This technique was then applied to the detection and identification of toxic lectins, bacterial samples and finally the life-stage specific detection of Plasmodium falciparum (one of the parasites responsible for human malaria). In this last case, drug resistance related to carbohydrate binding profile was also observed.
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Kathiria, Palak, and University of Lethbridge Faculty of Arts and Science. "Incompatible and compatible plant pathogen interactions." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2006, 2006. http://hdl.handle.net/10133/351.
Full textAbstract:
Pathogens are one of the prevalent stresses to plants. Resistance mediated by the
resistance genes is efficient mechanism for evading the pathogens. To understand the
influence of various biotic and abiotic factors on resistance gene promoters, plants having
N gene promoter fused with reporter genes were developed. Experiments with tobacco
plants revealed that on tobacco mosaic virus infection, the N protein may increase in the
cells. Also, extreme temperature may result in decrease in the N protein. The salicylic
acid produced during the development of systemic acquired resistance does not hinder the
N promoter function. Hence, it can be concluded that the promoter region of resistance
genes can be influenced by many biotic and abiotic factors. In the tobacco plants lacking
the N gene, infection with tobacco mosaic virus leads to generation of systemic
recombination signal. Experiments suggest that this signal can lead to better tolerance of
the pathogen in next generation. Also, in the plants which received systemic
recombination signal, the resistance gene loci are hypermethylated and the frequency of
rearrangement in these loci increases. Hence, the signal results in higher tolerance to
pathogen and increased genetic variability in resistance genes.xvi, 147 leaves : ill. (some col.) ; 29 cm.
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Hann, Dagmar R. "Early events in plant-pathogen interactions." Thesis, University of East Anglia, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502371.
Full textAbstract:
Plants are in constant contact with a wide range of microbes. Although many of them are potential pathogens, disease is the exception. This is partly due to a very effective immune response mounted by the plant. This immune response consists of two layers, both of which are innate. The first layer perceives the microbe directly after invasion, through recognition of pathogenassociated molecular patterns (PAMPs) by membrane localized pattern recognition receptors (PRRs). Microbes have developed specialized secretion systems for the delivery of effector proteins into the host cytoplasm, some of which act as suppressors PAMP-triggered immunity. In a second layer of immunity, some of these effectors are recognized in a cultivar specific manner by plant resistance genes. The defence response associated with effector recognition is also called effector-triggered immunity and usually leads to a very strong defence response in the form of localized cell death. In addition, some effector proteins were shown to suppress effector-triggered immunity. During my thesis I worked on several aspects of PAMP-triggered immunity and effector mediated suppression. Firstly, I identified and characterized the putative flagellin receptor in N. benthamiana termed NbF/s2 (Hann, DR and Rathjen, JP, The Plant Journal, 2007). Secondly, I performed a reverse genetic screen based on virus-induced gene silencing (VIGS) of a protein kinase gene library, to identify components involved in flagellin-elicited active oxygen generation in: N. benthamiana. With this screen I identified several kinases involved in defence signaling. One of these candidates encodes NbSerk3/BAK1, which was shown elsewhere to interact with the f1agellin receptor AtFLS2 in Arabidopsis (Heese, A, Hann DR, et al. PNAS, 2007). Thirdly, I investigated effector-mediated suppression of host defence responses. I focused on the P. syringae pv tomato DC3000 effectors AvrPto and AvrPtoB, which suppress a wide range of defence responses induced by various elicitors (Hann, DR and Rathjen, JP, The Plant Journal, 2007). I screened a library of P. syringae pv tabaci 11528 effectors and identified several suppressors of PAMP-triggered immunity. Interestingly, the range and specificity of defence-response suppression varied amongst the effectors tested, suggesting different host targets for each effector.
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Mastorakis, Emmanouil. "Chromatin remodelling during plant-pathogen interactions." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/101423/.
