LIMA, Beatriz de Sousa e. Tolerância de genótipos de Vigna unguiculata ao estresse salino: integração dos mecanismos moleculares, fisiológicos e bioquímicos. 2017. 139 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Coordenação PGBioquímica (pg@bioquimica.ufc.br) on 2017-11-08T19:30:39Z No. of bitstreams: 1 2017_tese_bselima.pdf: 8023473 bytes, checksum: 3a7160a365e703abea25a6d6c7065072 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-11-14T17:40:39Z (GMT) No. of bitstreams: 1 2017_tese_bselima.pdf: 8023473 bytes, checksum: 3a7160a365e703abea25a6d6c7065072 (MD5) / Made available in DSpace on 2017-11-14T17:40:39Z (GMT). No. of bitstreams: 1 2017_tese_bselima.pdf: 8023473 bytes, checksum: 3a7160a365e703abea25a6d6c7065072 (MD5) Previous issue date: 2017 / Agricultural production has become a challenge in the arid and semi-arid regions of the world, as a result of the constant incidence of adverse environmental factors, such as abiotic stresses. Among them, the excess of salts in the soil is characterized by severely limiting the growth and development of the plants, causing serious damages due to the low yield of the crops. In this scenario, the selection of cultivars tolerant to salinity and / or genetic engineering of plants emerge as strategies of great importance. However, because stress tolerance is a multigenic phenomenon, the selection / identification of metabolic pathways, molecular markers and potential gene products constitute a challenge for studies on saline stress in plants. The present research was developed with the objective of integrating the molecular, biochemical and physiological mechanisms involved in the tolerance of cowpea (Vigna unguiculata) plants to saline stress. For this, plants of two genotypes of V. unguiculata endowed with differential tolerance to saline stress, Pitiúba and TVU, were used as experimental model. Saline stress promoted severe reductions in plant growth of both genotypes; however, increased biomass production under salinity was observed in TVU genotypes throughout the experimental period (8 and 16 days). The best performance of the TVU plants was attributed to the higher efficiency of the photosynthetic machinery, evidenced by the higher CO2 assimilation rates and by the higher photochemical efficiency of photosystem II (↑ electron transport rate - ETR and ↑ photochemical quenching - qP). In addition, in the presence of NaCl, the high photosynthetic efficiency coincided with the higher pigment content involved in the energy absorption of both chlorophyll a, b and total as well as carotenoids. Such responses were accompanied by increases in the expression of the PGR5-like protein 1A, HEMA (Glutamyl-tRNA reductase-binding protein), Light-harvesting complex I chlorophyll, as well as several structural genes of the thylakoidal CTE. TVU plants also limited the excessive accumulation of Na + in the stems and leaves and, consequently, established a higher K + / Na + ratio under stress. The results suggest that the control of ionic homeostasis was due to the efficient activation of defense routes, involving mechanisms of exclusion and Na + compartmentalization, since there was an increase in the expression of genes encoding SOS route components and V-ATPase genes. In addition, lower oxidative damages (↓ MDA) were observed in plant tissues of the TVU genotype, a result of the effective action of enzymatic and non-enzymatic antioxidants. The higher activity of the SOD, APX and GPX enzymes was correlated with the expression of the DEAD-box and Glutathione peroxidase genes. In this genotype, genes involved in auxin signaling (IAA), jasmonic acid (JA), gibberellins (GA), ethylene (ETHY), abscisic acid (ABA), H2O2, CIPK (CIPK3 and CIPK14) and innumerable transcription factors for example WRKY, MYB and bZIP) may have operated in the intricate network of responses that culminated in greater tolerance to salt stress. In contrast, the higher sensitivity of the Pitiúba genotype to saline stress was associated with the lower efficiency of the photosynthetic machinery and the excessive accumulation of toxic ions in the aerial tissues. Under salinity, decreases in CO2 assimilation rates were due to severe damage to the photosynthetic apparatus, since the maximum quantum efficiency of PSII (Fv / Fm) was seriously compromised shortly after the onset of stress, and this effect was intensified with the imposition of saline treatment. Associated with this, there was a progressive reduction in the effective quantum efficiency of the PSII (φPSII), which was accompanied by decreases in the ETR, as well as by the greater non-photochemical dissipation of electrons, especially in the last times of analysis. Another determinant factor for the lower efficiency of the photosynthetic