Le cancer du sein est une pathologie hétérogène au plan clinique et au moins 5 sous-types moléculaires ont pu être définis sur la base de différences d’expression ARNm. Ces sous-types présentent des différences de profils d’anomalies génomiques et de méthylation des cytosines. Ces différences génétiques et épigénétiques s’expliqueraient par des types cellulaires d’origines distincts au sein de l’épithélium mammaire, toutefois, ceci n’a pas été confirmé clairement à ce jour. Alternativement, il a été proposé que l’activation de voies oncogéniques différentes pouvait avoir un impact significatif sur les modifications génétiques ou épigénétiques. Dans ce travail nous avons voulu vérifier cette hypothèse en l’appliquant à un modèle de cellules épithéliales mammaires normales primaires humaines, que nous avons isolé des à partir de glandes mammaires. Ces cellules ont été transformées en deux étapes par transduction avec (i) un shARN ciblant TP53, (ii) un oncogène. Nous avons sélectionné 3 oncogènes qui activent des voies de signalisations distinctes CCNE1, HRAS-v12 et WNT1. Nous avons établi un modèle de transformation tumorale en trois étapes, cellules normales, immortalisées et transformées, permettant de suivre les modifications moléculaires associées à chaque étape et de vérifier si l’activation de voies oncogéniques distinctes produisait des profils d’anomalies différents. Les différents modèles ont été analysés par CGH-array, RRBS, transcriptome et miRNA à des temps de culture définis.Nos résultats montrent que l’activation de la voie RAS aboutit à des profils d’anomalies génétiques et de méthylation des CpG radicalement différents de ceux obtenus après surexpression des gènes CCNE1 et WNT1. Ces différences apparaissent très rapidement après transduction des oncogènes alors que les profils des cellules CCNE1 et WNT1 divergent plus tardivement. Enfin, l’inactivation de p53 n’induit pas par elle-même une instabilité élevée, mais produit un contexte de plasticité favorable aux modifications génétiques et épigénétique.Par ailleurs, nous avons noté des différences phénotypiques entre les HMEC RAS (mésenchymateuses) et les HMEC CCNE1 et les HMEC WNT1 (épithéliales). Dans ce travail, je montre que les HMEC shp53 immortalisées présentent une plasticité phénotypique, une partie des cellules entrant en EMT spontanément, l’autre restant épithéliales. J’ai montré que la transduction RAS sélectionnait les cellules ayant effectué une EMT, alors que la transduction de CCNE1 ou WNT1 sélectionnait les cellules épithéliales. J’ai cherché à identifier les déterminants de ces changements phénotypiques et mes résultats suggèrent qu’ils résultent d’une balance entre une signalisation TGFB1/BMP1, qui favorise l’EMT, et BMP4/WNT7 qui favorise la MET. / Breast cancer is a heterogeneous pathology. Based on the differences of mRNA expression, at least five molecular subtypes have been defined. These subtypes show differences in profiles of genomic abnormalities and CpG methylation. These molecular subtypes are thought to originate from different cell lineages in the mammary gland. However, this has not yet been clearly demonstrated. Alternatively, it has been proposed that the activation of different oncogenic pathways could have a significant impact on genetic or epigenetic changes.We wanted to verify this hypothesis by applying it to a normal primary human mammary epithelial cells (HMEC) model, which we isolated from normal mammary explants. These cells were transformed in two step process by sequential transduction of (i) a shRNA targeting TP53, (ii) an oncogene. We selected 3 oncogenes that activate distinct signaling pathways CCNE1, HRAS-v12 and WNT1. Our tumor transformation model was established in three-step, normal, immortalized and transformed cells, allowing us to monitor the molecular changes associated with each step and to verify whether the activation of distinct oncogenic pathways produced different profiles of genetic and epigenetic modifications. The different models were analyzed at defined culture times by CGH-array, RRBS, transcriptome and miRNA. Our results show that genetic abnormalities and CpG methylation profiles are different between cells where the RAS pathway was activated and cells overexpressing WNT1 or CCNE1. These differences appear rapidly after oncogene transduction, whereas the profiles of the CCNE1 and WNT1 cells diverged later. Finally, inactivation of p53 by itself does not induce high instability, but produces a context of plasticity favorable to genetic and epigenetic changes.In addition, we noted phenotypic differences between HMEC RAS (mesenchymal) and HMEC CCNE1 and HMEC WNT1 (epithelial). In this work, I show that the immortalized HMEC shp53 exhibit a phenotypic plasticity, where some cells enter a spontaneous EMT and the others remain epithelial. I showed that RAS transduction selected cells that are undergoing an EMT, whereas transduction with CCNE1 or WNT1 selected the epithelial cells. I have sought to identify the determinants of these phenotypic changes and my results suggest that a balance exists between TGFβ1 / BMP1 signaling, which promotes EMT, and BMP4 / WNT7a which promotes TEM.

