京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13221号 / 論医博第2168号 / 新制||医||1033(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 松田 道行, 教授 小川 誠司, 教授 武藤 学 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Functional genetics is the study of the genes present in a genome of an organism, the complex interplay of all genes and their environment being the primary focus of study. The motivation for such studies is the premise that gene expression patterns in a cell are characteristic of its current state. The availability of the entire genome for many organisms now allows scientists unparalleled opportunities to characterize, classify, and manipulate genes or gene networks involved in metabolism, cellular differentiation, development, and disease. System-wide studies of biological systems have been made possible by the advent of high-throughput and large-scale tools such as microarrays which are capable of measuring the mRNA levels of all genes in a genome. Tools and methods for the integration, visualization, and modeling of the large-scale data obtained in typical systems biology experiments are indispensable. Our work focuses on a method that integrates gene expression values obtained from microarray experiments with biological functional information related to the genes measured in order to make global comparisons of multiple experiments. In our method, the integrated data is represented as a lattice and, using appropriate measures, a reference experiment can be compared to samples from a database of similar experiments, and a ranking of similarity is returned. In this work, support for the validity of our method is demonstrated both theoretically and empirically: a mathematical description of the lattice structure with respect to the integrated information is developed and the method is applied to data sets of both simulated and reported microarray experiments. A fast algorithm for constructing the lattice representation is also developed. / Ph. D.

Ph.D. / Medical Informatics and Clinical Epidemiology / Tools to automatically summarize gene information from the literature have the potential to help genomics researchers better interpret gene expression data and investigate biological pathways. Even though several useful human-curated databases of information about genes already exist, these have significant limitations. First, their construction requires intensive human labor. Second, curation of genes lags behind the rapid publication rate of new research and discoveries. Finally, most of the curated knowledge is limited to information on single genes. As such, most original and up-to-date knowledge on genes can only be found in the immense amount of unstructured, free text biomedical literature. Genomic researchers frequently encounter the task of finding information on sets of differentially expressed genes from the results of common highthroughput technologies like microarray experiments. However, finding information on a set of genes by manually searching and scanning the literature is a time-consuming and daunting task for scientists. For example, PubMed, the first choice of literature research for biologists, usually returns hundreds of references for a search on a single gene in reverse chronological order. Therefore, a tool to summarize the available textual information on genes could be a valuable tool for scientists. In this study, we adapted automatic summarization technologies to the biomedical domain to build a query-based, task-specific automatic summarizer of information on mouse genes studied in microarray experiments - mouse Gene Information Clustering and Summarization System (GICSS). GICSS first clusters a set of differentially expressed genes by Medical Subject Heading (MeSH), Gene Ontology (GO), and free text features into functionally similar groups;next it presents summaries for each gene as ranked sentences extracted from MEDLINE abstracts, with the ranking emphasizing the relation between genes, similarity to the function cluster it belongs to, and recency. GICSS is available as a web application with links to the PubMed (www.pubmed.gov) website for each extracted sentence. It integrates two related steps, functional gene clustering and gene information gathering, of the microarray data analysis process. The information from the clustering step was used to construct the context for summarization. The evaluation of the system was conducted with scientists who were analyzing their real microarray datasets. The evaluation results showed that GICSS can provide meaningful clusters for real users in the genomic research area. In addition, the results also indicated that presenting sentences in the abstract can provide more important information to the user than just showing the title in the default PubMed format. Both domain-specific and non-domain-specific terminologies contributed in the informative sentences selection. Summarization may serve as a useful tool to help scientists to access information at the time of microarray data analysis. Further research includes setting up the automatic update of MEDLINE records; extending and fine-tuning of the feature parameters for sentence scoring using the available evaluation data; and expanding GICSS to incorporate textual information from other species. Finally, dissemination and integration of GICSS into the current workflow of the microarray analysis process will help to make GICSS a truly useful tool for the targeted users, biomedical genomics researchers.

