京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13162号 / 論医博第2149号 / 新制||医||1029(附属図書館) / (主査)教授 武田 俊一, 教授 松田 文彦, 教授 清水 章 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les canalopathies cardiaques congénitales constituent la principale hypothèse diagnostique dans les cas de mort subite inexpliquée chez les sujets de moins de 35 ans. Notre travail a eu pour objectif demettre au point une stratégie de détection post-mortem des mutations sur les gènes connus pour être impliqués dans les canalopathies cardiaques, applicable en routine, à partir de la principale source d’ADN post-mortem disponible en France à savoir les prélèvements fixés au formol et inclus en paraffine (FFIP). A partir d’une cohorte de 12 cas, deux techniques de détection de variants génétiques ont été évaluées, une technique de criblage par l’analyse des courbes de fusion haute résolution et une technique de génotypage par spectrométrie de masse MALDI-TOF, respectivement sur le gène KCNQ1 et le gène RyR2. Quelle que soit la technique utilisée, il n’est pas possible de s’affranchir du séquençage de type Sanger afin d’explorer les séquences d’intérêts qui n’ont pu être optimisées avec l’une ou l’autre des méthodes à la fois sur les prélèvements congelés et FFIP. L’arrivée des séquenceurs de nouvelles générations ouvrent ainsi de nouvelles perspectives dans ce domaine. / The congenital cardiac channelopathies constitute the principal diagnostic hypothesis in autopsynegative sudden unexplained death concerning people younger than 35 years old. The present study aimed to develop a strategy of mutations detection on known genes implicated in the cardiac channelopathies. This strategy of mutations detection had to be applicable to routine and has been studied on formalin-fixed and paraffin-embedded (FFPE) tissues which are the principal DNA source available in France. On a cohort of 12 cases, two technique of sequence variants detection wereevaluated: the screening method of High Resolution Melt and the genotyping method based on a MALDI-TOF mass spectrometry, respectively on KCNQ1 and RyR2 genes. Whatever the technique, there is a necessity of resorting to the Sanger sequencing to explore the sequence of interest none optimized with one or the other technology both on FFEP and frozen tissues. That’s why the next generation sequencing method should open new perspectives in the post-mortem diagnostic of cardiac channelopathies.

Focal segmental glomerulosclerosis is one of the commonest cause of the nephrotic syndrome in adults patients. It is a damage of glomerulus characterized by leakage of proteins to urine and oedemas which usually develops into the end-stage renal disease within 10 years. Recently have been described familial forms of this disease which arise from injury to proteins making up filtration barrier of kidney. In 2005 non-selective ion channel TRPC6 was assigned among these proteins. In this thesis I focused on summarizing existing knowledge of the nephrotic syndrome, focal segmental glomerulosclerosis and involvement of TRPC6 in their origin. Second part of this work is devoted to the screening analysis of TRPC6 gene to discover possible mutations and polymorfisms in 47 patients with histologically proven focal segmental glomerulosclerosis or minimal change disease. The used methods were high resolution melting and direct sequencing. In the group of patients was detected no pathogenic mutation, only 2 known polymorfisms P15S and A404V and few changes which do not result in alteration of amino acid. So it seems TRPC6 gene mutations are a rare cause of the focal segmental glomerulosclerosis in adult patients in the Czech Republic.

Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...

Several malignant diseases, such as colorectal, pancreatic, breast or ovarial cancers, are primarily treated with cytostatics 5-fluorouracil (5-FU). 5-FU undergoes biotransformation in human body and arising metabolites induce the damage and subsequent apoptosis in the target cells. The main aim of this diploma Thesis was the determination of methylation in promoter regions of 14 candidate genes participating on 5-FU biotransformation: TK1, PPAT, RRM1, RRM2, UCK2, UCK1, UMPS, TYMP, UPP1, UPP 2 SLC29A1, UPB1, DPYS and DPYD. We hypothesize that the methylation in promoter regions regulates mRNA transcription of the above candidate genes. We have conducted appropriate analyses in 128 colorectal cancer patients, for whom both tumor and nonmalignant adjacent tissues were available. Sample processing and analysis involved DNA isolation, bisulfite conversion of unmethylated cytosines to corresponding uracils, methylation-specific analysis of melting curves with high resolution for theproper methylation analysis and gel electrophoresis to separate PCR products. For the majority of the studied genes (TK1, PPAT, RRM1, RRM2, UCK2, UCK1, UMPS, TYMP, UPP1, SLC29A1 and DPYD) we did not detect any aberrant methylation in promoter regions. In genes DPYS, UPB1 and UPP2 we recorded various degree of promoter...

Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.

Immunomagnetic separation is based on binding of antibody with antigen, where antibody is bound to magnetic particle. In this thesis there were used particles of magnetic pearl cellulose with antiLactobacillus and antiBifidobacterium antibodies. Immunomagnetic separation method was optimalized and verified for its efficiency and specifity with bacterial and yeast cells. This cells were identified by polymerase chain reaction. Efficiency of immunomagnetic separation was verified on probiotic meat product, where Lactobacillus cells were isolated. With DNA from isolated Lactobacillus cells the high resolution melting was performed. The results show presence of several bacterial strains of Lactobacillus species.

The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.

Nephrotic syndrome (NS) is characterized by proteinuria, hypalbuminemia and edemas. It occurs during first and second glomerulopathies. This disease can be divided into two groups: primary (idiopathic) and secondary. The heredity of the familial nephrotic syndrome is autosomal dominant and autosomal recessive. There are four most important genes that condition the formation of hereditary nephrotic syndrome in adult patienst. These genes are ACTN4, CD2AP, NPHS2 and TRPC6. The gene ACTN4, which encodes protein α-actinin 4, is responsible for the autosomal dominant form of focal segmental glomerulosclerosis (FSGS). FSGS is included in first glomerulopathies. α-Actinin 4 was also researched for some types of carcinomas. There was performed the mutational analysis of the gene ACTN4 on the set of 48 patients with nephrotic syndrome in this diploma thesis. High resolution melting (HRM) analysis and sequencing selected samples were used during this mutation detection. During this process many published and unpublished SNPs and one unpublished candidate mutation that could have causal associations with FSGS were found.

>Magister Scientiae - MSc / As human beings we are curious about our origin and ancestry. A curiosity has led to an investigation of human evolution and expansion across the world by means of population genetics and phylo-genetics by evaluating a region in Southern Africa that is largely unknown. The objective of this study was to develop a quick, inexpensive and accurate hierarchical diagnostic screening system of the MtDNA phylogenetic tree, AI-SNPs in the mtDNA genome by using High Resolution Melting analysis to evaluate the population composition and ancestral haplogroups of Southern African populations in Limpopo. The admixture between the ‘Khoesan’ hunter-gatherers, herders and the Bantu speaking populations led to population growth and expansion in Limpopo. This has contributed to populations settling in Limpopo and has thus shaped the ancestral contemporary populations. No research on these individuals residing in Limpopo has been done before, thus an investigation of their ancestral origin was necessary. A total of 760 saliva samples were collected from individuals residing in Limpopo. Only 500 saliva samples were extracted by means of an optimized salting out technique. Five hundred extracted genomic samples were genotyped by means of a quick, inexpensive High-resolution melting analysis. Of the 500 samples, the genotyping results showed 95 individuals derived for the L3 haplogroup which gives a 19% ratio of individuals screened with Multiplex 1. Only 56 individuals were derived for the L1 haplogroup, which gives a percentage of 11%. A total of 249 individuals were derived for the L0 haplogroup, making up a 50% of the total individuals genotyped. Only 100 samples were derived for L0a, making up 20% of individuals screened with Multiplex 1. Of the 95 samples derived for the L3 haplogroup, the results showed 87 individuals to be ancestral for both M and N, making up 91.57% of individuals screened with Multiplex 2. http://etd.uwc.ac.za/. In population genetics using SNPs to infer population history and ancestral origin has become significant, this study allowed researchers to evaluate population groups by investigating their genetic markers and the application of the results allowed for downstream analyses. Finally, this study provides a quick and simple screening method for the selection of lineages that are of interest for further studies.