The aim of this study was to develop and examine the validity of using a phenotype scoring system (PTS), a new concept, in evaluating milk yield and constituents in different goat genotypes (Indigenous, British Alpines, Saanen and Toggenburg) raised in small scale production systems. Strategic decisions of small scale African farmers are mostly based on visual appraisal or body condition scoring (BCS) of their animals. BCS has been highly recommended as a means to evaluate both the energy and the health status of animals, especially in beef farming, but this method has been criticized for being too simple and too subjective because its evaluation is often done too late after the damage has already happened. Phenotype scoring (an approach which includes breed, udder size and BCS of the animal) is presented in this study as a better tool to evaluate milk yield in different goat genotypes raised under free range conditions. This has also been a good opportunity firstly to indicate which, among the three dairy breeds of goat under discussion, can adapt best to the African small scale farming system; secondly to review the relevance of some blood metabolites in characterizing milk production in different goat breeds and thirdly to study the milking capacity of the indigenous compared to the dairy goats raised under small scale production systems in South Africa. Thirty-two goats (8 Indigenous, 8 British Alpines, 8 Saanen and 8 Toggenburg) were raised in a free range system at the ARC-Irene experimental farm close to Pretoria. The experiment was a completely randomized experimental design with eight replicates per treatment group. Blood samples were collected by jugular venipuncture into 10 ml heparinised tubes in the morning before feeding on a weekly basis over a period of two months. Blood plasma was immediately aspirated after centrifugation (3000G), kept on ice and brought to the laboratory for the analysis of glucose, cholesterol, urea nitrogen (BUN) and free fatty acid (FFA) concentrations. Immediately after, all does were entirely milked (followed by 1ml oxytocin IM injection and the kids taken away for a period of four hours) before a second milking session took place to measure the daily milk yield of the does. Milk samples were analyzed for lactose, milk proteins, milk fat, milk urea-nitrogen (MUN) and milk somatic cell count (SCC). In addition body condition score (BCS), age and data related to the goat genotype (breed, udder characteristics) were recorded. Results confirmed that milk yield from dairy goats was higher (p<0.001) than the milk yield of indigenous goats during the entire period of study. Milk lactose values recorded in this study (between 3.9 and 4.9%) were the most stable constituent in goat’s milk. Milk protein concentration (between 3.1 and 4.5%) was significantly higher in the indigenous than in dairy breeds, especially in week one and from week four onwards. Milk fat values (between 3.3 and 7.7%) displayed a decline in all breeds; but as from week three, the fat in milk of indigenous does increased and from week five onwards, it remained significantly higher (p<0.001) compared to that of dairy breeds. In conclusion, the superiority of dairy breeds in milk yield was proven while the quality of indigenous goat milk was recognized. Studies on the characterization of milk production in different breeds revealed that the Toggenburg was superior to the other breeds, followed by the British Alpines and the Saanen; but the British Alpines showed a better adaptability to the environment followed by the Toggenburg and the Saanen. The latter could not produce milk without feed supplementation and lost most body condition as compared to the other breeds. Statistical analyses indicated that breed influenced milk yield, milk fat and the protein content of milk (especially in the Saanen and Toggenburg goats). BCS influenced fat content, lactose, milk proteins, MUN and SCC and also milk yield. Udder size influenced milk proteins and milk yield while udder attachment was associated