Ixodes ricinus is the most important human-biting tick in Europe and the principal vector of Lyme borreliosis. In addition, this hard tick species transmits a large number of microbial pathogens that are of importance to animal and human health. Little is known about the diversity and genetic population structure of I. ricinus across Europe. Genetic diversity of these tick populations may have implications on disease transmission. I. ricinus primers were designed for a number of mitochondrial genes and a Multilocus Sequence Typing-like Scheme (MLST) was devised. This was termed mitochondrial MLST (mtMLST). MLST has so far mainly been used for typing microbes, and the development of a MLST scheme for an arthropod vector is novel. Understanding the geographic structure of I. ricinus populations, in combination with studies regarding the migration of tick-borne microbial infections, e.g. Lyme borreliosis, is likely to illuminate important processes in the evolution and spread of tick-borne diseases.

Virtual reality (VR) headsets are becoming more common and will require evolving input mechanisms to support a growing range of applications. Because VR devices require users to wear head-mounted displays, there are accomodations that must be made in order to support specific input devices. One such device, a keyboard, serves as a useful tool for text entry. Many users will require assistance towards using a keyboard when wearing a head-mounted display. Developers have explored new mechanisms to overcome the challenges of text-entry for virtual reality. Several games have toyed with the idea of using motion controllers to provide a text entry mechanism, however few investigations have made on how to assist users in using a physical keyboard while wearing a head-mounted display. As an alternative to controller based text input, I propose that a software tool could facilitate the use of a physical keyboard in virtual reality. Using computer vision, a user€™s hands could be projected into the virtual world. With the ability to see the location of their hands relative to the keyboard, users will be able to type despite the obstruction caused by the head-mounted display (HMD). The viability of this approach was tested and the tool released as a plugin for the Unity development platform. The potential uses for the plugin go beyond text entry, and the project can be expanded to include many physical input devices.

The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.