Prognostic models have clinical appeal to aid therapeutic decision making. In the UK, the Nottingham Prognostic Index (NPI) has been used, for over two decades, to inform patient management. However, it has been commented that NPI is not capable of identifying a subgroup of patients with a prognosis so good that adjuvant therapy with potential harmful side effects can be withheld safely. Tissue Microarray Analysis (TMA) now makes possible measurement of biological tissue microarray features of frozen biopsies from breast cancer tumours. These give an insight to the biology of tumour and hence could have the potential to enhance prognostic modelling. I therefore wished to investigate whether biomarkers can add value to clinical predictors to provide improved prognostic stratification in terms of Recurrence Free Survival (RFS). However, there are very many biomarkers that could be measured, they usually exhibit skewed distribution and missing values are common. The statistical issues raised are thus number of variables being tested, form of the association, imputation of missing data, and assessment of the stability and internal validity of the model. Therefore the specific aim of this study was to develop and to demonstrate performance of statistical modelling techniques that will be useful in circumstances where there is a surfeit of explanatory variables and missing data; in particular to achieve useful and parsimonious models while guarding against instability and overfitting. I also sought to identify a subgroup of patients with a prognosis so good that a decision can be made to avoid adjuvant therapy. I aimed to provide statistically robust answers to a set of clinical question and develop strategies to be used in such data sets that would be useful and acceptable to clinicians. A unique data set of 401 Estrogen Receptor positive (ER+) tamoxifen treated breast cancer patients with measurement for a large panel of biomarkers (72 in total) was available. Taking a statistical approach, I applied a multi-faceted screening process to select a limited set of potentially informative variables and to detect the appropriate form of the association, followed by multiple imputations of missing data and bootstrapping. In comparison with the NPI, the final joint model derived assigned patients into more appropriate risk groups (14% of recurred and 4% of non-recurred cases). The actuarial 7-year RFS rate for patients in the lowest risk quartile was 95% (95% C.I.: 89%, 100%). To evaluate an alternative approach, biological knowledge was incorporated into the process of model development. Model building began with the use of biological expertise to divide the variables into substantive biomarker sets on the basis of presumed role in the pathway to cancer progression. For each biomarker family, an informative and parsimonious index was generated by combining family variables, to be offered to the final model as intermediate predictor. In comparison with NPI, patients into more appropriate risk groups (21% of recurred and 11% of non-recurred patients). This model identified a low-risk group with 7-year RFS rate at 98% (95% C.I.: 96%, 100%).

Orientador: Júlio Lopes Sequeira / Banca: Renee Laufer Amorim / Banca: Sara Maria de Carvalho e Suzano / Resumo: Os linfomas não Hodgkin (LNHs) são as neoplasias hematopoiéticas mais comuns nos cães, sendo o Linfoma Difuso de Grandes Células B (DLBCL) o subtipo mais frequente. Os DLBCLs são neoplasias formadas por células linfoides B com padrão de crescimento difuso e podem apresentar pelo menos cinco variantes. O presente trabalho teve como objetivos traçar o perfil imunoistoquímico dos DLBCLs dos cães, classificá-los de acordo com os critérios estabelecidos pela WHO (2008), WHO veterinária (2002) e Classificação de Kiel e efetuar a avaliação dos anticorpos utilizados pelo método de microarranjo de tecido (TMA), comparando-o com o corte convencional. Foi avaliada a expressão imunoistoquímica de marcadores pan B (CD79a, CD20 e PAX-5); a determinação dos índices proliferativo (Ki-67) e apoptótico (caspase-3) e também da expressão de p53 mutante. Foram observados 24 linfomas centroblásticos monomórficos, 4 imunoblásticos B e 1 centroblástico polimórfico de acordo com a classificação de Kiel. Este perfil é de 25 Linfomas de Grandes Células B Difusos/ DLBCL, NOS variante centroblástica e 4 Imunoblásticos de Grandes Células /DLBCL, NOS variante imunoblástica, pela WHO de 2002 e 2008, respectivamente. Houve marcação em 100%, 75,8% e 58,6% dos casos para o CD79a, CD20 e PAX-5, respectivamente. A mediana de porcentagem de marcação para o Ki-67 e para a caspase-3 foi de 45,9% e 10%, respectivamente. Já a expressão da proteína p53 mutante foi verificada em 16 tumores (55,1%). A análise destes marcadores utilizando-se o TMA resultou em perfil imunofenotípico idêntico e medianas de caspase-3, Ki67 e p53 significativamente semelhantes quando comparadas com o corte convencional. Concluiu-se que em nossas amostras não houve diferença estatística entre os diferentes subtipos histológicos em relação aos índices proliferativo e apoptótico e expressão da p53. Ainda, o TMA é uma técnica adequada para avaliação do... / Abstract: Non Hodgkin lymphomas (LNHs) are the most common hematopoietic tumors of dogs, among which Diffuse Large B Cell Lymphoma (DLBCL) is the most frequent subtype. DLBCLs are tumors composed of lymphoid B cell with a diffuse growth pattern and may present at least five variants. The present work intended to delineate the immunohistochemical profile of canine DLBCL, classify them according to the criteria proposed by WHO (2008); veterinary WHO (2002) and Updated Kiel Classification and also perform the evaluation of the used antibodies on tissue microarray (TMA) method in comparison to conventional histological sections. The immunohistochemical expression of pan B markers (anti CD79a, anti CD20 and PAX-5) was assessed; as well as the determination of proliferation index (Ki-67) and apoptosis (caspase 3) and also the expression of the mutant p53. There were 24 monomorphic centroblastic lymphomas, 4 imunoblastic B and 1 polimorphic centroblastic according to Kiel. That profile is 25 Diffuse Large B Cell Lymphoma/ DLBCL, NOS centroblastic variant and 4 Large Cell Imunoblastic/ DLBCL, NOS imunoblastic variant according to WHO 2002 and 2008 respectively. Immunolabeling was seen in 100%, 75.8% and 58.6% of the cases for CD79a, CD20 and PAX-5 respectively. The immunolabeling median percentage of Ki67 was 45.9% and for caspase-3 it was 10%. The expression of mutant p53 was observed in 16 tumors (55.1%). The analysis of those markers using TMA resulted in identical imunophenotype and significantly similar medians of caspase-3, Ki67 e p53 when compared to conventional sections. In conclusion there was no statistical difference among the histological subtypes regarding proliferation and apoptotic indexes and p53 expression in our samples. Also, TMA is an adequate technique for evaluating imunophenotype, proliferation and apoptotic indexes as well as presence of mutant p53 / Mestre