with milk yield only. These results show that PTS, because it takes into account BCS, breed and udder size, is a better tool for predicting milk yield of goats herded in small scale farming systems. Africans interested in dairy goat farming should adopt PTS as a means to evaluate milk yield especially since milk is sold per volume and not by quality in Africa. Finally, milk from the indigenous goats is superior in terms of lactose, fat and protein content. The latter quality attributes can be used as selection criteria since the milk industry pays premiums for the fat and protein content of milk. / Thesis (PhD)--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted

When provided with spatial niches by growth in static nutrient medium, Pseudomonas fluorescens diversifies through adaptive radiation into several well-defined phenotype classes. One of these classes, named wrinkly spreader (WS) for its morphology on agar medium, forms a biofilm at the air-liquid interface through mutations in one of several loci including the genes wspF and awsX. These genes code for negative regulators of di-guanylate cyclases (DGCs). These DGCs catalyse synthesis of cyclic-di-GMP, a second messenger, overproduction of which effects physiological changes leading to overproduction of a cellulose polymer and the WS phenotype. Intriguingly, a diverse range of wspF mutations leads to diversity both in colony morphology and strain fitness.In this study, I investigate genetic and fitness diversity in wrinkly spreaders with the aim of identifying the causal factors that link genetic diversity and physiological factors with diversity in fitness. I approach the subject from several directions, examining the historical context of genetic diversity in wspF and awsX, distribution of control over output in the Wsp pathway and overall fitness effects of different causal factors. I investigate the genetic basis of wrinkly spreader evolution through generation of a large number of novel WS strains and exploration of the distribution of mutations in the wspF and awsX genes. In combination with this I calculate estimates of the past rates of mutation in these genes, derived from a phylogenetic investigation of a group of orthologues. I examine the response of the Wsp pathway to change in WspF function through a novel computational analysis that is capable of revealing valuable information on control in a biological system based purely on model structure. In addition I show how this analysis can be refined through specification of broad estimates of system parameters, thereby avoiding issues related to over-reliance on specific parameter values. Finally, I investigate the fitness implications of these factors, as well as a variety of others, through assays of fitness in a group of WS strains combined with machine learning analyses of predictive relationships between protein and mutation characteristics and experimentally measured strain fitness, and consider the implications of this analysis in the context of intermediate physiological effects.I find that mutations in the WspF protein that lead to the WS phenotype tend to be located in regions of historically strong conservation, the first time that any such pattern to WS mutations has been identified. Mutations in AwsX, on the other hand, do not fit such a pattern. Computational analysis of the Wsp pathway shows that, regardless of model parameters, pathway output is always more sensitive to changes in methylesterase activity by WspF than to changes in phosphorylation of WspF, which may explain the greater frequency of mutations fixed in vivo seen in the methylesterase domain. Despite these patterns, none of a wide range of mutation and sequence-based biochemical characteristics, including local rates of past evolution and size and position of mutations, exhibited any predictive power over WS fitness. Overall, the findings in this study point towards an essential role for complex pleiotropic effects in strongly modulating the fitness effect of different mutations in wspF.