Haemophilus parasuis es l'agent etiológic de la malaltia de Glässer's, però aquesta bactèria pot causar altres manifestacions clíniques, a més a més de poder ser aïllat del tracte respiratori superior de porcs sans. Els aïllaments de H. parasuis poden presentar diferents fenotips (per exemple diferent perfil de proteïnes, morfologia de colònia o bé producció de càpsula) i diferent capacitat patogènica. Les diferencies entre soques també s'han demostrat a nivell genètic. S'han emprat varis mètodes de tipat per classificar soques de camp d' H. parasuis, però totes presentaven problemes de resolució o implementació. Per resoldre aquestes limitacions es van avaluar diferents tècniques basades en seqüenciació d'ADN. Conseqüentment l'objectiu d'aquest estudi fou millorar el tipat d' H. parasuis i examinar l'associació entre grups de soques i aparició de malaltia. En el primer capítol d'aquest treball s'estudià l'ús d'una seqüència parcial del gen "heat shock protein 60 KDa" (hsp60) com a marcador epidemiológic en un esquema de "single locus sequence typing" (SLST). Es compararen els resultats obtinguts emprant patrons de "enterobacterial repetitive intergenic consensus" (ERIC)-PCR, seqüències parcials de hsp60 i 16S rARN de 103 soques d' H. parasuis i altres espècies relacionades. En el segon capítol d'aquest treball es va desenvolupar un esquema de "multilocus sequence typing" (MLST) fent servir seqüències parcial del gens "house-keeping" mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB. Onze soques de referència i 120 soques de camp van ser incloses en aquest darrer estudi. Els nostres resultats mostren que la hsp60 es un marcador fiable per estudis epidemiológics d' H. parasuis, i que l'anàlisi d'aquesta seqüència es una aproximació millor que els mètodes basats en patrons de bandes. Sorprenentment els gen 16S rARN mostrà prou variabilitat com per ser emprat en el tipatge de H. parasuis enlloc de només en la identificació a nivell d'espècie. A més a més, l'anàlisi de les seqüències de hsp60 i 16S rARN revelaren l'existència de un llinatge divergent de soques associades a l'aparició de malaltia. Ambdós estudis, un amb SLST i l'altre amb MLST, indicaren l'existència de transferències laterals de gens entre soques de H. parasuis i Actinobacillus invalidant l'ús d'aproximacions basades en un sol gen en l'anàlisi filogenètic d'aquesta espècie. L'anàlisi amb MLST mostrà l'existència de 6 "clusters". Quan s'examina l'origen clínic dels aïllaments es veié que un dels "clusters" estava estadísticament associat amb l'aïllament nasal mentre que un altre era associat amb l'aïllament de lesions. El darrer "cluster" incloïa les mateixes soques que el llinatge associat amb l'aparició de malaltia prèviament descrit amb els gens hsp60 i 16S rARN. Finalment, tot i que H. parasuis presenta una estructura de població lliurement recombinant es van trobar dues branques divergents en construir un arbre "neighbour-joining" amb les seqüències del MLST concatenades. Aquesta troballa dona suport als resultats obtinguts amb el gen 16S rARN indicant que H. parasuis sembla tenir una especiació críptica enlloc de una estructura de població panmíctica. / Haemophilus parasuis is the etiological agent of Glässer's disease, but this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates of H. parasuis differ in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classify H. parasuis field strains, but they showed resolution or implementation problems. To overcome these limitations, different DNA sequence based techniques were evaluated. Consequently, the final goal of this study was to improve H. parasuis typing and examine the association of groups of strains with disease outcome. In the first chapter of this work, a partial sequence from the heat shock protein 60 KDa (hsp60) gene was assessed as epidemiological marker in a single locus sequence typing (SLST). We compared enterobacterial repetitive intergenic consensus (ERIC)-PCR patterns, partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species. In the second chapter of this work, we developed a multilocus sequence typing (MLST) system using partial sequences of the house-keeping genes mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB. Eleven reference strains and 120 field strains were included in this latter study. Our results showed that hsp60 is a reliable marker for epidemiological studies in H. parasuis, and the analysis of its sequence is a better approach than fingerprinting methods. Surprisingly, the 16S rRNA gene showed enough variability to be used, not only for species identification, but also for typing. Furthermore, the analysis of the hsp60 and 16S rRNA sequences revealed the presence of a separated lineage of disease-associated strains. Both SLST and MLST studies indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains invalidating the use of single gene approaches in the phylogenetic analysis of these species. MLST analysis revealed the existence of 6 clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation, while another cluster was associated with isolation from lesions. The latter cluster was the same disease-associated cluster identified by hsp60 and 16S rRNA gene analysis. Finally, although a freely recombining population structure was reported, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. The latter, supports the results obtained by 16S rRNA gene sequencing and indicate that H. parasuis is more likely to have a cryptic speciation than a true panmictic population structure.

La enfermedad celíaca (EC) es una inflamación del intestino delgado, que afecta a individuos genéticamente susceptibles, provocada por la ingestión de algunos cereales. La prevalencia de EC en Europa y los EE.UU. es de aproximadamente 1%. La mayoría de los casos de CD sin diagnosticar durante muchos años porque de espectro clínico es muy variable y presentación atípica. La prueba estándar de oro para el diagnóstico de EC es la biopsia del intestino delgado, una prueba invasiva y costosa. Fuerte relación entre la CD y antígenos leucocitarios humanos (HLA) se ha demostrado, con un 95% de los pacientes con EC portadores del antígeno HLA DQ2 heterodímero y el 5% llevan DQ8 heterodímero. Personas negativos DQ2 y DQ8 han demostrado ser muy poco probable que desarrollen CD. El objetivo general de la tesis es el desarrollo de una manera rápida, fácil de utilizar, rentable matriz genosensor de diagnóstico para DQ2/DQ8 escribir como una herramienta de diagnóstico para la predisposición de CD. Dos métodos diferentes han sido investigados en paralelo. En el primer método, la matriz genosensor se desarrolló empleando el método SSOP, mediante el diseño de sondas diferentes alelos específicos. Mientras que en el segundo enfoque, la técnica SSP explotar un nuevo enfoque para la detección directa y rápida de una doble cadena de amplificación de PCR se investigó. En ambos métodos, condiciones de ensayo fueron optimizadas y finalmente el análisis de muestras clínicas reales se realizó. / Coeliac disease (CD) is a small intestinal inflammation, affecting genetically susceptible individuals, triggered by ingestion of certain cereals. Prevalence of CD in Europe and US is about 1%. Most of CD cases remain undiagnosed for many years because of highly variable clinical spectrum and atypical presentation. The gold standard test for CD diagnosis is the small-intestinal biopsy, an invasive and expensive test. Strong relation between CD and Human Leukocyte Antigens (HLA) has been proved, as 95% of the CD patients carrying HLA DQ2 heterodimer and 5% carrying DQ8 heterodimer. DQ2 and DQ8 negative individuals have been shown to be very unlikely to develop CD. The overall objective of the thesis is to develop a rapid, ease to use, cost effective diagnostic genosensor array for DQ2/DQ8 typing as a diagnostic tool for CD predisposition. Two different methods have been investigated in parallel. In the first method, the genosensor array was developed employing the SSOP method, by designing different allele specific probes. Whilst in the second approach, the SSP technique exploiting a novel approach for the direct and rapid detection of a double stranded PCR amplicon was investigated. In both approaches, assay conditions were optimised and finally analysis of real clinical samples was performed.

