京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22036号 / 医博第4521号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 松田 道行, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20989号 / 医博第4335号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 松田 文彦, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Clonal evolution is a multistep process characterized by progression of the disease, adverse prognosis and shortening of overall survival. The aim of the dissertation was a detailed characterization of identified changes in patients with myelodysplastic syndromes (MDS) and clonal evolution and evaluation of their prognostic impact. We performed detail cytogenomic analyses in 36/469 (8%) patients with confirmed linear clonal evolution. We described 57 primary abnormalities (32% MDS-specific) at the time of diagnosis, the most frequent was deletion of long arm of chromosome 5. We proved 156 secondary aberrations (21% MDS-specific) during the course of the clonal evolution, the most frequent were trisomies/tetrasomies of chromosome 8. We identified acquired uniparental disomies (aUPD) in 19% of patients. In MDS-specific aUPDs 4q, 11q and 17p, we proved homozygous mutations of TET2, c-CBL and TP53 genes. We found a statistically significant difference in overall survival between the groups of patients divided according to their diagnostic cytogenomic findings. In patients with clonal evolution before treatment 54% of aberrations were gains of whole chromosomes, by contrast 44% of abnormalities identified in patients with clonal evolution after treatment were monosomies or deletions. The study of clonal...

Polyclonal plantings of Populus deltoides are expected to display increased site resource use, productivity, and tolerance to stress through plasticity changes leading to niche differentiation (i.e changes to crown/canopy structures). In the present study, P. deltoides Clones S7C8, 110412, and polyclonal plots were tested for differentially expressed genes and enriched biological pathways between planting schemes. Transcriptomic analysis of leaves revealed upregulation of an active growth gene and gene family members that play important roles in plant stress and stress tolerance in polyclonal plantings. A gene associated with oxidative stress was upregulated in polyclonal plantings across all treatments. Secondary metabolic pathways including arginine and proline metabolism were upregulated in monoclonal plantings and downregulated in polyclonal plantings. Phenotypic results displayed greater aboveground biomass in polyclonal plantings. Results suggested a potential increased tolerance in polyclonal plantings to water and heat stress, including increased productivity and resource usage.

The human gamma-delta (gd) TCR+ T-cell subset may undergo specific antigen-driven activation and clonal expansion, in the context of systemic sclerosis (SSc) pathogenesis. The purpose of this study was; 1) To determine whether gd TCR+ T-cells are clonally expanded in skin biopsies and peripheral blood from patients with SSc; and 2) To develop approaches for identification of the antigens recognized by these clonally-expanded gd TCR+ T-cells. Total RNA was isolated from the skin biopsies and peripheral blood of patients with SSc (n=8). After cDNA synthesis, the g- and d-chain TCR transcripts were amplified by PCR, cloned and sequenced for analysis. Full length copies of the TCR transcripts were constructed, expressed in a TCR-negative Jurkat T-cell line using retroviral gene transduction, and verified by RT-PCR and flow cytometry for gd TCR expression. Putative antigen recognition, by the transduced gd TCR+ Jurkat T-cell lines, was assessed via; 1) Measuring intracellular calcium flux in the transduced cells after stimulation with putative SSc antigens, including DNA topoisomerase I, centromere proteins A and B, hsp 27, hsp 90 and the viral lysate of human cytomegalovirus; and 2) Cytotoxicity against human endothelial cell lines (HUVEC and HLMVEC) via measurement of lactate dehydrogenase release from the targets. We report the presence of substantial, statistically-significant, proportions of identical g- and d-chain transcripts in skin biopsies and PBMC of patients with SSc, demonstrating the presence of antigen-driven clonal expansions. Jurkat T-cells, transduced with the clonally-expanded gd TCR transcripts from a patient, showed no evidence of cytotoxicity against the human endothelial cell lines, or calcium flux in response to stimulation with the putative SSc antigens assessed. In conclusion, extensive clonal expansions of g- and d-chain TCR transcripts were identified in skin biopsies and peripheral blood of patients with SSc, demonstrating the presence of oligoclonal populations of gd TCR+ T-cells in these patients. These gd TCR+ T-cells have undergone proliferation and clonal expansion in vivo in response to as yet unidentified antigens. Furthermore, an approach has been developed for the identification of the antigens recognized by the clonally-expanded gd TCR transcripts, which can be expanded to additional patients with SSc. / Microbiology and Immunology