Les antigènes de groupes sanguins érythrocytaires peuvent être responsables d’une allo-immunisation anti-érythrocytaire et d’accidents immuno-hémolytiques lors de transfusion ou de grossesse. La transfusion des populations d’Afrique sub-saharienne est complexifiée par l’absence d’expression d’antigènes publics, l’expression d’antigènes privés et l’expression d’antigènes partiels en particulier pour le système RH. L’étude des systèmes de groupes sanguins d’intérêt transfusionnel chez les donneurs de Bamako a confirmé l’efficacité de la stratégie du génotypage multiplex incluant des polymorphismes d’appels pour identifier des donneurs rares, qui permet d’accéder aux phénotypes déduits des prélèvements. L’exploration du système RH réalisée par séquençage chez les Dogons et les Peulhs de Mopti met clairement en évidence que la diversité allélique et la fréquence de certains allèles RH sont fonction de l’ethnicité. Un nouvel haplotype associant un allèle RHD*DIVa codant pour un antigène D partiel, des antigènes ce potentiellement partiels, et une réactivité partielle anti-C, a été identifié chez les Dogons. L’exploration des allèles codant pour les antigènes de haute et basse fréquence en Afrique subsaharienne d’Est en Ouest constitue un exemple d’étude qui distingue clairement les populations d’Afrique subsaharienne de celles d’Europe par des différences de fréquences des allèles définissant la diversité génétique d’une population par rapport à une autre. Des orientations stratégiques en fonction du contexte local ont été identifiées pour l’évolution de la transfusion au Mali dans les prochaines années. / Blood group antigens may be responsible for alloimmunization and immuno-hemolytic accidents during transfusion or pregnancy. The transfusion of of sub-Saharan Africa populations is complex due to absence of high antigens expression, low antigens expression and partial antigens expression particularly for RH system.The study of blood group for transfusion of interest among donors in Bamako confirmed the effectiveness of multiplex genotyping strategy including polymorphisms calls to identify the rare donors, which permit access to phenotypes derived samples. In a second phase, the exploration of RH blood group system by sequencing among Dogon and Fulani in Mopti clearly shows that the allelic diversity and the frequency of some alleles RH depend on the ethnicity. A new haplotype RHD*DIVa/RHCE*ceTI(D2) combining an RHD*DIVa allele encoding a partial D antigen, potentially partial ce antigens, and a partial reactivity with anti-C, was identified among Dogon. In a third phase, the exploration of alleles encoding of the high and low frequency antigens in sub-Saharan Africa from East to West is an example of a study that clearly makes a difference between the populations of sub-Saharan African and those of Europe in terms of frequencies of alleles that define genetic diversity of one population compared to another. Thus, knowledge of ethnicity is more relevant than knowing the geographical origin in order to optimize transfusion in Saharan Africa and in European countries where some of these populations live. Guidelines strategic in relation with the local context have been identified for development of transfusion for next years in Mali.

Back pain is a highly prevalent condition whose pathogenesis is associated with intervertebral disc (IVD) degeneration. Degeneration is driven by abnormal cell biology, particularly within the IVD’s inner core, the nucleus pulposus (NP). In recent years, there has been an ever-increasing search for cell-based therapies aimed at correcting the cell biology and thus repairing/regenerating the degenerate IVD. The success of these novel therapies, however, requires a thorough understanding of IVD development and of the phenotype of its cells. The embryonic, foetal and juvenile NP is populated by large vacuolated notochordal cells that with skeletal maturity are replaced by smaller NP cells. Since notochordal cells have been shown to display protective and anabolic roles in the IVD their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of notochordal cells and their regulatory pathways may help identify factors involved in IVD homeostasis and aid the development of novel cell-based therapies targeting IVD degeneration. The study of human notochordal cells has, however, been hindered by ethical, logistical and technical difficulties in obtaining suitable samples and, as such, the human notochordal cell phenotype is, to date, unknown, constituting a major limitation in the field. The work presented