© 2015 Springer. http://dx.doi.org/10.1007/978-3-319-26529-2_11© 2015 ACM, Inc. http://doi.acm.org/10.1145/2676726.2676996 / 京都大学 / 0048 / 新制・課程博士 / 博士(情報学) / 甲第19863号 / 情博第614号 / 新制||情||107(附属図書館) / 32899 / 京都大学大学院情報学研究科通信情報システム専攻 / (主査)教授 五十嵐 淳, 教授 山本 章博, 教授 岡部 寿男 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21024号 / 医科博第85号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 川上 浩司, 教授 黒田 知宏, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of <I>Haemophilus</I> and <I>Neisseria</I>. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of <I>H. somnus</I>, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to <I>H. somnus</I> LOS were produced and used with cross-reacting MAbs to <I>H. aegyptius</I> LOS (MAb 5F5) and <I>Neisseria</I> <I>gonorrhoeae</I> LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of <I>H. somnus</I>. Using three MAbs, thirty-nine of these <I>H. somnus</I> strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of <I>H. somnus</I> strains. The anti-<I>H. somnus</I> LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the <I>H. somnus</I> strains. Six strains (13%) were not recognized by any of these MAbs. Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in <I>H. somnus</I> LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed. / Master of Science

Recently, many full-sized keyboards have been designed to fold in various ways in an attempt to make them more transportable. The flexible membrane keyboard, one type of full sized keyboard, is unique because it is made from silicon rubber, thus it is fully flexible and water resistant. Although a number of flexible keyboard characteristics are the same as standard keyboards (i.e. key size, shape and spacing), key-switch and key clicking mechanisms are inherently different. Since there is little or no existing research on flexible keyboards, there is a current need for data to facilitate design of such keyboards for use. Typing performance and perceived usability of several flexible keyboards that differed in terms of material hardness (hard, medium, or soft) and key contact point shape (circular or square) were studied. The results supported the hypothesis that both typing performance and usability of the flexible membrane keyboard were affected by material hardness and contact point shape. Square shaped contact points led to increased typing speed and decreased error rates, and medium or soft hardness led to increased typing speed. The best flexible keyboard (perceived by participants) in general received neutral usability ratings. However, ratings for mobility and design were much higher than neutral. Overall, subjective and objective measures of performance and usability indicated that flexible keyboards that are made of silicon of a soft or medium hardness and with a square shaped contact points are preferred. / Master of Science

Nas últimas décadas observou-se um número crescente de relatos de infecções invasivas por Trichosporon em ambientes hospitalares, devido ao aumento da população suscetível e a melhoria dos métodos diagnósticos. Leveduras do gênero Trichosporon, depois de Candida, são as mais relacionadas à infecção fúngica invasiva em pacientes hematológicos, sendo Trichosporon asahii responsável por 90% dos casos. A identificação de espécies de Trichosporon é realizada através do sequenciamento da região IGS1 do DNA ribossomal, técnica considerada padrão-ouro. Através do estudo dos polimorfismos da região IGS1 do DNA ribossomal, diversos genótipos de T. asahii têm sido descritos, entretanto sem relação com a distribuição geográfica, perfil de suscetibilidade aos antifúngicos ou patogenicidade. O presente estudo teve como objetivo padronizar um método de análise por sequenciamento multilocus para a espécie T. asahii, definindo novos genes (loci) para melhor descrever a filogenia da espécie. Foram analisadas 21 cepas de T. asahii de diferentes origens (Brasil, Europa, Ásia) e genótipos (1,3,4,5,6,7). As sequências de genes estruturais (housekeeping genes) dos genomas de T. asahii (CBS2479 e CBS8904) disponíveis no GenBank foram alinhadas e analisadas in silico para o delineamento e avaliação dos novos primers. Após as reações de PCR e análise das sequências de DNA, quatro novos loci foram selecionados para a análise filogenética multilocus: phosphate carrier protein, topoisomerase 1 (TOP1), beta-1-tubulin, copper-exporting ATPase. As árvores filogenéticas demonstraram dois clados bem distintos, com altos valores de bootstraps. Além disso, os genótipos 1 e 3 foram alocados em clados diferentes. Nossos resultados sugerem uma reclassificação genética para a espécie T. asahii. Novos estudos, incluindo um maior número de cepas e outros marcadores genéticos, são necessários para melhor abordar a filogenia atual de T. asahii / In the last decades there has been a significant increase of the reported cases of invasive fungal infections by Trichosporon in hospital settings, related to the increase of the susceptible population and to the improvement of diagnostic methods. Trichosporon are the most frequent yeast related to invasive fungal infection in hematological patients after Candida, with Trichosporon asahii accounting for 90% of the cases. The gold standard method for Trichosporon species identification is the sequence analysis of the intergenic spacer region 1 (IGS1) from the ribosomal DNA. Based on the polymorphisms of the IGS region of ribosomal DNA, several T. asahii genotypes have been described, without relation with geographical distribution, antifungal