machinery was the low accumulation of photosynthetic pigments, which probably resulted in the accumulation of energy in PSII and the higher production of reactive oxygen species (ROS). This phenomenon may have caused damage to cell membranes (↑ MDA), which intensified the degradation of the photosynthetic pigments and other structural components of the photosystems. As a result of the lower photochemical efficiency, photosynthetic rates (CO2 assimilation) were seriously compromised by salinity, which consequently restricted plant growth. The results of RNAseq demonstrated that Pitiúba plants also triggered an arsenal of genes in response to stress, including those focused on the control of ionic and redox homeostasis, photosystem repair, hormones, transcription factors, among others. However, such mechanisms were not sufficient to mitigate the deleterious effects of NaCl and the plants were highly sensitive to salt stress. In the present study, the main metabolic pathways involved in the tolerance to salinity of V. unguiculata plants were identified, as well as potential genes for genetic improvement studies. / A produção agrícola tem se tornado um desafio nas regiões áridas e semi-áridas de todo o mundo, resultado da incidência constante de fatores ambientais adversos, como é o caso dos estresses abióticos. Dentre eles, o excesso de sais no solo caracteriza-se por limitar severamente o crescimento e desenvolvimento das plantas, acarretando graves prejuízos devido ao baixo rendimento das culturas. Nesse cenário, a seleção de cultivares tolerantes à salinidade e/ou a engenharia genética de plantas emergem como estratégias de grande importância. Todavia, como a tolerância ao estresse é um fenômeno de natureza multigênica, a seleção/identificação das vias metabólicas, marcadores moleculares e de potenciais produtos gênicos constituem um desafio para os estudos sobre estresse salino em plantas. A presente pesquisa foi desenvolvida com o objetivo de integrar os mecanismos moleculares, bioquímicos e fisiológicos envolvidos na tolerância de plantas de feijão-caupi (Vigna unguiculata) ao estresse salino. Para isso, plantas de dois genótipos de V. unguiculata dotados de tolerância diferencial ao estresse salino, Pitiúba e TVU, foram utilizadas como modelo experimental. O estresse salino promoveu reduções severas no crescimento das plantas de ambos os genótipos, contudo, uma maior produção de biomassa sob salinidade foi observada nas plantas do genótipo TVU, ao longo de todo o período experimental (8 e 16 dias). O melhor desempenho das plantas TVU foi atribuído a maior eficiência da maquinaria fotossintética, evidenciada pelas maiores taxas de assimilação de CO2 e pela maior eficiência fotoquímica do fotossistema II (↑ taxa de transporte de elétrons – ETR e ↑ quenching fotoquímico - qP). Além disso, na presença de NaCl, a elevada eficiência fotossintética coincidiu com o maior conteúdo de pigmentos envolvidos na absorção de energia, tanto de clorofila a, b e total quanto de carotenoides. Tais respostas foram acompanhadas por incrementos na expressão dos genes PGR5-like protein 1A, HEMA (Glutamyl-tRNA reductase-binding protein), Light-harvesting complex I chlorophyll, bem como de vários genes estruturais da CTE tilacoidal. Plantas TVU também limitaram o acúmulo excessivo de Na+ nos caules e folhas e, consequentemente, estabeleceram maior relação K+/Na+ sob estresse. Os resultados sugerem que o controle da homeostase iônica foi decorrente da ativação eficiente de rotas de defesa, envolvendo mecanismos de exclusão e compartimentalização de Na+, pois houve aumento na expressão de genes que codificam componentes da rota SOS e de genes da V-ATPase. Aliado a isso, observou-se menores danos oxidativos (↓ MDA) nos tecidos das plantas do genótipo TVU, resultado da ação efetiva de antioxidantes enzimáticos e não enzimáticos. A maior atividade das enzimas SOD, APX e GPX foi correlacionada com a expressão dos genes DEAD-box e Glutathione peroxidase. Nesse genótipo, os genes envolvidos na sinalização por auxinas (IAA), ácido jasmônico (JA), giberelinas (GA), etileno (ETHY), ácido abscísico (ABA), H2O2, CIPK (CIPK3 e CIPK14) e inúmeros fatores de transcrição (por exemplo, WRKY, MYB e bZIP) podem ter atuado na rede intricada de respostas que culminou na maior tolerância ao estresse salino. Contrariamente, a maior sensibilidade das plantas do genótipo Pitiúba ao estresse salino foi associada com a menor eficiência da maquinaria fotossintética e o acúmulo excessivo de íons tóxicos nos tecidos aéreos. Sob salinidade, os decréscimos nas taxas de assimilação de CO2 foram resultado de danos severos ao aparato fotossintético, pois a eficiência quântica máxima do PSII (Fv/Fm) foi seriamente comprometida logo após o início do