here was conducted with the objective of developing a methodology to isolate human developing notochordal cells (NP progenitors) from adjacent sclerotomal cells (annulus fibrosus and vertebral body progenitors), to characterise the notochordal cell phenotype and identify potential factors involved in notochordal cell biology. Initially, human embryonic and foetal spines were characterised to assess their suitability as a source of notochordal cells and to identify a notochord-specific marker that could be used to isolate notochordal cells for microarray studies. The human developing spine contained large vacuolated notochordal cells in all stages analysed (3.5-18 weeks post-conception (WPC)) that specifically expressed KRT8, KRT18 and KRT19 at all stages and CD24 between 5.5-18 WPC. KRT18 and CD24 were independently used to label notochordal cells (7.5-14 weeks post-conception) and separate them from sclerotomal cells. Methodologies were developed to allow extraction of RNA of sufficient quality for microarray analysis from fixed, permeabilised (in the case of KRT18) and/or, labelled and sorted cells (CD24). Microarray analysis identified and real-time qPCR and, for some markers, immunohistochemistry, validated GRB14, SLC19A1, FGF10, ADORA3, TBXA2R, CDH6, ANPEP, CD69, CD24, RTN1, PRPH, MAP1B, ISL1 and CLDN1 as human notochordal cell markers. Ingenuity pathway analysis was performed to investigate the pathways/networks and upstream regulators and downstream effectors of notochordal cells. Inhibition of inflammation and angiogenesis were identified as relevant to notochordal cell biology, function and, possibly, to the known protective and anabolic role notochordal cells display in the IVD. Notochordal marker gene expression was identified in adult NP tissue, and negatively correlated with degeneration. Proteins encoded by ADORA3 and MAP1B were expressed by a proportion of adult NP cells, suggesting the presence of notochord-derived cells in the adult NP.Importantly, this is the first study to detail a methodology and successfully isolate human notochordal cells. Such methodology has the potential to be used to culture and investigate the biology of viable human notochordal cells (CD24+ve). Future studies aimed at developing cell-based therapies for IVD degeneration could also use these identified markers to assess appropriate stem cell differentiation to notochordal cells.

Orientador: André Santanchè / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Computação / Made available in DSpace on 2018-08-24T12:38:30Z (GMT). No. of bitstreams: 1 Miranda_EduardodePaula_M.pdf: 3021722 bytes, checksum: 93a67943f673753c003a021060a55b6c (MD5) Previous issue date: 2013 / Resumo: Um grande número de estudos em biologia, incluindo os que envolvem a reconstrução de árvores filogenéticas, resultam na produção de uma enorme quantidade de dados -- por exemplo, descrições fenotípicas , matrizes de dados morfológicos , árvores filogenéticas, etc. Biólogos enfrentam cada vez mais o desafio e a oportunidade de efetivamente descobrir conhecimento a partir do cruzamento e comparação de vários conjuntos de dados, nem sempre conectados e integrados. Neste trabalho, estamos interessados em um contexto específico da biologia em que biólogos aplicam ferramentas computacionais para construir e compartilhar descrições digitais dos seres vivos. Nós propomos um processo que parte de fontes de dados fragmentadas, que nós mapeamos para grafos, em direção a uma plena integração das descrições através de ontologias. Os bancos de dados de grafos intermediam o processo de evolução. Eles são menos dependentes de esquema e, uma vez que ontologias também são grafos, o processo de mapeamento do grafo inicial para uma ontologia torna-se uma sequência de transformações no grafo. Nossa motivação parte da ideia de que a conversão de descrições fenotípicas em uma rede de relações e a busca de conexões entre elementos relacionados irá aumentar a capacidade de resolver problemas mais complexos suportados por computadores. Este trabalho detalha os princípios de concepção por trás do nosso processo e duas implementações práticas como prova de conceito / Abstract: A large number of studies in biology, including those involving phylogenetic trees reconstruction, result in the production of a huge amount of data -- e.g., phenotype descriptions, morphological data matrices, phylogenetic trees, etc. Biologists increasingly face a challenge and opportunity of effectively discovering useful knowledge crossing and comparing several pieces of information, not always linked and