Full textAbstract:
Plants - including commercially important crops - are exposed to numerous pathogens often resulting in significant loss of yield. Understanding the underlying mechanisms of pathogen recognition and defence strategies is key in successfully ensuring food security. Research on plant-pathogen interactions has mainly focused on the gene networks after pathogen perception as well the identification of resistance genes. Latest research suggests that chromatin remodelling, including nucleosome displacement and DNA or histone-modifying enzymes are important in plant immunity. This thesis focuses on chromatin remodelling as the mechanism by which plants mount an effective immune response. The thesis also investigates the role of histone acetylation as one of several chromatin remodelling mechanisms. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two classes of histone modifying enzymes that antagonistically govern the acetylation levels of histones in gene promoters and gene bodies ultimately affecting gene expression. HAG1 was identified as an important positive regulator of plant immunity in the interaction with Pst DC3000. A proteomic approach allowed the identification of TOPLESS family members as HAG1 interactors. Considering that chromatin remodelling is an important aspect of plant immunity, it was hypothesised that pathogens have evolved mechanisms to interfere with such processes. To this end, this thesis will present a comprehensive approach towards identifying Pst DC3000 Type-III effectors with the ability to interfere with chromatin remodelling. HopO1-1 was initially identified as an effector with chromatin binding properties, however, further experiments pointed more strongly towards this effector’s involvement in processes such as translation and photosynthesis. Overall, this thesis contributes towards a better understanding of the roles of histone acetylation and HAG1 histone acetyltransferase in plant immunity and sheds light into which Pst DC3000 effectors could be potentially involved in chromatin remodelling processes.
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Gupta, Saurabh Ph D. Massachusetts Institute of Technology Dept of Biological Engineering. "Genetically programmable pathogen sense and destroy/." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/75839.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references (p. 123-134).
Twenty five percent of all the deaths worldwide are caused by infectious diseases. They are also the biggest cause of mortality among children under five years of age. Among them diarrheal diseases alone cause as many deaths as AIDS or TB and malaria combined. Also up to 80% of traveler's diarrhea is bacterial in nature. Vibrio cholerae (cholera), Salmonella spp (typhoid fever), Shigella spp (shigellosis) and a variety of enteropathogenic Escherichia coli strains are among the principle bacterial agents that cause this type of diarrhea. Improvements in hygiene and access to adequate nutrition are good strategies but immunization against specific diseases still offers the best solution to fight these infections. Unfortunately the wide diversity of bacterial and viral agents that cause diarrhea complicates accurate diagnosis and makes the development of vaccines difficult. Antibiotics used in timely manner and in appropriate doses can be effective but the diagnosis is usually made too late for the therapy to be effective. Moreover frequent use of over-the-counter drugs without any medical supervision has led to multidrug resistance in most of the bacterial strains. To counter this problem I demonstrate a proof of principle of a novel cell therapy against Pseudomonas Aeruginosa (major cause of urinary tract disease and hospital infections). Using principles of Synthetic Biology I genetically modified a probiotic strain of E. coli to specifically detect PAO₁ and respond by secreting a novel, pathogen-specific engineered toxin. Additionally, I translated the bacterial system into mammalian cells and established a foundation for an adaptive system where the sentinel cells secrete an alternate toxin if the pathogen becomes resistant to the first one. Finally, based on this system I proposed designs against highly pathogenic strains of Shigella, Salmonella and Vibrio cholerae. This cell therapy remains inert until a threat is detected, and then serves as an early detection and rapid response agent. Furthermore this platform can be tuned to release minimum but sufficient amounts of very narrow spectrum antimicrobial proteins to control the early stages of infection before the disease becomes systemic. Therefore this system's rapid, automated and highly specific response can be helpful in reducing the occurrence of dose dependent resistance. This approach offers a single integrated solution to eradicating multiple threats with a strategy that is a rapid, selective, and highly sensitive.
by Saurabh Gupta.
Ph.D.
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Arnold, Markus F. F. "Host-pathogen interactions in chronic infections." Thesis, University of Aberdeen, 2012. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=192267.