estresse, sendo esse efeito intensificado com a imposição do tratamento salino. Associado a isso, houve uma redução progressiva na eficiência quântica efetiva do PSII (ϕPSII), que foi acompanhada por decréscimos na ETR, bem como pela maior dissipação não fotoquímica de elétrons, principalmente nos últimos tempos de análise. Outro fator determinante para a menor eficiência da maquinaria fotossintética foi o baixo acúmulo de pigmentos fotossintéticos, o que provavelmente resultou no acúmulo de energia no PSII e na maior produção de espécies reativas de oxigênio (EROs). Esse fenômeno pode ter ocasionado danos as membranas celulares (↑ MDA), o que intensificou a degradação dos pigmentos fotossintéticos e de outros componentes estruturais dos fotossistemas. Como resultado da menor eficiência fotoquímica, as taxas fotossintéticas (assimilação de CO2) foram seriamente comprometidas pela salinidade o que, consequentemente, restringiu o crescimento das plantas. Os resultados do RNAseq demonstraram que as plantas Pitiúba também acionaram um arsenal de genes em resposta ao estresse, incluindo aqueles voltados para o controle da homeostase iônica e redox, reparo dos fotossistemas, hormônios, fatores de transcrição, dentre outros. Entretanto, tais mecanismos não foram suficientes para mitigar os efeitos deletérios do NaCl e as plantas foram altamente sensíveis ao estresse salino. No presente estudo foram identificadas as principais vias metabólicas possivelmente envolvidas na tolerância à salinidade de plantas de V. unguiculata, além de genes potenciais para estudos de melhoramento genético de plantas

AN ABSTRACT OF THE DISSERTATION OF SOLOMON G. HAILU, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry, presented on August 22, 2017, at Southern Illinois University, School of Medicine. TITLE: DISTINCT GENOME WIDE FUNCTIONS OF CHROMATIN REMODELERS IN NUCLEOSOME ORGANIZATION AND TRANSCRIPTION REGULATION MAJOR PROFESSOR: Dr. Blaine Bartholomew Chromatin remodelers are conserved from yeast to humans and are the gatekeepers of chromatin. They regulate transcription by occluding or exposing DNA regulatory elements globally. They are crucial for DNA processes such as DNA replication, repair and recombination. In addition, they are critical in developmental processes and differentiation. Chromatin remodelers are categorized into several families based on their conserved ATPase domain, an essential component required for their DNA translocation ability. In this study, we investigated the role yeast ISWI and SWI/SNF family of chromatin remodelers play on nucleosome rearrangement and transcription regulation by targeted mutagenesis of domains in accessory subunits and at the C-terminus of the catalytic subunit. All members of the ISWI family (ISW1a, ISW1b, ISW2) share a conserved C-terminal HAND, SANT and SLIDE domains, which are important for sensing linker DNA. We find an auto-regulation of ISWI complexes by the SLIDE domain, independent of the histone H4 Nterminal tail. Our protein-protein chemical crosslinking and mass spectrometry (CX-MS) analysis indicate that the SLIDE domain regulates the ATPase core through N terminal domains of the accessory subunit Itc1. Moreover, we show that the accessory subunits of ISWI modulate the ATPase activity and specificity of ISWI complexes. The DNA sensing ability of the SLIDE domain is required for the in vivo nucleosome spacing and transcription regulation by ISWI. We find that while ISW2 primarily regulates transcription at the 5’ end of genes, ISW1a is important in transcription elongation by rearranging nucleosomes starting at the +2 nucleosome and through the rest of the body of genes towards the 3’ end. ISW1b on the other hand rearrange nucleosomes in the gene body to facilitate suppression of cryptic transcription. For the first time, we show the potential division of labor between ISW1a and ISW1b during transcription elongation. On the other hand, SWI/SNF chromatin remodelers are essential epigenetic factors that are frequently mutated in cancer and neurological disorders. They harbor a C-terminal SnAC and AT hook domains that positively regulate their DNA dependent ATPase activity and nucleosome mobilizing capabilities. By deleting the AT hook motifs, we have identified the role of SWI/SNF in organizing the -1 and +1 nucleosomes at transcription start sites flanking the nucleosome free region (NFR). Our RNA-seq analysis shows SWI/SNF positively regulates the bi-directional transcription of non-coding RNA (ncRNA) which are activated when the AT hook motifs are deleted. Moreover, AT hooks regulate such activities of SWI/SNF through direct protein-protein interactions with the ATPase core as evidenced by our chemical crosslinking and mass spectrometry (CX-MS) analysis.