integrated. In this work, we are interested in a specific biology context, in which biologists apply computational tools to build and share digital descriptions of living beings. We propose a process that departs from fragmentary data sources, which we map to graphs, towards a full integration of descriptions through ontologies. Graph databases mediate this evolvement process. They are less schema dependent and, since an ontology is also a graph, the mapping process from the initial graph towards an ontology becomes a sequence of graph transformations. Our motivation stems from the idea that transforming phenotypical descriptions in a network of relationships and looking for links among related elements will enhance the ability of solving more complex problems supported by machines. This work details the design principles behind our process and two practical implementations as proof of concept / Mestrado / Ciência da Computação / Mestre em Ciência da Computação

A Síndrome de Marfan (SMF) é uma doença de caráter autossômico dominante que acomete o tecido conjuntivo. As principais manifestações clínicas afetam o sistema cardiovascular, ótico e ósseo. A SMF é causada por mutações no gene FBN1, que codifica a proteína extracelular fibrilina-1, componente principal das microfibrilas, que formam as fibras elásticas. Estudos mostraram que mutações no gene da fibrilina-1 levam a um aumento indiscriminado do TGF-&Beta; ativo na matriz, que resulta nos principais fenótipos da doença. O Sistema Renina Angiotensina (RAS) tem como produto principal a Angiotensina II (Ang-II), envolvida na regulação da massa óssea e da atividade do TGF-&Beta;. Estratégias terapêuticas para a SMF utilizando fármacos que agem no RAS têm sido alvos de estudos em modelos animais. Em camundongos, Ramipril, inibidor da ACE (Angiotensin-converting enzyme inhibitor, ACEi), aumenta a transcrição do gene Fbn1 em 35% e melhora a cifose, característica do fenótipo ósseo no modelo animal mg&Delta;loxPneo. O mecanismo de ação do Ramipril no sistema ósseo ainda não está totalmente elucidado, sendo que pode agir por diminuição na produção de Ang-II e consequente diminuição nos níveis de TGF-&Beta;, ou pela inibição de degradação da bradicinina (BK) pela Ang-II. A bradicinina ativa diretamente seu receptor B2R, que induz ações fisiológicas opostas às da Ang-II. O objetivo deste trabalho foi avaliar e compreender os mecanismos gerais da patogênese óssea do modelo murino mg&Delta;loxPneo para a SMF. Para tanto, foi analisado o fenótipo ósseo dos animais mg&Delta;loxPneo e selvagens controle e tratados com Ramipril. Foi verificado que, além da cifose, os animais mutantes apresentaram pior estrutura óssea. O tratamento melhorou a cifose, porém não alterou a qualidade óssea dos animais mutantes. Portanto, o efeito benéfico do Ramipril na cifose dos animais mg&Delta;loxPneo não se deve a uma melhora da estrutura óssea, e pode estar relacionado à integridade do ligamento que sustenta a coluna vertebral. Com o intuito de testar a hipótese de que a sinalização pelo receptor B2R da BK possa estar envolvida no desenvolvimento do fenótipo ósseo dos animais mg&Delta;loxPneo, foram gerados animais mg&Delta;loxPneo e knockout para o receptor B2R. Os resultados mostram que o receptor B2R não interfere no desenvolvimento da cifose, sendo apenas o genótipo para Fbn1 o fator determinante para a manifestação desse fenótipo. Foi realizada a análise de RNA-seq para verificar genes e vias diferencialmente expressas que possam explicar o mecanismo de desenvolvimento do fenótipo ósseo dos animais mg&Delta;loxPneo. Foram encontradas vias como da adesão focal, interação receptor-meio extracelular (MEC), junção ocludente, reparação por excisão de nucleotídeo e de reparação missmatch, que podem explicar alterações no metabolismo de células ósseas. Além disso, foram encontradas diferenças de expressão de genes relacionados ao metabolismo muscular esquelético, o que está de acordo com a hipótese de regulação parácrina entre o tecido muscular e ósseo, levando a uma pior estrutura óssea / Marfan syndrome (MFS) is an autosomal dominant disease that affects the connective tissue. The main clinical manifestations affect the cardiovascular, optical and bone systems. MFS is caused by mutations in the FBN1 gene, that encodes the extracellular protein fibrillin-1, a major component of microfibrils, which form elastic fibers. Studies have shown that mutations in the fibrillin-1 gene lead to an indiscriminate increase in active TGF-&Beta; in the matrix, which results in the major phenotypes of the disease. The