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23772号 / 医博第4818号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 松田 文彦, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

All members of the genus Xanthomonas are considered to be plant pathogenic, with specific pathovars infecting several high value agricultural crops. One of these pathovars, X. campestris pv. zeae (as this is only a proposed name it will further on be referred to as Xanthomonas BLSD) the causal agent of bacterial leaf steak of maize, has established itself as a widespread significant maize pathogen within South Africa. Insufficient information about the present distribution of the pathogen is available. The main aim of the study was thus to isolate and characterise the pathogen using molecular methods. Results demonstrated that the causal agent of bacterial leaf streak disease (Xanthomonas BLSD: potentially X. campestris pv. zeae) was widely distributed within the major maize cultivation regions of South Africa. Most of the isolates collected originated from the Highveld maize production provinces (North West, Free State, Gauteng and Mpumalanga provinces) as well as from irrigated maize fields in the Northern Cape province. The XgumD gene marker was used to determine if the isolates belonged to the genus Xanthomonas. The gumD gene fragment is located within the gumB-gumM region of the operon and is conserved among Xanthomonas species. This gene fragment is partially responsible for xanthan production. This marker was amplified from all isolates and a selected number were sequenced. The marker was only able to confirm that the causal agent was a member of the genus Xanthomonas. PCR methods were used for the characterisation of the isolates. This included PCR and sequencing of ribosomal RNA- gyraseB and gumD genes. A fingerprinting method BOX-PCR was also employed. Good quality DNA of sufficient quantities was obtained from the various isolates. Amplification produced no non-specific amplification products. This resulted in good quality sequences that could be analysed using bioinformatics tools. Phylogenetic analyses of the ribosomal RNA and gyraseB genes could not detect differences amongst the 47 Xanthomonas BLSD isolates. However, these genes were able to distinguish between the type strain of these isolates and various Xanthomonas species and pathovars. From all three neighbour joining trees the Xanthomonas BLSD isolates had close association with X. axonopodis pv. vasculorum strain ATCC 35938. For the 16S rRNA gene there exists no sequence differences between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. A single nucleotide difference was observed between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938 for the 23S rRNA gene. The gyraseB gene detected a total of six nucleotide variations between these two Xanthomonas species. For all of the phylogenetic trees there was no clustering of Xanthomonas BLSD with X. campestris pathovars. Genetic profiling (via BOX-PCR) based on present/absent analysis revealed no variations amongst the Xanthomonas BLSD isolates. All isolates shared an identical pattern produced by 12 distinct PCR products. This profiling technique did differentiate between the isolates of Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. Their profiles shared common bands, but differed in the number and overall pattern of the bands. These results suggest two main conclusions: (i) Xanthomonas BLSD has a clonal origin with geographical separation not impacting genetic variation. The fact that all the isolates appear to be clonal may imply that when resistant maize cultivars are developed these should be resistant to all isolates of the pathovar irrespective of their geographical origin. This is a suggestion that will have to be corroborated using more isolates and additional genetic fingerprinting techniques (ii) the Xanthomonas BLSD isolates from this study may not belong to X. campestris. Further studies using other markers should be conducted to determine the real identity of Xanthomonas BLSD. / MSc Environmental Sciences, North-West University, Potchefstroom Campus, 2015