Submitted by la_simielli@yahoo.com.br (la_simielli@yahoo.com.br) on 2016-04-05T13:12:40Z No. of bitstreams: 1 TESE_LARISSA_FERNANDA_SIMIELLI_FONSECA.pdf: 1971633 bytes, checksum: 177f6a3044467459f559bbf217ba550f (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-06T20:32:27Z (GMT) No. of bitstreams: 1 fonseca_lfs_dr_jabo.pdf: 1971633 bytes, checksum: 177f6a3044467459f559bbf217ba550f (MD5) / Made available in DSpace on 2016-04-06T20:32:27Z (GMT). No. of bitstreams: 1 fonseca_lfs_dr_jabo.pdf: 1971633 bytes, checksum: 177f6a3044467459f559bbf217ba550f (MD5) Previous issue date: 2016-03-07 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Mais de 80% da carne bovina brasileira é proveniente de animais de origem zebuína. Este produto é considerado de baixa maciez e reduzido teor de gordura entremeada quando comparado à carne de animais de raças taurinas. Investir na melhoria das características organolépticas como marmoreio e maciez da carne torna-se importante do ponto de vista econômico, pois tratam-se de atributos muito apreciados pelo consumidor. A maciez pode ser medida por meio de análises sensoriais, índice de fragmentação miofibrilar ou força de cisalhamento. Nesse último, um aparelho é utilizado para medir a força necessária para o cisalhamento de uma seção transversal de carne e, quanto maior a força dispensada, menor é a maciez apresentada pelo corte de carne. Outra característica relacionada à qualidade da carne é o marmoreio, que pode ser analisada por meio de escala de graduação visual denominada Quality Grade. No entanto, estas metodologias só podem ser utilizadas após o abate do animal. As técnicas de análise do transcriptoma, como o RNA-Seq, permitem a identificação de genes cuja expressão está relacionada com o controle de características quantitativas e podem ser alternativas na busca pelo melhoramento destes atributos. Com isso, o objetivo deste trabalho foi identificar genes diferencialmente expressos utilizando a técnica RNA-Seq, em tecido muscular de bovinos da raça Nelore, visando contribuir para o conhecimento dos fatores genéticos que estão relacionados com a qualidade da carne. Para isso, foi sequenciado o mRNA de 40 amostras divergentes para a maciez da carne (20 com carne dura e 20 com carne macia) e 40 amostras divergentes para marmoreio (20 com alto e 20 com baixo marmoreio). Essas amostras foram escolhidas com base na análise de força de cisalhamento e índice de marmorização realizadas em 132 e 80 amostras de músculo longissimus dorsi respectivamente, coletadas da meia carcaça esquerda de cada animal. As análises dos resultados obtidos pela técnica de RNA-Seq revelaram genes diferencialmente expressos relativos à maciez e marmoreio da carne em gado Nelore. Os genes referentes à maciez estão relacionados ao metabolismo de colágeno, à actina e miosina, ao transporte de cálcio, dentre outros. Por outro lado, nos resultados das análises de marmoreio, os genes que se mostraram diferencialmente expressos estão relacionados à ocitocina, ao transporte de oxigênio, ao crescimento e à síntese de lipídios e proteínas. Portanto, este estudo revelou a possibilidade de se utilizar alguns desses genes diferencialmente expressos como marcadores genéticos em bovinos Nelore para seleção precoce quanto à maciez e marmoreio da carne. / More than 80% of Brazilian beef comes from zebu animals. The Zebu meat show reduced marbling and is considered harder when compared to Taurine meat. The improvement of the organoleptic traits like marbling and meat tenderness is important from an economic point of view, because these attributes are highly appreciated by the consumer. The meat tenderness can be measured by sensory analysis, myofibril fragmentation index or by shear force. This method use an apparatus which measure the force required to shear a cross section of meat. The higher the strength dispensed, lower is the tenderness showed by the cut of meat. Another trait related to meat quality is marbling, which can be analyzed by Quality Grade visual graduation scale. However, these methods can only be performed after the animal was killed. The transcriptome analysis techniques, as RNA-Seq, are able to identify genes whose expression are related to quantitative traits control and can be used as alternative to help the improvement of these traits. Thus, the aim of this study was to identify differentially expressed genes using RNA-Seq, in Nelore cattle muscular tissue, to contribute to the knowledge of the genetic factors that are related to meat quality. We sequenced the mRNA of 40 samples divergently ranked to meat tenderness (20 with hard