Renin Angiotensin System (RAS) has as its main product Angiotensin II (Ang-II), involved in bone mass regulation and TGF-&Beta; activity. Therapeutic strategies using drugs targeting the RAS have been studied in animal models. Ramipril, an ACE inhibitor (Angiotensin-converting enzyme inhibitor, ACEi), increase Fbn1 gene expression in 35% and improve kyphosis index in the mg&Delta;loxPneo mouse model for SMF. Its mechanism of action in bone tissue is not completely elucidated, and it may act by decreasing Ang-II production and consequent reduction in TGF-&Beta; levels, or by inhibiting degradation of bradykinin (BK) by Ang II. BK directly activates its B2R receptor, which induces opposite physiological actions to Ang-II. This study aims to evaluate and understand the general mechanisms of bone pathogenesis in the mg&Delta;loxPneo mouse model. We analyzed the bone phenotype of mg&Delta;loxPneo and wildtype animals treated, or not, with Ramipril by measuring the kyphosis index (KI), micro computed tomography (&mu;CT) and Real-time PCR (RT-PCR). We found that mutant animals showed a greater degree of kyphosis and an altered bone structure. Ramipril improved kyphosis but did not alter bone quality of mutant animals, while in wild type animals Ramipril decreased bone structure without altering KI. Therefore, the beneficial effect of Ramipril on mg&Delta;loxPneo animals\' kyphosis is not due to an improvement in bone structure. In order to test the hypothesis where signaling through BK B2R receptor may be involved in the development of bone phenotype of mg&Delta;loxPneo animals, a mouse model with the mg&Delta;loxPneo mutation and knockout for B2R receptor was generated. The analysis of these animals show that the B2R receptor does not interfere with the development of kyphosis, with Fbn1 genotype as sole determinant for this phenotype manifestation. RNA-seq analysis was performed to verify differential expression of genes and altered cellular pathways, which could reveal mechanisms of bone phenotype development in mg&Delta;loxPneo animals. Altered pathways found included focal adhesion, receptor- extracellular matrix (ECM) interaction, tight junction, nucleotide excision repair and missmatch repair, which may explain changes in bone cells metabolism. In addition, there were differences in gene expression related to skeletal muscle metabolism, which is in agreement with the paracrine regulation of bone and muscle tissue, leading to worst bone structure

Ample studies have shown that perturbation of metabolic phenotype is correlated with gene mutation and pathogenesis of colorectal cancer (CRC) and rheumatoid arthritis (RA). Mass spectrometry (MS)-based metabolomics as a powerful and stable approach is widely applied to bridge the gap from genotype/metabolites to phenotype. In CRC suffers, KRAS mutation accounts for 35%-45%. In previous study, SLC25A22 that encodes the mitochondrial glutamate transporter was found to be overexpressed in CRC tumor and thus to be essential for the proliferation of CRC cells harboring KRAS mutations. However, the role of SLC25A22 on metabolic regulation in KRAS-mutant CRC cells has not been comprehensively characterized. We performed non-targeted metabolomics, targeted metabolomics and isotope kinetic analysis of KRAS-mutant DLD1 cells with or without SLC25A22 knockdown using ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap MS and tandem MS (MS/MS). In global metabolomics analysis, 35 differentially regulated metabolites were identified, which were primarily involved in alanine, aspartate and glutamate metabolism, urea cycle and polyamine metabolism. Then targeted metabolomics analysis on intracellular metabolites, including tricarboxylic acid (TCA) cycle intermediates, amino acids and polyamines, was established by using LC-MS/MS coupled with an Amide BEH column. Targeted metabolomics analysis revealed that most TCA cycle intermediates, aspartate (Asp)-derived asparagine, alanine and ornithine (Orn)-derived polyamines were strongly down-regulated in SLC25A22 knockdown cells. Moreover, the targeted kinetic isotope analysis using [U-13C5]-glutamine as isotope tracer showed that most of the 13C-labeled TCA cycle intermediates were down-regulated in SLC25A22-silencing cells. Orn-derived polyamines were significantly decreased in SLC25A22 knockdown cells and culture medium. Meanwhile, accumulation of Asp in knockdown of GOT1 cells indicated that oxaloacetate (OAA) was majorly converted from Asp through GOT1. Exogenous addition of polyamines could significantly promote cell proliferation in DLD1 