The development of microarray technology has provided an unprecedented wealth of data on gene expression in various tissue and cell types. Few studies have, however, taken full advantage of these data by selecting and then extensively characterising the functions of particular genes chosen from these microarray datasets. In this study, after analysing differentially-regulated genes revealed by microarray analysis of human mast cells activated via Fc??RI cross-linking, we chose two promising gene candidates for further research, A20 and Gem. Our group??s extensive gene expression database of major leukocytes showed that both A20 and Gem were up-regulated in other leukocyte types, and yet neither of these genes has been extensively explored in mast cells or in the immune system prior to our study. In order to investigate the first of these genes selected for further study, A20, we utilised both A20-deficient mast cells and mast cells in which A20 was over-expressed. Our findings establish for the first time that A20 is an important regulator of mast cell inflammatory responses to both LPS and Fc??RI cross-linking, and that it plays a novel role in mast cell proliferation. Our study of the second gene chosen for investigation, Gem, was conducted in a Gemdeficient mouse model developed by our group. In this study, we investigated the effect of Gem deficiency in two key immune cell types, macrophages and T-cells, complementing the work of a previous group member who investigated Gem deficiency in mast cells. Our results clearly exclude a role for Gem in macrophage and T-cell effector responses, and further establish that Gem is dispensable for in vivo inflammatory responses in models of delayed-type hypersensitivity and allergic airway inflammation. In addition to these findings, and given that the physiological role of Gem was not yet understood prior to our study, we extended our investigation to explore a potential function for Gem in the metabolic system. Using Gem-deficient mice, we found that Gem is necessary for insulin secretion from pancreatic islets. These findings confirm the potential for microarray expression data to reveal excellent gene candidates for further research and functional characterisation.

Schizophrenia is a devastating psychiatric disorder that affects approximately 1% of the population. The main treatments for schizophrenia are antipsychotic drugs that target dopamine receptors, yet the underlying biological mechanisms through which they alleviate the symptoms of schizophrenia remain ill defined. In this study, we used microarray analysis to profile the expression changes of thousands of genes simultaneously, following antipsychotic drug treatment of mice. Mice were treated chronically (28 days), or for a novel intermediate time-point (7 days), with one of three antipsychotic drugs: clozapine, haloperidol or olanzapine. The use of three drugs enabled us to discern antipsychotic-specific effects co-regulated by multiple drugs, rather than the side effects of individual compounds. Transcript profiling and validation by quantitative PCR of whole brain tissue revealed antipsychotic drug regulation of genes in diverse biological pathways, including: dopamine metabolism, neuropeptide and second-messenger signalling, neurogenesis, synaptic plasticity, cell adhesion, myelination, and voltage-gated ion channels. The regulation of voltage-gated channels by antipsychotic drugs has been suggested previously by electrophysiological studies, although thorough analysis has not been undertaken in vivo. Therefore, the second aim of this study was to characterise the regional mRNA and protein expression of two genes altered by multiple APDs, the voltage-gated potassium channel ??-subunit (Kcna1) and voltage-gated potassium channel interacting protein (Kchip3). Regional characterisation and expression analyses were carried out by immunohistochemistry, in situ hybridisation, and Western blot analysis of mouse brain regions of interest to schizophrenia and its treatment. Following 7-day haloperidol treatment we observed up-regulation of Kcna1 in the striatum and dentate gyrus, with increased protein in the striatum, hippocampus and midbrain; and down-regulation of Kchip3 in the striatum, with decreased protein in the cortex, hippocampus and midbrain. These studies implicate voltage-gated potassium channels in the antipsychotic drug regulation of midbrain dopaminergic neuronal activity, adult neurogenesis and/or striatothalamic GABAergic neuronal inhibition. These findings indicate that regulation of potassium channels may underlie some of the mechanisms of action of antipsychotic drugs, and that voltage-gated ion channels may provide alternative drug targets for the treatment of schizophrenia.

Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events. The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to twoin vitromodel systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice usingin vivotissue samples as starting material. A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results. Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis. <b>Keywords:</b>representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation

Bartonella, a group of Gram negative facultative intracellular bacteria, are known to cause diseases, such as Cat Scratch Disease, Trench Fever and Carrion’s Disease, that involve angiogenesis during the infective cycle. B. bacilliformis, the etiological agent of Carrion’s Disease, causes a bi-phasic infection resulting in the formation of blood-filled angiogenic proliferative cutaneous nodules called verruga peruana. The work presented here was undertaken to characterize the mechanism by which these nodules are produced. Previous work in our laboratory suggested that the Bartonella henselae genome contains a homologue to the virB operon, a set of genes coding for a Type IV Secretion System (TFSS) that has been implicated in the pathogenesis of other α-2-proteobacteria. We identified virB operons in two additional Bartonella pathogens, B. quintana and B. clarridgeiae. No corresponding operon sequences were detected in B. bacilliformis DNA, however. This finding suggests that virB gene products are not required for verruga peruana formation. To continue our search for factors involved in B. bacilliformis-induced angiogenesis, we conducted a microarray analysis of differential gene expression in infected and uninfected endothelial cells. The results suggest similarities between later stage (36 hours) B. bacilliformis infection and that of HHV-8, the causative agent of Kaposi’s Sarcoma, particularly in relation to the host immune response. Finally, our research focused on the secreted factors that B. bacilliformis produces during its host infective cycle. Our data suggest that the B. bacilliformis homologue to the molecular chaperone GroEL not only induces angiogenesis in endothelial cells, but also protects endothelial cell tubule from the degradation seen when these cells are in the presence of live B. bacilliformis. In summary, the induction of verruga peruana nodules via B. bacilliformis may be the result of multiple factors over the course of persistent infection. Early infection may cause vascular damage, which induces VEGF and hypoxia factors. As infection persists, bacterial secretion of a unique GroEL may result in continued angiogenesis and the ensuing activation of immune cells, producing a localized environment of continual incomplete angiogenesis in areas of cutaneous infection.

The genus Bartonella comprises emerging pathogens that are causative agents of a wide range of clinical manifestations such as cat scratch disease, bacillary angiomatosis, and Carrion’s disease. All species are transmitted by blood-sucking arthropods and infect erythrocytes and endothelial cells of hosts. Carrion’s disease is a bi-phasic infection caused by Bartonella bacilliformis which is characterized by hemolysis of infected erythrocytes followed by invasion of the vascular endothelium. This provokes pronounced cellular proliferation, angiogenesis and skin eruptions called verruga peruana. Endothelial cells are thought to be the primary niche wherein bacteria reside between inoculation and erythrocyte infection. This study aims to elucidate some of the endothelial factors involved during the verruga peruana phase of Carrion’s disease. In order to adhere to and invade human microvascular endothelial cells (HMEC-1), B. bacilliformis engages a family of cell receptors called integrins. We used anti-integrin antibodies to show that the primary integrin involved is the fibronectin receptor á5â1, although the vitronectin receptor áVâ3 also plays a minor role. We show B. bacilliformis invasion is also dependent on integrin ligands, fibronectin and vitronectin as antibodies against these proteins decreased invasion and attachment, whereas pre-treatment of the bacteria with these molecules enhanced infection of endothelial cells. Bacterial uptake requires various host cytoplasmic signaling pathways to work in tandem, and our study identified three mitogen activated protein kinases involved. Apart from MAPKs, phosphotidylinositol 3 kinase plays a role during invasion and cell survival. PI3K inhibitors blocked bacterial internalization and B. bacilliformis infected cells showed accelerated apoptosis. Lastly, microarray analysis was performed to study the gene expression profile of B. bacilliformis infected HMEC-1 cells. Numerous molecules of the integrin signaling pathways are involved, suggesting integrins as the major receptor recruited for the successful infection by B. bacilliformis. In summary this is the first study to demonstrate the role of integrins as B. bacilliformis receptors and integrin ligands as facilitators of infection. Gene expression analysis suggests the possibility that integrin mediated signaling pathways are the key modulators of cellular alterations during B. bacilliformis infection. This hypothesis is supported by the identification of some members of the integrin signaling pathway necessary for B. bacilliformis entry into endothelial cells.