meat and 20 with tender meat) and sequenced the mRNA of 40 samples divergently ranked to marbling (20 with high and 20 with low marbling). These samples were chosen based on shear force and marbling content analysis held at 132 and 80 longissimus dorsi muscle samples respectively, collected from each left half carcass. The analysis of the results obtained by RNA-Seq technique revealed differentially expressed genes related to meat tenderness and marbling in Nelore cattle. Regarding the meat tenderness, these genes are related to collagen metabolism, actin and myosin, the calcium transport, among others. Moreover, the marbling analysis results, revealed differentially expressed genes related to oxytocin, transport of oxygen, growth and lipids and proteins synthesis. Therefore, this study showed the possibility of using some of these differentially expressed genes as genetic markers in Nellore cattle for early selection to meat tenderness and marbling. / FAPESP: 2013/09190-7

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-04-27T19:34:22Z No. of bitstreams: 2 Dissertação - Vanessa Cristina da Silva Araújo - 2017.pdf: 2199979 bytes, checksum: f02e05314927339cf3c54225f8ad52db (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-03T12:09:43Z (GMT) No. of bitstreams: 2 Dissertação - Vanessa Cristina da Silva Araújo - 2017.pdf: 2199979 bytes, checksum: f02e05314927339cf3c54225f8ad52db (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-03T12:09:43Z (GMT). No. of bitstreams: 2 Dissertação - Vanessa Cristina da Silva Araújo - 2017.pdf: 2199979 bytes, checksum: f02e05314927339cf3c54225f8ad52db (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Large-scale sequencing of transcripts via RNA-Seq has been changing paradigms by demonstrating that transcription is prevalent throughout the eukaryotic genome. In these organisms, the vast majority of transcripts are non-coding (ncRNA). One type of RNA that has aroused great interest, given its prevalence, is long non-coding RNAs (lncRNAs), which are ncRNA with more than 200 nucleotides. However, little is known about the role and prevalence of these lncRNAs in plant genomes, even in model species such as Arabidopsis thaliana (L.) Heynh. The objective of this work was to identify lncRNAs in the Arabidopsis genome and to characterize their size, structure and nucleotide diversity. The sequences were obtained from previous work that sequenced total RNA from A. thaliana, grown under different light regimes, using Illumina Hiseq 2000 platform. These sequences were mapped into the reference genome with TopHat and assembled with Cufflinks. The assembled transcripts were compared with the genome annotation with Cuffcompare, to identify non-annotated transcripts. A total of 4,305 long putative RNAs were obtained, with 314 (7%) sense in relation to coding transcripts (mRNAs), 392 (9%) intergenic, 2,216 intronic (52%) and 1,383 (32%) antisense mRNAs. The lncRNAs obtained were filtered to eliminate those with coding potential, as well as those related to rRNA, tRNA and miRNA synthesis. A total of 3,710 high-confidence lncRNAs (HC-lncRNA) were obtained, of which 58.6% were not previously annotated. These HC-lncRNA emcompass a low proportion (~ 1%) lncRNAs in the genome of Arabidopsis thaliana. A functional enrichment analysis of Gene Ontology (GO) categories demonstrated that among genes containing lncRNAs there is a high proportion of categories linked to the localization and transport of proteins within the cell, as well as to nucleic acid binding. A gene expression analyses identified only 22 differentially expressed lncRNAs under the different light conditions in which samples were exposed. Using the SNP data from the 1001 genomes project, identified high nucleotide diversity within lncRNAs regions, indicating low conservation of the primary structure of these transcripts. The nucleotide diversity in regions of long noncoding RNAs is lower than in coding regions, but less than a diversity observed in neutral regions such as pseudogenes. / O sequenciamento em larga escala de transcritos, via RNA-Seq, vêm mudando paradigmas ao demonstrar que a transcrição é prevalente por todo o genoma dos eucariotos. Nesses organismos, a grande maioria dos transcritos não codificam proteínas (ncRNA). Um tipo de RNA que vêm despertando grande interesse, dado sua prevalência, são os RNAs longos não codificantes (lncRNAs), que são ncRNA com mais de 200 nucleotídeos. No entanto, pouco se sabe sobre o seu papel e prevalência nos genomas de plantas, mesmo em espécies modelo como Arabidopsis thaliana (L.) Heynh. O objetivo desse trabalho