cells, highlighting their potential role as oncogenic metabolites that function downstream of SLC25A22-mediated glutamine metabolism. SLC25A22 acts as an essential metabolic regulator during CRC progression as promotes the synthesis of TCA cycle intermediates, Asp-derived amino acids and polyamines in KRAS-mutant CRC cells. Moreover, OAA and polyamine could promote KRAS-mutant CRC cell growth and survival. Rheumatoid arthritis (RA) is a chronic, inflammatory and symmetric autoimmune disease and a major cause of disability. However, there is insufficient pathological evidence in term of metabolic signatures of rheumatoid arthritis, especially the metabolic perturbation associated with gut microbiota (GM). Based on consistent criteria without special diet and therapeutic intervention to GM, we enrolled 50 RA patients and 50 healthy controls. On basis of the platform of UHPLC-MS and GC-MS, were performed for the non-targeted metabolomics to investigate alterations of endogenous metabolites in response to RA inflammation and interaction with GM. 32 and 34 significantly changed metabolites were identified in urine and serum of patients with RA, respectively. The altered metabolites were identified by HMDB, METLIN database or authentic standards, and mostly metabolites were attributed into tryptophan and phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, aminoacyl-tRNA biosynthesis and citrate cycle. To obtain alterations of more components in tryptophan and phenylalanine metabolism, we developed and validated a targeted metabolomics method of 19 metabolites by using LC-QqQ MS. Combining the results of targeted metabolomics with global metabolomics, significantly up-regulated kynurenine (KYN), anthranilic acid (AA) and 5-hydroxylindoleacetic acid (HIAA) simultaneously in urine and serum was found to implicate the activation of tryptophan metabolism under the condition of RA, which acted pro-inflammatory roles in inflammation and was closely correlated with GM. IDO/TDO functioned as a pro-inflammation mediator was overexpressed in RA patients. Urinary kynurenic acid and serum serotonin that have impacts on anti-inflammation in immune system were down-regulated in RA patients. The levels of phenylacetic acid and phenyllactic acid serving as a pro-inflammatory and an anti-inflammatory agent, respectively, increased in serum of patients with RA. Moreover, certain essential amino acids (EAAs), and mostly conditional EAAs were decreased in RA patients, which have been reported to inhibit cell proliferation of immune cells. In particular, deficiency of branched chain amino acids (BCAAs, valine and isoleucine) was observed in serum of patients with RA, which may lead to muscle loss and cartilage damage. The specificity of all altered metabolites resulted from RA was considerably contributed through the GM-derived metabolites. The findings revealed that GM-modulated RA inflammation was mainly resulted from tryptophan and phenylalanine metabolism, and amino acid biosynthesis, which may provide more information for better understanding the RA mechanism.

In silico prioritization of undiscovered associations can help find causal genes of newly discovered diseases. Some existing methods are based on known associations, and side information of diseases and genes. We exploit the possibility of using a neural network model, Neural inductive matrix completion (NIMC), in disease-gene prediction. Comparing to the state-of-the-art inductive matrix completion method, using neural networks allows us to learn latent features from non-linear functions of input features. Previous methods use disease features only from mining text. Comparing to text mining, disease ontology is a more informative way of discovering correlation of dis- eases, from which we can calculate the similarities between diseases and help increase the performance of predicting disease-gene associations. We compare the proposed method with other state-of-the-art methods for pre- dicting associated genes for diseases from the Online Mendelian Inheritance in Man (OMIM) database. Results show that both new features and the proposed NIMC model can improve the chance of recovering an unknown associated gene in the top 100 predicted genes. Best results are obtained by using both the new features and the new model. Results also show the proposed method does better in predicting associated genes for newly discovered diseases.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13326号 / 論医博第2194号 / 新制||医||1043(附属図書館) / (主査)教授 小川 誠司, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM