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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

Espécies de Cryptococcus obtidas de isolados clínicos e ambientais da cidade de Campinas, SP : genotipagem e avaliação da suscetibilidade "in vitro" frente a agentes antifúngicos isolados e em diferentes combinações Cryptococcus species obtained from clinical and environmental isolates from the city of Campinas, SP : genotyping and assessment of in vitro susceptibility of antifungal agents alone and in different combinations

2014 (has links)
Orientador: Angélica Zaninelli Schreiber Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Made available in DSpace on 2017-04-02T08:52:47Z (GMT). No. of bitstreams: 1 Reichert Lima, Franqueline_M.pdf: 2034183 bytes, checksum: 203b846210e23f42a824280eb3cadda2 (MD5) Previous issue date: 2014 Made available in DSpace on 2017-07-20T12:26:45Z (GMT). No. of bitstreams: 1 ReichertLima_Franqueline_M.pdf: 2034183 bytes, checksum: 203b846210e23f42a824280eb3cadda2 (MD5) Previous issue date: 2014 Resumo: O gênero Cryptococcus engloba duas espécies consideradas patogênicas: C.neoformans e C.gattii. Apesar dos avanços na área médica, a criptococose permanece uma das infecções fúngicas sistêmicas mais importantes no Brasil. Anfotericina B (AMB) associada a flucitosina (5FC) é a terapia de indução indicada porém, no Brasil, 5FC não está disponível e o tratamento segue somente com AMB ou em associação com fluconazol (FCL). Este trabalho avaliou a ocorrência de espécies e os genótipos de isolados clínicos de pacientes atendidos no Hospital de Clínicas da UNICAMP em um período de 5 anos; isolados ambientais coletados na cidade de Campinas-SP e o perfil de suscetibilidade aos antifúngicos sozinhos contra isolados clínicos e ambientais e o efeito de combinações de antifúngicos frente a isolados clínicos de Cryptococcus spp.. A identificação de espécies e genótipos foi realizada por testes bioquímicos, Restriction Fragment Length Polymorphism do gene URA5 (URA5-RFLP) e sequenciamento da região Internal Transcribed Spacer (ITS) do DNA ribossomal. Testes de suscetibilidade para AMB, 5FC, FCL, voriconazol (VRC), itraconazol (ITC) e terbinafina (TRB) isolados foram realizados conforme CLSI M27-A3 (2008). Os testes com antifúngicos combinados (AMB+5FC; AMB+FCL; AMB+TRB; FCL+TRB), foram realizados pelo método do "tabuleiro de xadrez" para determinação do Coeficiente de Inibição Fracional (CIF) para avaliar o tipo de interação entre as substâncias (sinergismo, indiferença ou antagonismo). Dentre 75 isolados clínicos reativados, foram identificados 66 C.neoformans e 9 C.gattii. Todos C.gattii pertenceram ao genótipo VGII enquanto que 62 isolados de C.neoformans ao genótipo VNI e apenas 4 ao genótipo VNII. Foram obtidos 92 isolados ambientais de Cryptococcus, pertencentes às espécies C.neoformans (genótipo VNI), C.laurentii, C.albidus, C.flavescens e Cryptococcus spp.. Os valores dos intervalos de CIM para C.neoformans clínicos foram AMB: ? 0,125-1 µg/mL; 5FC: ? 0,125-2 µg/mL; FCL: 0,25-8 µg/mL; VRC: ? 0,015-0,125 µg/mL; ITC: 0,03-0,25 µg/mL e TRB: 0,125-2 µg/mL. Intervalos de CIM para C.gattii variaram de 0,25-1 µg/mL para AMB; 0,5-4 µg/mL para 5FC; 2-16 µg/mL para FCL; 0,06-0,25µg/mL para VRC; 0,06-0,5 µg/mL para ITC e 0,5-4 µg/mL para TRB. Foram observados elevados valores de CIM para os antifúngicos 5FC e FCL frente aos isolados ambientais de C.albidus e C.laurentii. O genótipo VNI de C.neoformans clínico mostrou 75,80% de interação sinérgica para AMB+5FC; 79,03% para AMB+FCL; 77,42% para AMB+TRB e 95,16% para FCL+TRB. O genótipo VNII apresentou 100% de sinergismo em todas as combinações. C.gattii (VGII) apresentou 88,9% de sinergismo nas combinações AMB+5FC e AMB+FCL; 100% para AMB+TRB e FCL+TRB. Não foi observado efeito antagônico nas combinações de antifúngicos. Foi observado bom desempenho nas combinações realizadas, especialmente naquelas envolvendo a TRB para ambas as espécies C.neoformans e C.gattii. O genótipo VNI foi o predominante entre os genótipos que afetam os pacientes com