foi identificar lncRNAs no genoma de Arabidopsis e caracterizar seus tamanhos, estruturas e diversidade genética. As sequências utilizadas foram obtidas de um trabalho que sequenciou RNA total de A. thaliana, sob diferentes regimes de luminosidade, utilizando a plataforma Illumina HiSeq 2000. Estas sequências foram mapeadas no genoma referência com o programa TopHat e montadas com o Cufflinks. Os transcritos montados foram comparados com a anotação do genoma com o Cuffcompare, afim de identificar transcritos ainda não anotados. Um total de 4.305 RNAs longos putativos foi obtido, sendo 314 (7%) senso em relação a transcritos codantes (mRNAs), 392 (9%) intergênicos, 2.216 intrônicos (52%) e 1.383 (32%) antisenso de mRNAs. Os lncRNAs obtidos foram filtrados para eliminar aqueles com potencial de codificação, bem como aqueles relacionados com a síntese rRNA, tRNA e miRNA. Após essa filtragem, foram obtidos 3.710 lncRNAs de alta cofiança (HC-lncRNA), sendo que desses 58,6% ainda não foram previamente anotados. Esses HC-lncRNA representam uma baixa proporção (~1%) do genoma de Arabidopsis thaliana. Uma análise de enriquecimento funcional de categorias do Gene Ontology (GO) demonstrou que os genes que contém lncRNAs apresentam enriquecimento para processos ligados à localização e transporte de proteínas dentro da célula, bem como para ligação a ácidos nucléicos. Uma análise de expressão gênica identificou apenas 22 lncRNAs diferencialmente expressos entre as diferentes condições de luminosidade em que as amostras foram expostas. Utilizando os SNPs do projeto 1001 genomes, identificou-se alta diversidade nucleotídica em regiões de lncRNAs, indicando baixa conservação da estrutura primária destes transcritos. A diversidade nucleotídica em regiões de RNAs longos não codificantes é menor do que em regiões codantes, mas menor do que a diversidade observada em regiões neutras como os pseudogenes.

L'ADN génomique des bactéries existe dans un complexe dynamique riche en protéines, le "nucléoïde'', très bien organisé à différentes échelles de longueur. Cette thèse décrit notre modélisation et analyse des données en mettant l'accent sur l'organisation du nucléoïde de \textit{E. coli}, et sur comment cette organisation affecte l'expression des gènes. La première partie du travail est une revue des progrès récents expérimentaux et théoriques quantifiant l'organisation physique (la géométrie et le compactage) du chromosome bactérien. En particulier, nous soulignons le rôle que la physique de la matière molle et la physique statistique jouent dans la description de ce système. Une deuxième partie de l'ouvrage traite d'un modèle de la physique des polymères inspiré par deux caractéristiques du nucléoïde: auto-adhérence osmotique et effet des protéines de pontage. Les résultats sont une caractérisation qualitative du diagramme de phase, qui montre que les nucléoïdes forment des domaines distincts sans interactions intra-spécifiques. La thèse couvre également plusieurs approches d'analyse de données pour tester les connexions entre l'organisation du nucléoïde avec l'expression des gènes (RNA-Seq) et des protéines (ChIP-Seq). Cette dernière partie contient une description de l'outil web NuST, qui permet d'effectuer de simples analyses statistiques sur de multiples échelles. En outre, nous présentons une étude de corrélation d'un grand nombre de mesures d'expression génomique dans différentes conditions de croissance, et nous le comparons avec les cartes d'interaction (Hi-C) spatiale entre le chromosome. / The genomic DNA of bacteria exists in a complex and dynamic protein-rich state, which is highly organized at various lengthscales. This thesis describes a work of physical modeling and data analysis focused on the E. coli genome organization, in the form of the "nucleoid'', and on how nucleoid organization affects gene expression.The first part of the work is a review of the recent experimental andt heoretical advances quantifying the physical organization (compactionand geometry) of the bacterial chromosome with its complement of proteins (the nucleoid). In particular, we highlight the role that statistical and soft condensed matter physics play in describing this system of fundamental biological importance.A second part of the work discusses a simple polymer physics model inspired by two main features of the nucleoid: osmotic self-adhesion and protein bridging. Results are summarized by a qualitative characterization of the phase diagram of this model which shows the general feature that distinct domains may form without the need forintra-specific interactions.The thesis also covers several data analysis approaches to test possible connections between the physical organization of the nucleoid with gene expression (RNA-Seq) and protein binding (ChIP-Seq) datasets. This latter part contains a description of the NuST webtool, which consists of a database which collect datasets from past experiments and an implementation of simple multi scale statistical analysis tools. Additionally, we introduce a correlation study of a large number (about 300) of genome-wide expression data-sets, also compared to the outcome to the published genome interaction map (Hi-C)data.