susceptibility profile or pathogenicity. The objective of the study was to evaluate a multilocus sequencing method for the T. asahii species, defining new loci to better describe the phylogeny of the species. Twenty-one strains of T. asahii from different origins (Brazil, Europe, Asia) and genotypes (1,3,4,5,6,7) were analyzed. Housekeeping genes from T. asahii genomes (CBS2479 and CBS8904) available in GenBank were aligned and in silico analyses were carried out to design and evaluate the new primers. After PCR reactions and DNA sequence analysis, four new loci were selected for the multilocus plylogenetic analysis along with the IGS1 region from the rDNA: topoisomerase 1 (TOP1), phosphate carrier protein, beta-1-tubulin, copper-exporting ATPase. Phylogenetic trees revealed two well-distinct clades, with high bootstraps values. Moreover, IGS genotypes 1 and 3 strains were split into the different clades. Our results suggest a different genetic background for the species T. asahii. Further studies including more T. asahii strains and other genetic markers are necessary to better address the current phylogeny of T. asahii

Descoberto e caracterizado em 1909 por Carlos Chagas, o Trypanosoma cruzi é o agente etiológico da Doença de Chagas. Durante a fase crônica da doença de Chagas, onde a parasitemia é baixa, o diagnóstico se baseia na busca de anticorpos contra os antígenos de T. cruzi no sangue. Para aumentar a certeza do resultado recomenda-se o uso de pelo menos dois métodos sorológicos (geralmente ensaio ELISA, Imunofluorescência Indireta ou Hemaglutinação Indireta) para a confirmação de diagnóstico. Embora os ensaios mencionados sejam de uso amplamente difundido, nenhum deles possui suficiente especificidade para definir o diagnóstico isoladamente, especialmente em pacientes provenientes de regiões onde há sobreposição geográfica com outros parasitos, especialmente do gênero Leishmania ou T. rangeli. Uma alternativa de interesse que foi levantada por alguns autores na década de oitenta é a busca de moléculas de interesse diagnóstico (anticorpos, antígenos ou imunocomplexos) na urina de pacientes. Durante os últimos anos técnicas de proteômica têm sido aplicadas a muitos campos da medicina. Uma das aplicações é na nefrologia para o melhor entendimento da fisiologia renal, explorar a complexidade de mecanismos de doenças e para identificar novos biomarcadores. Além do mais, a maioria das proteínas na urina são glicosiladas e suas propriedades são únicas fazendo delas uma importante fonte de biomarcadores. De maneira geral, percebe-se que apesar dos avanços significativos nos métodos de diagnósticos para a detecção da infecção por T. cruzi, há algumas lacunas que ainda precisam ser preenchidas. O fato de que a sorologia convencional para busca de anticorpos IgGs manter-se-á positiva ao longo da vida do paciente, mesmo após tratamento devido a persistência da resposta imune humoral, é uma limitação, já que não permite ter um critério confiável de cura. Por outro lado, a falta de um sistema rápido e confiável para acompanhar a evolução do tratamento, gera sérias limitações para avaliar o desempenho de novos protocolos de tratamento com fármacos já existentes ou de novos fármacos. Em função dessas limitações, propõe-se desenvolver e validar novos métodos de diagnóstico baseado em espectrometria de massas para a detecção da infecção pelo T. cruzi utilizando a urina como fonte de biomarcadores. / Discovered and characterized in 1909 by Carlos Chagas, Trypanosoma cruzi is the etiologic agent of Chagas\' disease. During the chronic phase of Chagas\' disease, where parasitemia is low, the diagnosis is based on the search for antibodies against T. cruzi antigens in the blood. To increase the certainty of the result, is recommended two serological methods (usually ELISA, Indirect Immunofluorescence or Indirect Hemagglutination) for diagnostic confirmation. Although the aforementioned assays are widely used, none of them has sufficient specificity to define the diagnosis alone, especially in patients from regions where there is geographical overlap with other parasites, especially of the genus Leishmania or T. rangeli. An interesting alternative that was raised by some authors in the 1980s is the use of molecules of diagnostic interest (antibodies, antigens or immunomplexes) in the patients\' urine. During the last years techniques of proteomics have been applied to many fields of medicine. One of the applications is nephrology for the better understanding of renal physiology, to explore the completeness of disease mechanisms and identify new biomarkers. Moreover, most of the proteins in the urine are glycosylated. Their properties are unique, making them an important source of biomarkers. In general, perceived that despite significant advances in diagnostic methods for the detection of T. cruzi infection, there are some gaps that need to be filled. The fact that conventional IgG antibody serology will remain positive over the life of the patient, despite the persistence of the humoral immune response, is a limitation, since it does not allow a reliable criterion of cure. On the other hand, the lack of a reliable system to follow the evolution of the treatment generates serious limitations to evaluate the performance of new treatment protocols with existing drugs or new drugs. Due to these limitations, we propose to develop new methods of diagnosis based on mass spectrometry for the detection of T. cruzi infection using urine as a source of biomarkers.

The possibility of identifying single nucleotide polymorphisms (SNPs) that would be useful for rapid bacterial typing was investigated. Neisseria meningitidis was the organism chosen for modelling the approach since informative SNPs could be found amongst the sequence data available for multi-locus sequence typing (MLST) at http://www.mlst.net. The hypothesis tested was that a small number of SNPs located within the seven gene fragments sequenced for MLST provide information equivalent to MLST. Preliminary investigations revealed that a small number of SNPs could be utilised to highly discriminate sequence types (STs) of clinical interest. Laboratory procedures demonstrated that SNP fingerprinting of N. meningitidis isolates is achievable. Further tests showed that laboratory identification of a defining SNP in the genome of isolates was to be a practical method of obtaining relevant typing information. Identification of the most discriminating SNPs amongst the ever-increasing amount of MLST sequence data summoned the need for computer-based assistance. Two methods of SNP selection devised by the author of this thesis were translated into computer-based algorithms by contributing team members. Software for two computer programs was produced. The algorithms facilitate the optimal selection of SNPs useful for (1) distinguishing specific STs and (2) differentiating non-specific STs. Current input information can be obtained from the MLST database and consequently the programs can be applied to any bacterial species for which MLST data have been entered. The two algorithms for the selection of SNPs were designed to serve contrasting purposes. The first of these was to determine the ST identity of isolates from an outbreak of disease. In this case, isolates would be tested for their membership to any of the STs known to be associated with disease. It was shown that one SNP per ST could distinguish each of four hyperinvasive STs of N. meningitidis from between 92.5% and 97.5% of all other STs. With two SNPs per ST, between 96.7% and 99.0% discrimination is achieved. The SNPs were selected from MLST loci with the assistance of the first algorithm which scores SNPs according to the number of base mismatches in a sequence alignment between an allele of an ST of interest and alleles belonging to all other STs at a specified locus. The second purpose was to determine whether or not isolates from different sources belong to the same ST, regardless of their actual ST identity. It was shown that with seven SNPs, four sample STs of N. meningitidis could, on average, be discriminated from 97.1% of all other STs. The SNPs were selected with the aid of the second algorithm which scores SNPs at MLST loci for the relative frequency of each nucleotide base in a sequence alignment as a measure of the extent of their polymorphism. A third algorithm for selecting SNPs has been discussed. By altering the method of scoring SNPs, it is possible to overcome the limitations inherent in the two algorithms that were utilised for finding SNPs. In addition, the third approach caters for finding SNPs that distinguish members of a complex from non-members.

Infecções causadas por Candida tropicalis (C. tropicalis) são associados à elevada morbi-mortalidade, e foram consideradas como importantes causas de infecção de corrente sanguínea no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) de março de 1998 a março de 2001. Adicionalmente, são responsáveis pelo aumento do tempo e dos custos de hospitalização e necessidade de cuidados intensivos. Esse estudo tem como objetivo a caracterização molecular e perfil de sensibilidade de 61 isolados de C. tropicalis a partir de candidemias no HCFMUSP e Universidade Estadual de Campinas (UNICAMP), através das técnicas de amplificação aleatória do DNA polimórfico (RAPD), eletroforese em campo pulsátil (PFGE), tipagem de sequências de múltiplos locus gênicos (MLST) e antifungigrama por microdiluição pelos métodos propostos, Clinical and Laboratory Standards Institute (CLSI) e European Committee on Antibiotic Susceptibility Testing (EUCAST). A análise filogenética por RAPD evidenciou que os iniciadores P1 e P2 mostraram maior capacidade de discriminação que P3. Na análise por PFGE com enzimas de restrição SfiI, SmaI, BssHII e NaeI, a enzima BssHII mostrou maior poder discriminatório. MLST contribuiu com 36 novas diploid sequence type (DSTs) e 23 novos alelos, de acordo com o banco de dados oficial do MLST (http://pubmlst.org/ctropicalis/), representando o primeiro estudo que caracterizaram isolados sequenciais na América do Sul. Entre os isolados sequenciais de um mesmo paciente, as microvariações foram mais frequentes no fragmento de gene XYR1 em 8 pacientes e macrovariações ocorreram em quatro pacientes com mais de um isolado, destacando-se três que apresentaram diferença nos seis alelos estudados. A análise comparativa entre os métodos evidenciou diferenças entre os isolados múltiplos dos pacientes 3, 7 e 11, considerados diferentes pelos três métodos. O poder discriminatório foi de 83,47% para RAPD, 82,18% para PFGE e 97,4 % para MLST. Os resultados do antifungigrana mostraram concordância entre os métodos CLSI e EUCAST de 73,8% para o fluconazol, 67,2% para o itraconazol e 80,3% para o voriconazol. Do total de 61 isolados estudados, 3 isolados de diferentes pacientes foram resistentes ao fluconazol, com MIC de 64 g/mL. O fenômeno de trailing foi observado em 50% das amostras testadas frente ao fluconazol, 23% ao voriconazol e 21,3% ao itraconazol. O uso de pH 5,0 para re-análise do CLSI frente ao fluconazol revelou-se como uma ferramenta útil para esclarecer o perfil de sensibilidade de isolados que apresentaram o fenômeno de trailing. Não houve correlação entre perfil genético gerado pelas técnicas de caracterização molecular estudadas e o perfil fenotípico através do teste de sensibilidade aos antifúngicos / Infections caused by Candida tropicalis (C. tropicalis) have been characterized as important causes of candidemia at the Hospital of the Medical school, University of São Paulo (HCFMUSP) from March 1998 to March 2001 and are associated with high morbidity and mortality. Additionally, they have been related to higher hospitalization costs because of longer hospitalization times and intensive care needs. This study aims to analyze the molecular typing and antifungal susceptibility profile of 61 isolates of C. tropicalis from 41 patients with candidemia in HCFMUSP and University of Campinas (UNICAMP), through Random Amplified Polymorphic DNA (RAPD), Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and broth microdilution antifungal susceptibility methodologies proposed by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antibiotic Susceptibility Testing (EUCAST). Phylogenetic analysis showed higher discriminatory power index of P1 and P2 primers than P3 by RAPD analysis. PFGE was performed with restriction enzymes SfiI, SmaI, NaeI and BssHII) and the enzyme BssHII presented the best performance. MLST analyses revealed 36 new diploid sequence type (DSTs) and 23 new alleles according to the C. tropicalis MLST database (http://pubmlst.org/ctropicalis/), representing the first study to characterize the sequential isolates of C. tropicalis candidemia in South America. Microvariation in a single gene was found in the sequential isolates from 8 patients. The main polymorphisms occurred in the alleles of the XYR1 gene. Macrovariation was detected in isolates from four patients, where 3 patients presented polimorphisms in six gene fragments. The comparative analysis revealed differences among sequential isolates from patients 3, 7 and 11, considered by three different methods. The discriminatory power was 83.47% for RAPD, 82.18% for PFGE and 97.4% for MLST. The agreement between the CLSI and EUCAST methods was 73.8% to fluconazole susceptibility, 67.2% to itraconazole and 80.3% to voriconazole. Of the 61 isolates tested, 3 isolates from different patients were resistant to fluconazole, MIC of 64 mg/mL. The trailing phenomenon was observed in 50% to fluconazole, 23% to voriconazole and 21.3% to itraconazole. Among the isolates studied, the use of pH 5.0 facilitated the determination of minimum inhibitory concentrations (MICs) for the re-analysis of fluconazole by CLSI, proving to be an important tool for the trailing phenomenon. No correlation was observed between genetic profile generated by the techniques of molecular characterization and phenotypic profile determined by susceptibility tests to antifungal drugs