criptococose na região de Campinas. Em infecções de difícil tratamento ou que não respondem aos antifúngicos convencionais, a combinação de diferentes antifúngicos como AMB+TRB ou FCL+TRB podem vir a ser uma alternativa em países onde 5FC não está disponível, como no Brasil. Mais estudos são necessários para avaliar o papel dos genótipos na sensibilidade aos antifúngicos, assim como estudos de combinações de antifúngicos in vitro e in vivo para que novas estratégias possam ser empregadas no tratamento da criptococose Abstract: Cryptococcus genus comprises two species considered pathogenic: C.neoformans and C.gattii. Despite advances in the medical field, cryptococcosis remains one of the most important systemic fungal infections in Brazil. Amphotericin B (AMB) associated with flucytosine (5FC) induction therapy is indicated but, in Brazil, 5FC is not available and treatment follows only with AMB or in combination with fluconazole (FCL). This study evaluated the prevalence of species and molecular subtypes of clinical isolates from patients treated at the Hospital of UNICAMP in a period of 5 years; environmental isolates collected in Campinas ¿ SP and in vitro antifungal susceptibility profile of antifungal agents alone against clinical and enviromental isolates of Cryptococcus spp. and the effect of combinations of antifungal agents against clinical isolates of Cryptococcus spp.. The species identification and subtyping was performed by biochemical tests, Restriction Fragment Length Polymorphism of URA5 gene (RFLP - URA5) and sequencing of the Internal Transcribed Spacer region (ITS) of ribosomal DNA. Susceptibility tests for AMB, 5FC, FCL, voriconazole (VRC), itraconazole (ITC) and terbinafine (TRB) isolated were performed according to CLSI M27-A3 (2008). Combined antifungals tests (AMB +5FC; AMB+FCL; AMB +TRB, FCL+TRB), were performed by the "Checkerboard" method to determine the Fractional Inhibitory Coefficient index (FIC) to assess the type of interaction between substances (synergism, indifference or antagonism). Among the 75 viable clinical isolates, 66 were identified as C.neoformans and 9 as C.gattii. All C.gattii belonged to subtype VGII while 62 isolates belonged C.neoformans VNI genotype and only 4 VNII. 92 environmental isolates of Cryptococcus were obtained. The species C.neoformans (subtype VNI) C.laurentii, C.albidus, C.flavescens and Cryptococcus spp.were identified. The MIC range values of clinical C.neoformans for AMB were: ? 0.125 -1 µg/mL; 5FC: ? 0.125 to 2 µg/mL; FCL: 0.25-8 µg/mL; VRC: ? 0.015 to 0.125 µg/mL; ITC: 0.03 to 0.25 µg/mL and TRB: 0.125 to 2 µg/mL. MIC for C.gattii ranged from 0.25-1 µg/mL for AMB; 0.5-4 µg/mL for 5FC; 2-16 µg/mL for FCL; 0.06 to 0.25 µg/mL for VRC; 0.06 to 0.5 µg/mL for ITC and 0.5-4 µg/mL for TRB. High MIC values were observed for FCL and 5FC against environmental isolates of C.albidus and C.laurentii. The VNI C.neoformans genotype showed 75.80 % of synergistic interaction for AMB+5FC; 79.03 % for AMB + FCL; 77.42 % for AMB + TRB and 95.16 % for FCL+TRB. The VNII genotype showed 100% synergism in all combinations. C.gattii (VGII) showed 88.9 % of synergism in combinations AMB+5 FC and AMB+FCL; 100 % for AMB+TRB and TRB+FCL. No antagonistic effect was observed in all evaluated antifungal combinations. Good performance was observed in all combinations performed, especially those involving TRB for both C.neoformans and C.gattii species. The VNI was the predominant genotype among genotypes affecting patients with cryptococcosis in Campinas region. In difficult to treat infections or unresponsive to conventional antifungal agents, the combination of different antifungals so as AMB+FCL or TRB+TRB may become an alternative in countries where 5FC is not available, as in Brazil. More studies are needed to evaluate the role of genotypes in sensitivity to antifungal agents, as well as antifungal agents combination studies in vitro and in vivo so that new strategies can be employed in the treatment of cryptococcosis Mestrado Ciencias Biomedicas Mestra em Ciências Médicas
42

Cellular adhesion gene SELP is associated with rheumatoid arthritis and displays differential allelic expression

Burkhardt, Jana, Blume, Mechthild, Petit-Teixeira, Elisabeth, Teixeira, Vitor Hugo, Steiner, Anke, Quente, Elfi, Wolfram, Grit, Scholz, Markus, Pierlot, Céline, Migliorini, Paola, Bombardieri, Stefano, Balsa, Alejandro, Westhovens, René, Barrera, Pilar, Radstake, Timothy R. D. J., Alves, Helena, Bardin, Thomas, Prum, Bernard, Emmrich, Frank, Cornelis, Francois, Ahnert, Peter, Kirsten, Holger 5 September 2014 (has links) (PDF)
In rheumatoid arthritis (RA), a key event is infiltration of inflammatory immune cells into the synovial lining, possibly aggravated by dysregulation of cellular adhesion molecules. Therefore, single nucleotide polymorphisms of 14 genes involved in cellular adhesion processes (CAST, ITGA4, ITGB1, ITGB2, PECAM1, PTEN, PTPN11, PTPRC, PXN, SELE, SELP, SRC, TYK2, and VCAM1) were analyzed for association with RA. Association analysis was performed consecutively in three European RA family sample groups (Nfamilies = 407). Additionally, we investigated differential allelic expression, a possible functional consequence of genetic variants. SELP (selectin P, CD62P) SNP-allele rs6136-T was associated with risk for RA in two RA family sample groups as well as in global analysis of all three groups (ptotal = 0.003). This allele was also expressed preferentially (p,1026) with a two- fold average increase in regulated samples. Differential expression is supported by data from Genevar MuTHER (p1 = 0.004; p2 = 0.0177). Evidence for influence of rs6136 on transcription factor binding was also found in silico and in public datasets reporting in vitro data. In summary, we found SELP rs6136-T to be associated with RA and with increased expression of SELP mRNA. SELP is located on the surface of endothelial cells and crucial for recruitment, adhesion, and migration of inflammatory cells into the joint. Genetically determined increased SELP expression levels might thus be a novel additional risk factor for RA.
43

Duffy ir Kidd antigenų sistemų fenotipavimo ir genotipavimo reikšmė, atliekant dažnas eritrocitų transfuzijas The value of phenotyping and genotyping of Duffy and Kidd antigen systems in case of frequent red blood cell transfusions

Remeikienė, Diana 18 June 2014 (has links)
Nors yra žinoma, kad pacientams, kuriems neseniai atliktos eritrocitų transfuzijos, kraujo grupių nustatymo hemagliutinacijos reakcija rezultatai gali būti nepatikimi dėl kraujyje cirkuliuojančių donoro ertrocitų, tačiau literatūroje nėra aiškių rekomendacijų, kuriais atvejais reikėtų naudoti molekulinius tyrimo metodus. Serologinis Duffy ir Kidd sistemų antigenų ir antikūnų prieš juos nustatymas – viena svarbiausių imunohematologinių problemų transfuzinėje medicinoje. Šiame darbe pirmą kartą Lietuvoje atlikti moksliniai tyrimai imunohematologijos srityje ir panaudoti genetiniai Duffy ir Kidd antigenų sistemų tyrimo metodai. Siekiant įvertinti Duffy ir Kidd antigenų sistemų fenotipavimo ir genotipavimo reikšmę, mes nagrinėjome klinikinių, demografinių, imunohematologinių bei su transfuzija susijusių veiksnių įtaką tyrimų rezultatams. Mūsų tyrimo metu nustatytas didesnis nei 30 proc. nesutapimų dažnis tarp Duffy ir Kidd antigenų sistemų fenotipavimo ir genotipavimo rezultatų patvirtino genetinių tyrimų naudą, atliekant dažnas eritrocitų transfuzijas. Atliekant šį darbą, pirmą kartą Lietuvoje nustatytas Duffy ir Kidd antigenų sistemų fenotipų paplitimas. Nustatytas laiko tarpas, per kurį įprastai klinikinėje praktikoje naudojamų serologinių tyrimų (hemagliutinacijos reakcijos) rezultatai gali būti patikimi, bei pateiktos atitinkamos rekomendacijos didina šio tyrimo praktinę vertę ir yra novatoriška šiuolaikinėje transfuzinėje medicinoje. Accurate phenotyping of multitransfused patients is often complicated - mostly due to the presence of circulating transfused donor’s RBCs in the recipient’s blood, leading to discrepancies in the assessment of test results. The question when genotyping including Duffy and Kidd systems should be used for patients undergoing chronic RBC transfusions is still being discussed. Serological testing and evaluation of the antigens and antibodies of Duffy and Kidd systems are among the main problems in multitransfused patients. The research on immunohematology and blood group genetics has been caried out for the first time in Lithuania. A high rate (more than 30%) of disagreements between the results of phenotyping and genotyping in our study demonstrates the benefit of DNA-based testing for chronically-transfused patients. In order to estimate the value of phenotyping and genotyping of Duffy and Kidd antigen systems in patients undergoing long-term RBC transfusions, the impact of demographic, clinical, immunohaematological or transfusion-related factors, on the discrepancy of the results, was investigated. Time frame that could be appropriate to obtain reliable results of conventionaly used serologic tests (hemmagglutination reaction) after the last transfusion was established as well as appropriate recommendations were made. We believe that these studies could be helpful for clinical practice as well as in decreasing the risk of transfusion of red blood cells.
44

HPV and p16 in head and neck cancer

Sailan, Ahmad Tarmidi 2010 (has links)
There is some evidence to suggest that human papilloma virus (HPV) may play a causal role in head and neck carcinoma (HNSCC). The aim of this study was to investigate the prevalence of HPV DNA in HNSCC and to determine whether any correlation exists with p16 or survival. An initial pilot study of sixty formalin-fixed HNSCC was carried out in order to optimise the methodology for the PCR and immunohistochemistry. A further 84 benign lesions, 12 dysplasias and additional 80 HNSCC were also included. In the pilot study the prevalence of all HPV types was 67% of which 18% were high risk-HPV (HR-HPV) and for the combined carcinoma sample it was 59% of which 25% were HR-HPV. The overall HPV prevalence was 51% and 42% for benign lesions and dysplasias with HR-HPV accounting for 14% and 8% respectively. A total of four alpha HPV types were identified and eleven beta HPV types. Multiple HPV types co-existed in the same tissue and in some cases both alpha and beta HPV. The results may suggest that HR-HPV may play a role in a small subset of HNSCC. An association was found between HPV status and gender, age group, survival, nodal metastasis and T3 tumour size and smoking. HPV16 was predominantly present in female patients and was associated with an improved overall survival and recurrence free survival. p16 positivity varied from 76-78% in carcinomas, 51% in benign lesions and 66% in dysplasias. p16 status was not associated with disease recurrence or nodal metastasis. Positive p16 staining and high staining intensity was associated with a poorer overall survival and the male gender, an older age group, anatomic site, and T2 tumour size. Overall HPV status was not correlated with p16 expression but a correlation found between p16 and HPV16 may suggest that p16 could potentially act as a surrogate marker of HPV16. However, the lack of concordance would suggest that in isolation p16 may not be a reliable marker for HR-HPV and should not be relied upon in isolation. Our findings could suggest that HPV16 and p16 status may be independent predictors for prognosis and disease recurrence.
45

Etude du polymorphisme associé aux répétitions en tandem pour le typage de bactéries pathogènes : Pseudomonas aeruginosa et Staphylococcus aureus

Onteniente, Lucie 13 February 2004 (has links) (PDF)
Les répétitions en tandem sont constituées de successions de motifs d'ADN. Ces structures présentes dans tous les organismes, procaryotes comme eucaryotes, ont des applications dans de nombreux domaines. Depuis quelques années seulement, les répétitions en tandem sont étudiées chez les bactéries. Le polymorphisme associé à ces séquences peut être utilisé pour le génotypage de bactéries pathogènes, permettant une identification précise au niveau de la souche. Le polymorphisme des séquences répétées est de deux types : polymorphisme de longueur et mutations internes aux motifs. Les génomes des deux bactéries pathogènes responsables d'infections nosocomiales, Staphylococcus aureus et Pseudomonas aeruginosa, ont été étudiés dans le but d'identifier des séquences répétées polymorphes. Un ensemble de marqueurs polymorphes a été validé expérimentalement pour ces deux espèces permettant un typage dit MLVA (pour « Multiple Locus VNTR Analysis »). Le travail plus classique de typage par la taille de la répétition a été complété par un travail de séquençage de certains allèles. Les résultats obtenus montrent comment le typage « MLVA » complété si nécessaire par le séquençage d'allèles, pourraient constituer de nouvelles méthodes peu coûteuses participant au contrôle des infections bactériennes.
46

Caracterización genética y molecular del linfoma de células del manto y sus implicaciones clínicas

Royo Moreno, Cristina 15 January 2013 (has links)
Los resultados de esta tesis doctoral han permitido profundizar en el reconocimiento y clasificación del linfoma de células del manto (MCL), basándonos en dos subtipos poco habituales. Por un lado, hemos estudiado las muestras de los pacientes de MCL que no presentan la traslocación principal t(11;14), característica que ha sido objeto de discusión al pensar que el diagnóstico de MCL podía no ser el correcto. Por otro lado, hemos estudiado los MCL con presentación clínica indolente, larga supervivencia, y que no requieren tratamiento durante largos períodos de tiempo, particularidad llamativa en los pacientes de MCL. En las dos primeras publicaciones de esta tesis hemos profundizado en el estudio de los MCL ciclina D1 negativos. Hemos demostrado que el factor de transcripción SOX11, además de sobreexpresarse en MCL ciclina D1 positivos, es un biomarcador útil para identificar los MCL ciclina D1 negativos. Además, caracterizamos genéticamente estos casos y mostramos que el 55% presenta traslocaciones de la CCND2 predominantemente con cadenas ligeras de los genes de las inmunoglobulinas. El perfil de alteraciones genómicas estudiado con arrays de alta densidad ha demostrado que las alteraciones genéticas secundarias entre los MCL ciclina D1 negativos y positivos son muy similares. Los casos de MCL ciclina D1 negativos tienen características clínicas y patológicas similares a los ciclina D1 positivos. Las translocaciones de CCND2 junto con la expresión de SOX11 son herramientas de diagnóstico muy útiles en la identificación de los MCL ciclina D1 negativos para que éstos puedan beneficiarse de los mismos protocolos terapéuticos usados en los MCL convencionales. En el tercer trabajo de esta tesis, nos hemos centrado en el reconocimiento del grupo de pacientes de MCL que no necesita tratamiento durante largos periodos de tiempo, idea que está modificando la manera de entender el MCL, ya que en general está considerado un linfoma agresivo que requiere un tratamiento inmediato. En este trabajo nos basamos en el estudio de Fernandez et al.1 en que se comparó un grupo de pacientes de MCL con comportamiento clínico indolente con un grupo de pacientes con MCL convencional (agresivo). Ambos grupos de muestras tenían un perfil de expresión global similar, sugiriendo que pertenecen a la misma enfermedad, pero también diferían en la expresión de una pequeña firma de 13 genes que estaba altamente expresada en los casos convencionales de MCL pero era negativa en los tumores de los pacientes con comportamiento indolente. Por lo que en el presente estudio, diseñamos y validamos un ensayo de PCR cuantitativa, seleccionando tres genes de la firma de perfil de expresión génica: SOX11, HDGFRP3 y DBN1 que permiten clasificar los casos en uno de estos dos grupos. El subgrupo con baja expresión de los tres genes incluye principalmente los pacientes con clínica indolente, afectación no-ganglionar, genes de las inmunoglobulinas hipermutados, y una supervivencia significativamente superior en comparación a los casos con expresión alta. Además, los MCL leucémicos sin afectación ganglionar y con alta expresión de los tres genes tienen una supervivencia significativamente inferior y un comportamiento clínico más agresivo que los pacientes con firma de expresión baja sin afectación ganglionar. En cuanto a las alteraciones genéticas, tanto los MCL con alta o baja expresión de los tres genes tenían una supervivencia significativamente inferior si presentan alteraciones en 17p. Por tanto, concluimos que la evaluación de la firma de los tres genes junto con el estudio de 17p/TP53 en muestras leucémicas puede ayudar a identificar un subgrupo particular de MCL y determinar la evolución clínica de estos los pacientes. Por tanto, estos estudios pueden proporcionar una evaluación más precisa del MCL y una ayuda en la decisión de tratamiento más adecuado para cada paciente. In the first publication of this thesis we have studied the cyclin D1-negative mantle cell lymphomas (MCL). These cases are not well characterized due to the difficulties in their recognition. Overexpression of the transcription factor SOX11 has been observed in conventional MCL. In this study we demonstrated that SOX11 was highly expressed in cyclin D1-negative MCL, thus SOX11 expression is a highly specific marker for both cyclin D1-positive and negative MCL. In the second study we investigated 40 cyclin D1-negative MCL. Chromosomal rearrangements of CCND2 were detected in 55% of the cases and frequently with light chain immunoglobulin (IG) genes as partners. The genomic profile of the cyclin D1-negative cases analyzed by high resolution arrays were similar to the cyclin D1-positive MCL. This characterization of a large series of cyclin D1-negative MCL indicates that these tumors are clinically and biologically similar to the cyclin D1-positive MCL and provides a basis for the proper identification and clinical management of these patients. In the third publication, we studied the clinical and biological features of mantle cell lymphoma with a more indolent disease and long survival. The expression of 3 genes (SOX11, HDGFRP3, DBN1) selected from a gene expression signature distinguishing conventional an indolent MCL was studied in 68 leukemic MCL by quantitative PCR. An unsupervised analysis segregated two groups of MCL based on the expression levels of these genes. The tumors with low expression presented mainly with a non-nodal disease, had more frequently mutated IG, fewer genomic alterations, remained untreated more frequently and longer time, and had a better outcome than the tumors with high expression. The presence of 17p/TP53 alterations had an adverse effect on the outcome of both subgroups of MCL. The detection of the 3-gene signature may help to identify this particular subtype of MCL.
47

The genetic composition and diversity of Francisella tularensis

Larsson, Pär 2007 (has links)

Francisella tularensis is the causative agent of the debilitating, sometimes fatal zoonotic disease tularemia. To date, little information has been available on the genetic makeup of this pathogen, its evolution, and the genetic differences which characterize subspecific lineages. These are the main areas addressed in this thesis.

The work indicated a high degree of genetic conservation of F. tularensis, both on the sequence level as determined by sequencing and on the compositional level, determined by array-based comparative genomic hybridizations (aCGH). One striking finding was that subsp. mediasiatica was most similar to subsp. tularensis, despite their natural confinement to Central Asia and North America, respectively. All genetic Regions of Difference RD found by aCGH distinguishing lineages were had resulted from repeat-mediated excision of DNA. This was used to identify additional RDs. Such data along with a multiple locus sequence analysis suggested an evolutionary scenario for F. tularensis.

Based on genomic information, a novel typing scheme for F. tularensis was furthermore devised and evaluated. This method provided increased robustness compared to previously used methods for F. tularensis typing, while retaining a capacity for high resolution.

Finally, the genomic sequence of the highly virulent F. tularensis strain SCHU S4 was determined and analysed. Evidenced by numerous pseudogenes and disrupted metabolic pathways, the bacterium appears to be undergoing a genome reduction process whereby a large proportion of the genetic capacity gradually is lost. It is likely that F. tularensis has irreversibly has evolved into an obligate host-dependent bacterium, incapable of a free-living existence. Unexpectedly, the bacterium was found to be devoid of common virulence mechanisms such as classic toxins, or type III and IV secretion systems. Instead, the virulence of this bacterium is probably largely the result of specific and unusual mechanisms.

48

The genetic composition and diversity of Francisella tularensis

Larsson, Pär 2007 (has links)
Francisella tularensis is the causative agent of the debilitating, sometimes fatal zoonotic disease tularemia. To date, little information has been available on the genetic makeup of this pathogen, its evolution, and the genetic differences which characterize subspecific lineages. These are the main areas addressed in this thesis. The work indicated a high degree of genetic conservation of F. tularensis, both on the sequence level as determined by sequencing and on the compositional level, determined by array-based comparative genomic hybridizations (aCGH). One striking finding was that subsp. mediasiatica was most similar to subsp. tularensis, despite their natural confinement to Central Asia and North America, respectively. All genetic Regions of Difference RD found by aCGH distinguishing lineages were had resulted from repeat-mediated excision of DNA. This was used to identify additional RDs. Such data along with a multiple locus sequence analysis suggested an evolutionary scenario for F. tularensis. Based on genomic information, a novel typing scheme for F. tularensis was furthermore devised and evaluated. This method provided increased robustness compared to previously used methods for F. tularensis typing, while retaining a capacity for high resolution. Finally, the genomic sequence of the highly virulent F. tularensis strain SCHU S4 was determined and analysed. Evidenced by numerous pseudogenes and disrupted metabolic pathways, the bacterium appears to be undergoing a genome reduction process whereby a large proportion of the genetic capacity gradually is lost. It is likely that F. tularensis has irreversibly has evolved into an obligate host-dependent bacterium, incapable of a free-living existence. Unexpectedly, the bacterium was found to be devoid of common virulence mechanisms such as classic toxins, or type III and IV secretion systems. Instead, the virulence of this bacterium is probably largely the result of specific and unusual mechanisms.
49

Chlamydia trachomatis: Development of molecular typing methods and applications in epidemiology

Klint, Markus 2009 (has links)
A general aim was to combine molecular typing methods with clinical background information to increase epidemiological knowledge about Chlamydia trachomatis infections. An outbreak of Lymfogranuloma venereum (LGV), caused by a more invasive variant of C. trachomatis, was reported from the Netherlands in 2003 among men who have sex with men (MSM). All Chlamydia positive specimens from a venereal disease clinic for MSM in Stockholm during one year were genotyped. No spread of LGV was found, apart from three symptomatic cases. The same ompA genotypes were found among MSM in Melbourne, but the genotype distribution was different compared to findings among the heterosexual population in Sweden. The standard method for genotyping of Chlamydia is ompA-sequencing, but it has low resolution because one genotype predominates. A multilocus sequence typing (MLST) system based on five targets was developed. In a sample of 47 specimens, 32 variants were found with MLST, but only 12 variants with ompA-sequencing. The polymorphisms in the hctB gene, one MLST target, are caused by an element of 108 bp that is present in two to four repetitions and in different variants. Although the DNA-binding function of Hc2 that is encoded by hctB has been studied, our findings of a considerable size variation show that new studies are needed. In 2006, specimens with a 377 bp deletion in the cryptic plasmid covering the target region for diagnostic test systems from Abbott and Roche were discovered in Sweden. Applying MLST to these specimens indicated that there was a single clone, denoted nvCT. The proportion of nvCT in all detected Chlamydia cases was higher (20% to 65%) in counties using Abbott/Roche compared to counties using the BectonDickinson test system (7% to 20%). The proportions of nvCT converge in counties with high or low levels when detection systems were adjusted to detect nvCT.
50

Tagging systems for sequencing large cohorts

Neiman, Mårten 2010 (has links)
Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets. This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized. The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set. In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained. QC20100907

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