No description available.

The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.

Freie Universiẗat, Diss., 2006--Berlin.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Clinical Laboratory Sciences. Title from title-page of electronic thesis. Bibliography: leaves 127-148.

Orientador: Vera Lúcia Mores Rall / Resumo: Staphylococcus aureus é um agente comum de mastite bovina, responsável por grandes perdas econômicas na pecuária mundial. Esse micro-organismo possui fatores de virulência bastante conhecidos como a produção de hemolisinas, leucotoxinas e superantígenos como a toxina do síndrome do choque tóxico e enterotoxinas. O objetivo do estudo foi caracterizar molecularmente os isolados de S. aureus provenientes de leite de vacas com mastite subclínica, de várias regiões do estado de São Paulo, através de Multilocus Sequence Typing (MLST), spa typing, Pulse Field Gel Electrophoresis (PFGE) e agr. Também fizeram parte dos objetivos, testes fenótipicos e genotípicos de resistência a antimicrobianos, além da pesquisa de genes de alguns fatores de virulência como o pvl (toxina de Panton Valentine), tst (toxina da Síndrome do Choque Tóxico, , sea-see, seg, seh e sei, a fim de verificar quais os fatores de virulência mais envolvidos nesse quadro. Todos os isolados de S. aureus foram sensíveis a meticilina (MSSA), pois os genes mecA e mecC não foram encontrados. Entre os 12 antibióticos testados, observou-se resistência intermediaria à eritromicina e nenhuma das cepas foram resistentes à oxacilina, vancomicina e gentamicina. Os isolados resistentes à tetraciclina apresentaram o gene tetK . Na caracterização molecular, observou-se 23 spa types diferentes com prevalência dos tipos t605 e t127. A maioria das cepas (48,1%) eram pertencentes ao grupo agr II, seguido de 20,1% do grupo III e 8,1%... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Staphylococcus aureus is a common agent of bovine mastitis, causing economic losses in Brazilian livestock. This microorganism has well known virulence factors such as the production of hemolysins, leukotoxins and superantigens such as toxic shock syndrome toxin and enterotoxins. The objective of the study was to characterize molecularly isolates of S. aureus from milk of cows with subclinical mastitis, of the several regions of São Paulo State, through Multilocus sequence typing (MLST), spatyping, Pulse Field Electrophoresis Gel (PFGE) and agr. Phenotypic and genotypic tests of antimicrobial resistance, as well as the search for genes of some virulence factors such as pvl (Panton Valentine toxin), tst (Toxic Shock Syndrome toxin). In the present study, all isolates of S. aureus were sensitive to methicillin (MSSA), as the mecA and mecC genes were not found. About 12 antibiotics tested, intermediate resistance to erythromicyn was observed and none resistance to oxacillin, vancomycin and gentamicin. The tetracycline resistant isolates showed the tetK gene. Molecular characterization showed 23 different spa types with prevalence of t605 and t127. The most of the strains (48.1%) presented as belonging to the agr II group, followed by 20.1% of the agr III group and 8.1% of the agrI group, and the agr IV group was not found. About the virulence factors studied, the pvl gene was not observed. In relation to super antigens, the tst gene was observed in 105 of the 285 strains (37.1%)... (Complete abstract click electronic access below) / Doutor