All members of the genus Xanthomonas are considered to be plant pathogenic, with specific pathovars infecting several high value agricultural crops. One of these pathovars, X. campestris pv. zeae (as this is only a proposed name it will further on be referred to as Xanthomonas BLSD) the causal agent of bacterial leaf steak of maize, has established itself as a widespread significant maize pathogen within South Africa. Insufficient information about the present distribution of the pathogen is available. The main aim of the study was thus to isolate and characterise the pathogen using molecular methods. Results demonstrated that the causal agent of bacterial leaf streak disease (Xanthomonas BLSD: potentially X. campestris pv. zeae) was widely distributed within the major maize cultivation regions of South Africa. Most of the isolates collected originated from the Highveld maize production provinces (North West, Free State, Gauteng and Mpumalanga provinces) as well as from irrigated maize fields in the Northern Cape province. The XgumD gene marker was used to determine if the isolates belonged to the genus Xanthomonas. The gumD gene fragment is located within the gumB-gumM region of the operon and is conserved among Xanthomonas species. This gene fragment is partially responsible for xanthan production. This marker was amplified from all isolates and a selected number were sequenced. The marker was only able to confirm that the causal agent was a member of the genus Xanthomonas. PCR methods were used for the characterisation of the isolates. This included PCR and sequencing of ribosomal RNA- gyraseB and gumD genes. A fingerprinting method BOX-PCR was also employed. Good quality DNA of sufficient quantities was obtained from the various isolates. Amplification produced no non-specific amplification products. This resulted in good quality sequences that could be analysed using bioinformatics tools. Phylogenetic analyses of the ribosomal RNA and gyraseB genes could not detect differences amongst the 47 Xanthomonas BLSD isolates. However, these genes were able to distinguish between the type strain of these isolates and various Xanthomonas species and pathovars. From all three neighbour joining trees the Xanthomonas BLSD isolates had close association with X. axonopodis pv. vasculorum strain ATCC 35938. For the 16S rRNA gene there exists no sequence differences between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. A single nucleotide difference was observed between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938 for the 23S rRNA gene. The gyraseB gene detected a total of six nucleotide variations between these two Xanthomonas species. For all of the phylogenetic trees there was no clustering of Xanthomonas BLSD with X. campestris pathovars. Genetic profiling (via BOX-PCR) based on present/absent analysis revealed no variations amongst the Xanthomonas BLSD isolates. All isolates shared an identical pattern produced by 12 distinct PCR products. This profiling technique did differentiate between the isolates of Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. Their profiles shared common bands, but differed in the number and overall pattern of the bands. These results suggest two main conclusions: (i) Xanthomonas BLSD has a clonal origin with geographical separation not impacting genetic variation. The fact that all the isolates appear to be clonal may imply that when resistant maize cultivars are developed these should be resistant to all isolates of the pathovar irrespective of their geographical origin. This is a suggestion that will have to be corroborated using more isolates and additional genetic fingerprinting techniques (ii) the Xanthomonas BLSD isolates from this study may not belong to X. campestris. Further studies using other markers should be conducted to determine the real identity of Xanthomonas BLSD. / MSc Environmental Sciences, North-West University, Potchefstroom Campus, 2015

About 70% of the plant species in the temperate zone are characterised by clonal growth, clonal species are also in majority in the Arctic and Subarctic where they affect the structure and composition of the vegetation. It is therefore of great importance to increase our knowledge about clonal plants and their growth and life histories. I have investigated how ramets of the stoloniferous plant Linnaea borealis are affected by the naturally occurring variation in environmental factors, such as: light, nutrient and water availability. Moreover, I examined the seed set and how supplemental hand pollination affects seed set in L. borealis, and also investigated the significance of the apical meristem for shoot population fitness. All studies were performed under field conditions in northern Sweden in a Subarctic environment and most are experimental. The results show that nutrient resorption from senescing leaves is not significantly affecting the growth and nutrient pools of the ramet. This implies that the growth of L. borealis ramets is not governed by micro-site resource availability. However, removal of light competition resulted in increased branching and number of lateral meristems produced, reduced growth, and decreased root:shoot ratio on a per ramet basis. Thus, ramets of L. borealis can efficiently exploit favourable light patches through plastic growth. Apical dominance exerts a significant effect on shoot population fitness and can be lost through rodent grazing. However, loss of apical dominance is dependent on the timing of grazing, if the apical meristem is removed early in the autumn the ramet can repair the loss until the next summer. If grazing occur during spring the dry weight and leaf area production is affected negatively. Seed production in L. borealis in the Abisko area varies between years and sites, and was unaffected by supplemental hand pollination treatment, implying that there is no lack of pollinator activity.

The aim of this study is to provide a further insight into influence of environment on clonal plants. The study focuses particularly on effects of fertilization level and light availability on production and growth of clonal organs. Three experiments were carried out within the study, targeted to elicit influence of these environmental conditions or clonal interactions on six species of clonal plants. Interspecies dependencies on these conditions was compared, regarding habitat occurrence of these species. A comparison was made also between species producing rhizomes and stolons. The experiments revealed that five of six studied species show significant relationship among at least one environmental condition and parameters of clonal reproduction. Most of the species showed higher elongation and production of clonal organs in relation to fertilization level. On the contrary, only three species reacted significantly to the light availability level by alternation of at least one parameter of clonal reproduction and the light availability level affected each species differently. A strong influence on production and elongation of clonal organs had also a size of a plant. There was not found significant difference in influence of environmental conditions on clonal reproduction among plant families. It...