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The BacA protein plays a key role in the symbiosis of Sinorhizobium meliloti with the leguminous plant alfalfa (Medicago sativa) and is proposed to be the transmembrane subunit of an ATP‐binding cassette (ABC) transporter. BacA homologues are also present in Brucella species, enteric bacteria (known as SbmA) and Mycobacterium tuberculosis. The S. meliloti‐alfalfa symbiosis, although beneficial to both partners, can also be viewed as a chronic infection. The M. tuberculosis BacA protein is important for M. tuberculosis for the maintenance of a chronic infection. In order to study the M. tuberculosis BacA protein a codon optimised M. tuberculosis bacA gene was synthesised and cloned into vectors for expression in an S. meliloti ΔbacA mutant. Evidence is presented that M. tuberculosis BacA sensitises an S. meliloti ΔbacA mutant towards the glycopeptide bleomycin and the truncated proline rich peptide Bac71‐16, and further that a functional ATPase domain is essential to perform BacA mediated peptide transport. The M. tuberculosis BacA protein protected an S. meliloti ΔbacA mutant from being killed by host defensins. In addition, it was determined that M. tuberculosis BacA‐mediated protection of the legume symbiont S. meliloti against legume defensins as well as mouse alveolar lavages and human ‐defensin 2 is dependent on an ATPase domain which is present in the M. tuberculosis BacA protein. M. tuberculosis encounters ‐defensins during mammalian infections in the host’s lungs and my data show that BacA is likely to be important in conferring immunity to these peptides. The mechanism of persistent infection by M. tuberculosis is therefore very reminiscent of the Sinorhizobium ‐ legume interaction. Also Salmonella enterica is able to cause asymptomatic infections and about 5% of these develop a chronic carrier state and are able to spread the pathogen. In enteric bacterial species SbmA is in close proximity to the putative lipoprotein YaiW. In this study it was determined that YaiW is exposed on the cell surface and that it is involved in the protection of E. coli and potentially Salmonella spp. against a cysteine rich host peptide. YaiW is potentially involved in swarming motility. It was also determined that an alfalfa plant infection model can distinguish between Salmonella strains forming acute and chronic infections. Staphylococcus aureus is one of the major hospital acquired and opportunistic pathogens. Asymptomatic carriers, infected with multi‐drug resistant strains (MRSA) pose a threat to immunocompromised individuals. Here, novel compounds and ways from a variety of sources have been tested for their potential antimicrobial activity against a range of multi‐drug resistant clinical S. aureus isolates. This project significantly advanced the molecular understanding of asymptomatic bacteria‐host infections and helped to understand and establish novel ways to treat infections with multi‐drug resistant clinical pathogens.
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Ross, David. "Data mining for exotic pathogen spread." Thesis, University of Strathclyde, 2016. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=27557.
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Major disease outbreaks command worldwide attention. Many recent outbreaks were caused by pathogens that were considered 'exotic' with severe implications from a health or economic standpoint. As such, there is need for an in depth examination of these threats and the means by which they might be introduced to effectively manage future risk. This thesis examines a means of identifying key emerging threats and, once identified, then modelling techniques are used to estimate the risk of introduction. To determine the relevant exotic pathogens, data from a survey of experts were examined. In 2010 the 4th Annual Meeting of the EPIZONE network was held at which work was carried out to elicit the opinions of delegates on current and future epidemic threats to the EU. Data from this study were examined using both univariate and multivariate analytical techniques to fully explore and understand what might become an emerging threat. This found that a particular group of zoonotic arboviruses are viewed as important potential emerging threats for Europe. Increasingly realistic and complex modelling approaches were utilised to give an increasingly accurate estimate of the risk of introduction of one of these viruses, Crimean-Congo Haemorrhagic Fever Virus (CCHFV),by means of migratory birds - a potentially key means of introduction. Evaluating this risk must take into account not just disease related factors but also geographic factors especially the migration distance. To model this risk, spatially explicit models that correctly re ect bird migratory behaviour were used in contrast to models published previously. The approaches in this thesis show that for CCHFV there is a de nite risk of introduction but it is smaller than has been estimated previously. Results also show that the bird species that should be focused on are not those intuitively identi ed. The migratory speed of birds is a key factor in identifying the species that represent the greatest risk of introducing CCHFV positive ticks into Europe.
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Asif, Muhammad. "Acanthamoeba and the bacterial pathogen interactions." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/20427.
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The present study investigates Acanthamoeba-bacteria interaction and how this relation can influence human health aiming at the influence of bacteria on Acanthamoeba in terms of their isolation and diversity, and the effect of Acanthamoeba on bacteria focusing on two emerging human bacterial pathogens Arcobacter butzleri and Rhodococcus equi. To first objective was investigated by the test question “can the presence of a particular type of bacteria play role in the diversity of Acanthamoeba by masking and/or favouring certain genotypes of Acanthamoeba?” To answer this, two different bacteria the Gram+ve Enterococcus the Gram-ve Arcobacter were used as food source for isolation of Acanthamoeba from 102 soil samples while E. coli was used as control. It was found that the presence of different bacteria could affect the isolation of genotypes specially the subgroups and subtypes of Acanthamoeba as manifested by greater diversity of 18S rRNA sequences of Acanthamoeba isolated from environmental samples on Arcobacter (Arc) and Enterococcus (Ent) than those isolated on E. coli (Eco). The Eco isolates consisted of only T4 > T11=T13 compared to Ent isolates with T4 > T16 > T13/16 and the Arc isolates which comprised of T4 > T2 > T2/6 = T13 > T13/16. The T13/16 were the intermediate sequence types with no match to any T types. There were also considerable differences among the T4 subgroups; the Eco isolates consisted of T4-A > T4-B > T4-N > T4- E > T4-D > T4-C while Ent isolates comprised of T4-A > T4-C=T4-D=T4-E=T4-N > T4-B and the Arc isolates had only T4-E > T4-A > T4-B > T4-N. In both Eco and Ent isolates 11 subtypes were recovered with T4-36 being the most abundant, however, in Arc isolates eight subtypes were recovered with T4-12 as the most abundant. The non-Eco isolates were also different in their bacterial endosymiotic profile from Eco isolates with Arc isolates having the greatest proportion of bacterial endosymbionts (15.7%) as compared to 7.8% of Eco and 12.9% of Ent isolates. Together these results indicate a prominent role of prey bacteria on favouring certain genotypes and thus compelling consideration for use of different types of bacteria for isolation of Acanthamoeba to help surface the masked populations as well for more realistic prevalence that will help in better designing of prevention and control strategies. The influence of Acanthamoeba on bacteria was investigated for A. butzleri and R. equi both of which appeared to exploit the former as an environmental reservoir and for modulation of their pathogenic potential. A. butzleri which are closely related to Campylobacter, appeared to have a smooth interaction with Acanthamoeba. They were shown to be easily located through chemotaxis, readily attached and internalized using monosaccharide receptors and a complex phagocytic process, and could survive/proliferate in Acanthamoeba by defying the intra-vacuolar killing processes. Intracellular survival in Acanthamoeba did play a role in promoting the pathogenicity of these bacteria enabling them to survive more than three times longer. Co-culturing of the two organisms also seemed to benefit the bacteria but not Acanthamoeba. A. butzleri were found to be able to sense the environmental changes and thus modulate their virulence, a feature that together with selection pressure for intracellular survival in Acanthamoeba can cause rapid adaptation to intra-amoebal environment and enhance the pathogenic potential of these bacteria for humans and animals. Exploitation of Acanthamoeba for survival was also found to be exhibited by the Mycobacterium-resembling Gram+ve R. equi by utilizing similar strategies for survival/proliferation as used for macrophages, which involved the definite presence of virulence plasmid and its activation at higher temperatures. Moreover, similar genes (vapA, vapC and vapF) were found to play role in intracellular survival in both the macrophages and amoeba cells. The intra-amoebal survival/proliferation capabilities of A. butzleri and R. equi appear to support the notion that free living protists like Acanthamoeba act as environmental reservoirs/virulence trait selectors and are strong candidates for the “missing link” between the ecology and pathology of these emerging pathogenic pathogens. Overall, the observations made in this study explore the vital role of Acanthamoeba-bacteria interaction not only mutually on each other but as a consequence the impact on human health either as a result of masked genotypes in clinical diagnosis of Acanthamoeba or due to environmental reservoir role of Acanthamoeba in selecting virulence traits of bacteria, can pose serious challenges leaving ample opportunities for more emerging bacterial pathogens. These observations call for revising the protocols for Acanthamoeba prevalence, eradication and control strategies.
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Lim, Elisa X. "Host-pathogen interactions during alphavirus infection." Thesis, Griffith University, 2021. http://hdl.handle.net/10072/410163.
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Arthritogenic alphavirus infection causes debilitating pain in the joints and muscles with many patients experiencing such symptoms chronically. However, there is insufficient evidence to explain the underlying causes behind symptoms of persistent arthralgia and myalgia. Joint-associated tissues are the main site of inflammation during alphavirus infection, and it has been shown that alphaviruses induce damage to the cartilage and synovium. Therefore, the cell types present in these tissues play critical roles in disease pathogenesis. The findings described in this thesis contribute to the general understanding of host-pathogen interactions during alphavirus infection of joint-associated cell types. Here, the analysis of murine joints revealed chondrocytes as a target of RRV infection (Chapter 1). Further evaluation of human primary chondrocytes and skeletal muscle cells through short-term in vitro cell culture showed that these cell types could support productive RRV infection. Our study presents the first evidence of the role of chondrocytes in alphavirus disease pathogenesis. Currently, there are gaps in our understanding of chronic alphavirus disease, especially in the absence of detectable viraemia after recovery from infection. Here, we have investigated the r sponses of several cell types in joint-associated tissues during chronic infection. Human primary cells and their corresponding cell line counterparts for chondrocytes, muscle cells and fibroblast-like synoviocytes (FLS) were infected with four alphaviruses of clinical importance, namely Ross River virus (RRV), Barmah Forest virus (BFV), chikungunya virus (CHIKV) and o’nyong’nyong virus (ONNV). We found that all cell types studied were able to retain residual alphaviral nucleic acids after recovery from infection despite several passages in culture (up to 10 weeks), indicating the potential of these cell types as reservoirs for the virus and/or viral RNA (Chapter 2). Regretfully, we were unable to determine the roles of the lingering viral nucleic acids though we hypothesise that they may play roles in causing chronic inflammation. During this study, we also established persistent alphavirus infection in chondrocyte C28/I2 and muscle RD cell lines (Chapter 2) and hypothesise that these two cell types could act as potential harbours for virus evasion from the immune system. The characterisation of genetic variants present in samples from persistent infections led to the identification of several mutations which could potentially be important for alphavirus persistence. We speculate that C28/I2 and RD cell lines are suitable candidates for exploring alphavirus evolution through selective pressures applied by in vitro serial passaging of infected cells. Our findings indicate that infected chondrocytes, muscle cells and FLS contribute to alphavirus disease pathogenesis through increased expression of pro-inflammatory cytokines associated with clinical disease such as IL-6, MCP-1 and IL-8 (Chapter 1 and 2). However, further studies are required to determine if the presence of residual alphaviral nucleic acids serves as PAMPs that are responsible for eliciting chronic inflammatory responses. While we have shown that RRV-infected chondrocytes play a role in causing alphavirus-induced inflammation, we also observed that these cells cause cartilage damage through disruption of ECM homeostasis. As the main cell type of the cartilage, chondrocytes are responsible for the regulation of ECM synthesis and degradation. During RRV-infection of chondrocytes, we observed reduced gene expression of key ECM constituents COL1A1, COL2A1 and ACAN and elevated gene expression of ECM breakdown enzymes like HPSE, ADAMTS4 and MMP9 (Chapter 1, 2 and 3). We also observed evidence of this through our transcriptomic analysis of RRVinfected and uninfected bystander chondrocytes. This is also the first study that investigates the direct and indirect responses to alphavirus infection of chondrocyte (Chapter 3). As an avascular tissue type, chondrocytes are not easily accessible to virus infection. However, we found evidence of RRV RNA in the chondrocytes of infected mice (Chapter 1) and have shown that these cells are susceptible to alphavirus infection (Chapters 1-4). Therefore, we can only speculate on the possible routes of alphavirus infection of chondrocytes. The use of in vitro chondrocyte models with complex ECM architecture allows for greater physiological relevance in the study of cartilage and their responses to alphavirus infection. The synovium is a neighbouring tissue with access to the blood supply network and provides nutrients to the cartilage. Therefore, it is possible that chondrocytes can acquire alphavirus infection via the synovium. Fibroblast-like synoviocytes (FLS) are the main resident cell type of the synovium and maintains the synovial fluid through the expression of ECM components and breakdown enzymes like MMP3. We found that interactions between chondrocytes and FLS result in increased viral infectivity profiles (Chapter 4). Our study also demonstrates that the ECM surrounding the chondrocytes acts as a physical barrier that prevents access to virus particles. Treatment of cells with MMP3 was able to loosen the interactions of the ECM and expose the chondrocytes to virus infection, resulting in greater virus attachment and infectivity compared to non-treated cells. Taken together, this thesis presents key findings on the possible mechanisms involved in alphavirus disease pathogenesis and the roles of cell types of joint-associated tissues in causing the chronic symptoms of joint and muscle pain felt by the majority of infected patients.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
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Olive, Andrew James. "Immunity to Chlamydia trachomatis and Host-Pathogen Interactions During Infection." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11263.
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Infections with the bacterial pathogen Chlamydia trachomatis are a critical public health problem. Chlamydia remains the number one cause of preventable blindness worldwide and the leading cause of bacterial sexually transmitted infections in the United States. In humans, repeat and persistent infections with Chlamydia result in severe inflammation. Inflammation in the conjunctiva can result in blindness, while inflammation in the genital tract can result in pelvic inflammatory disease, ectopic pregnancy or infertility. In order to curb the increasing incidence of Chlamydia infections worldwide it will be necessary to develop a protective vaccine that affords long-term protection and prevents pathologies. To better inform vaccine development we must understand the mechanisms that drive long-term immunity in the genital tract and elucidate critical interactions between Chlamydia and host cells to uncover potential mechanisms of immune evasion.
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Dauch, Amélie L. "Velvetleaf-Colletotrichum coccodes pathosystem : molecular monitoring of the pathogen and gene expression analysis during plant pathogen interaction." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102492.
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Colletotrichum coccodes strain DAOM 183088 is considered a potential bioherbicide for velvetleaf (Abutilon theophrasti), a devastating weed in North American corn and soybeans. Risk assessment studies have created a demand for an accurate and robust method to monitor this strain, and to distinguish it from indigenous background population of microorganisms present in the field. Safe biological control management of velvetleaf also requires comprehensive understanding of the pathogenicity determinants employed by this host-specific fungus to establish infection on velvetleaf, an aspect central to a safe biocontrol strategy task. In this study, molecular markers were designed that allow strain specific identification of the bioherbicide strain of C. coccodes and its identification within complex plant and soil matrices. An assay was developed to quantify C. coccodes from deliberate release field soil samples, in which biases caused by soil-originating PCR inhibitors were monitored on a sample per sample basis. The developed external control assay allowed for the estimation of target C. coccodes DNA quantities with normalization for the presence of PCR inhibitory compounds. Kinetic growth curves of disease development were performed for C. coccodes wild-type and T20-a (genetically engineered for hypervirulence with the NEP1 (necrosis and ethylene inducing peptide) gene) strains on velvetleaf leaves over a period of 14 days after C. coccodes infection. The wild-type strain was more efficient at infecting velvetleaf than the transgenic T-20a strain, while expression of NEP1 could not be detected suggesting that the introduced gene may not be transcriptionally active in the transformed strain, a result in conflict with previous observations. Velvetleaf and C. coccodes genes specifically upregulated at 12 and 24 h after fungal infection were cloned and differentially screened by microarrays. The resulting EST collection was sequenced and assigned to putative functions. Early gene up-regulation was confirmed by QRT-PCR analysis for type 3 metal lothionein, EREB, WRKY, and bZIP transcription factors, reticuline oxidase, ascorbate peroxidase, and ACC oxidase gene candidates. In addition, type 2, type 3 metallothionein, and bZIP gene expression profiles were investigated over a period of 14 days after C. coccodes infection, and the results indicated that C. coccodes altered the expression of all three gene analyzed.
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Sastry, J. S. S. G. M. "Molecular studies on genetic variability and plant pathogen interactions in pearl millet downy mildew(sclerospora graminicola) pathogen." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 1998. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/3384.
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Crossley, Brian E. "Role of the Exopolysaccharide Alginate in Adherence to and Inflammation of Pulmonary Epithelial Cells." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4473.
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Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.
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Terry, Karianne. "Chemotactic Signaling in the human gastric pathogen /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2005. http://uclibs.org/PID/11984.
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Marotta, Nicole Ella. "Patterned nanoarray sers substrates for pathogen detection." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/37274.
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The objectives of the work presented were to 1) fabricate reproducible nanorod array SERS substrates, 2) detection of bacteria using nanorod substrates, 3) detection of DNA hybridization using nanorod substrates and 4) critically evaluate the sensing method.
Important findings from this work are as follows. A novel method for batch fabrication of substrates for surface enhanced Raman scattering (SERS) has been developed using a modified platen machined to fit in a commercial electron beam evaporator. The use of this holder enables simultaneous deposition of silver nanorod (AgNR) arrays onto six microscope slide substrates utilizing glancing angle deposition. In addition to multiple substrate fabrication, patterning of the AgNR substrates with 36 wells allows for physical isolation of low volume samples. The well-to-well, slide-to-slide, and batch-to-batch variability in both physical characteristics and SERS response of substrates prepared via this method was nominal. A critical issue in the continued development of AgNR substrates is their stability over time, and the potential impact on the SERS response. The thermal stability of the arrays was investigated and changes in surface morphology were evaluated using scanning electron microscopy and x-ray diffraction and correlated with changes in SERS enhancement. The findings suggest that the shelf-life of AgNR arrays is limited by migration of silver on the surface. Continued characterization of the AgNR arrays was carried out using fluorescent polystyrene microspheres of two different sizes. Theory suggests that enhancement between nanorods would be significantly greater than at the tops due to contributing electromagnetic fields from each nanostructure. In contrast to the theory, SERS response of microspheres confined to the tops of the AgNR array was significantly greater than that for beads located within the array. The location of the microspheres was established using optical fluorescence and scanning electron microscopy.
The application of SERS to characterizing pathogens such as bacteria and viruses is an active area of investigation. AgNR array-based SERS substrates have enabled detection of pathogens present in biofluids. Specifically, several publications have focused on determining the spectral bands characteristic of bacteria from different species and cell lines. Studies were carried out on three strains of bacteria as well as the medium in which the bacteria were grown. The spectra of the bacteria and medium were surprisingly similar, so additional spectra were acquired for commonly used bacterial growth media. In many instances, these spectra were similar to published spectra purportedly characteristic of specific bacterial species.
In addition to bacterial samples, nucleic acid hybridization assays were investigated. Oligonucleotide pairs specifically designed to detect respiratory syncytial virus (RSV) in nasal fluids were prepared and evaluated. SERS spectra acquired on oligos, alone or in combination, contain the known spectral signatures of the nucleosides that comprise the oligo. However, spectra acquired on an oligo with a 5'- or 3' thiol were distinctly different from that acquired on the identical oligo without a thiol pendant group suggesting some control over the orientation of the oligo on the nanorod surface. The signal enhancement in SERS depends markedly upon the location of the probe relative to the substrate surface. By systematic placement of nucleotide markers along the oligo chain, the point at which the nucleotide disappears from the spectrum was identified.
The overall findings for AgNR SERS substrates suggest that the applicability of SERS for detecting nucleic acid hybridization is limited. The strong distance dependence coupled with the lack of substrate stability at temperatures required for annealing oligos during hybridization suggest that AgNRs are not the platform to use for hybridization assays.
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Dourou, Dimitra. "Pathogen responses in foods : underestimated ecophysiological factors." Thesis, Cranfield University, 2009. http://dspace.lib.cranfield.ac.uk/handle/1826/3809.
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Accurate prediction of the fate of microbial foodborne pathogens in foods is of great concern for anyone involved in the food chain. Factors that may influence microbial responses in foods and food environments, such as food structure and composition, microbial interactions and mode of growth were identified and assessed in the present study. The fate of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7 was monitored both in and on teewurst, a raw spreadable sausage, at different storage temperatures. Regardless of the storage temperature and inoculation type, pathogen numbers decreased during storage. The increase of endogenous lactic acid bacteria and the concomitant reduction of pH mostly accounted for this reduction. The inactivation of all three pathogens inoculated into batter or onto slices varied considerably among trials possibly due to variations in the initial batch-to-batch levels of lactic acid microflora and the associated microbial interactions. The effect of structure, composition and microbial interactions on the growth kinetics of L. monocytogenes was evaluated in different growth substrates, including broth, agar, sterile meat blocks, naturally contaminated meat blocks and minced meat. The growth responses of L. monocytogenes were significantly different in the different growth media and food products tested. These differences were more pronounced at low temperatures. The validation of a model based on data from broth against the observed growth of the pathogen in the rest of the tested media showed that broth models may result in significant prediction errors. The potential for mono- or multi-strain cultures of Escherichia coli O157:H7 to attach and form biofilm in combinations of food-contact surfaces, growth substrates and storage temperatures was examined. The susceptibility of biofilms to sanitizers was also evaluated. Attachment and biofilm formation was strain dependent. The presence of food residues (liquid or solid) facilitated the attachment/transfer of E. coli O157:H7 on food-contact surfaces. At moderately cold temperatures culture broth was more conducive to subsequent growth. At chill temperatures the presence of natural microflora in liquid residues enhanced further attachment of the pathogen. Biofilms were less susceptible to sanitation treatments as compared to planktonic cells. Biofilm cells surviving sanitation were able to survive and present slight increases at refrigeration and abuse temperatures, respectively, in cross-contaminated ground meat. Acylated homoserine lactones (AHLs) and autoinducer-2 (AI-2) signalling molecules in the cell-free supernatants of Pseudomonas aeruginosa, Yersinia enterocolitica-like, Serratia proteamaculans and a mixture of two Yersinia enterocolitica strains were found to affect the growth kinetics of two Salmonella Enteritidis and S. Typhimurium strains, respectively. P. aeruginosa synthesized quorum sensing signals that accelerated the metabolic activity of Salmonella strains. All other quorated bacteria tested had a negative effect on both initiation of growth and metabolic activity. The effect seems to be strain and QS signal dependent.
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Carlin, Aaron Foster. "Siglec interactions with a sialylated bacterial pathogen." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3263070.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed April 9, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Buckee, Caroline O'Flaherty. "The evolution and maintenance of pathogen diversity." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433383.
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Lipsitch, Marc. "Pathogen transmission and the evolution of virulence." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294342.
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Hitchman, Richard B. "Pathogen variability and dynamics in insect populations." Thesis, Oxford Brookes University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247600.
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Zhang, Hongliang. "The pathogen construct in project risk analysis." Thesis, Lancaster University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507279.
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Patel, Trupti. "Mechanisms of Pathogen Sensing in Cardiac Myocytes." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486584.
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Bacterial sepsis and septic shock are major causes ofdeath in the critically ill. The majority ofthese deaths are due to the development ofmyocardial contractile dysfunction. The pathophysiology ofthis phenomenon is incompletely understood,. with previous work focussing on the influence ofcirculating inflammatory mediators ofnon-cardiac origin. Despite the recognised importance ofthese factors in contrit>uting to myocardial dysfunction, how the heart itselfresponds to bacterial pathogens directly has not been fully elucidated. The aims ofthis study were, first, to characterise the functional changes induced in isolated cardiac myocytes by whole bacteria. With the recent identification ofToll-like receptors (TLR)s (pattern recognition receptors (PRR)s) within the cardiac compartment, the second aim was to examine their roles in any changes seen. Finally, the role ofkey inflammatory mediators was examined. A novel method ofevaluating how Gram positive Staphylococcus aureus (S. aureus) or Gram negative Escherichia coli (E. call) directly modified 'populations' ofcardiac rat and mouse myocytes is described. Bacteria not' only decreased the propot:tion ofviable rod-shaped cells, they also decreased the proportion ofcells able to contract to electrical stimulation. S. aureus was found to have more pronounced effects than E. coli. In separate experiments, extracellular oxidants mimicked the effects ofbacteria on cardiac myocytes. The effects ofS. aureus and E. coli were mediated by TLR2ffLR6 and TLR4 respectively. Although no role for nitric oxide was found in bacteria-induced changes in myocyte function, the adverse effects ofS. aureus were partly prevented by specific cyclo-oygenase-2 inhibitors. However, the central hormone mediating the effects of bacteria (and oxidants) was found to be endothelin-l (ET-l), acting on ETA receptors. Caspase activation, without leading to apoptosis, was also implicated in mediating the phenotype changes induced by bacteria. Finally, cardiac myocytes ofthe noncontracting phenotype showed a reduced myofilament sensitivity to calcium, explaining the functional changes seen. Although the data are limited, a similar phenomenon was seen in failing human myocytes.
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Marchione, Wesley A. "Pathogen resistance genes and proteins in orchids." Virtual Press, 2003. http://liblink.bsu.edu/uhtbin/catkey/1260625.
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To study resistance (R) genes that are expressed when Sophrolaeliacattleya Ginny Champion 'Riverbend' orchid tissue was infected with the tobacco mosaic virus (TMV0), a subtraction library of cDNA clones was previously constructed using mRNA isolated before and after infection (Shuck, unpublished). From 200 clones collected, 5 clones were randomly selected, DNA was isolated, and the cDNA insert was sequenced. These sequences were imported into BLAST to search for homology to other R genes. This search revealed clone 4A to have an 84% homology to a 54 nucleotide region from the Arabidopsis thaliana oligouridylate binding protein which is highly expressed and known to bind RNA Polymerase III transcripts and adenovirus associated RNAs. Further bioinformatics analysis was performed utilizing databases and analysis packages available on the Internet, software such as Vector NTI (Informax, Bethesda, MD), and manual searches. However, no additional domains or motifs indicative of pathogen resistance genes were located in any of the 5 clones. Subsequently, total proteins expressed at various time points following infection were examined on denaturing 5-20% gradient polyacrylamide gels stained with the ProteoSilver Plus TM silver stain kit (Sigma, St. Louis, MO) in order to examine the timing and duration of expression of proteins involved in TMV-O resistance. One protein of-18 kDa was highly expressed at 4 hr after infection that was not seen in the negative control. By 8 hr the band was no longer expressed, it was expressed again from 30 - 48 hr, but was not seen again in later time points. Finally, total mRNA isolated from pooled time points and subjected to in vitro translation indicated a reduction in translation products after infection, providing evidence of posttranscriptional gene silencing (PTGS) following TMV-O infection.Department of Biology
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Weaver, Louise. "Protozoan pathogen removal by wastewater treatment systems." Thesis, University of Portsmouth, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419025.
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Reade, Brian. "The population dynamics of mixed pathogen infections." Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264017.
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