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23046号 / 理博第4723号 / 新制||理||1677(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)講師 嶋田 知生, 教授 松下 智直, 教授 鹿内 利治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Salinity is a major abiotic stress in plants that causes significant reductions in crop yield. The need for improvement of food production has driven research to understand factors underlying plant responses to salt and mechanisms of salt tolerance. The aim of improving tolerance in traditional crops has been initiated but most crops can only tolerate a limited amount of salt in their systems to survive and produce biomass. Studies of naturally occurring high salt-tolerant plants (halophytes) are now being promoted for economic interests such as food, fodder or ecological reasons. Suaeda fruticosa, a member of the family Chenopodiaceae, belongs to a potential model halophyte genus for studying salt tolerance. However, published reports on the identification of genes, expression patterns and mechanisms of salinity tolerance in succulent halophytes are very limited. Next generation RNA-sequencing techniques are now available to help characterize genes involved in salinity response, along with expression patterns and functions of responsive genes. In this study, we have optimized the assembly of the transcriptome of S. fruticosa. We have annotated the genes based on their gene ontology characteristics and analyzed differential expression to identify genes that are up- and down-regulated in the presence of salt and have grouped the genes based on their putative functions. We also have provided evidence for groups of transcription factors that are involved in salt tolerance of this species and have identified those that may affect the regulation of salt tolerance. This work elucidates the characterization of genes involved in salinity tolerance to increase our understanding of the regulation of salt in a succulent halophyte.

The components contributing to cancer progression, especially the transition from early to invasive are unknown. Consequently, the biological reasons are unclear as to why some patients diagnosed with atypia and ductal carcinoma in situ (DCIS) never progress into invasive breast cancer. The “one gene at a time” approach does not sufficiently predict progression. To elucidate the early stage progression to invasive ductal cancer, expression signature of transcripts and transposable elements in micropunched samples of formalin-fixed, paraffin embedded (FFPE) tissue was conducted. A bioinformatics pipeline to analyze poor quality, short reads (>36 nts) from RNA-Seq data was created to compare the most common tools for alignment and differential expression. Most samples from patients prepared for RNA-seq analysis are acquired through archived FFPE tissue collections, which have low RNA quality. The pipeline analytics revealed that STAR alignment software outperformed others. Furthermore, our comparison revealed both DESeq2 and edgeR, with the estimateDisp function applied, both perform well when analyzing greater than 12 replicates. Transcriptome analysis revealed progressive diversification into known oncogenic pathways, a few novel biochemical pathways, in addition to antiviral and interferon activation. Furthermore, the transposable element (TE) signature during breast cancer progression at early stages indicated long terminal repeat (LTRs) as the most abundantly differentially expressed TEs. LTRs belong to endogenous retroviruses (ERV), a subclass of TEs. The retroviral and innate immune response activity in DCIS, which indirectly corroborates the increase in ERV expression in this pre-malignant stage. Finally, to demonstrate the potential role of TEs in the transition from pre-malignant to malignant breast cancer we used pharmacological approaches to alter global TE expression and inhibit retrotransposition activity in control and breast cancer cell lines. It was expected that dysregulation of TEs be associated with increased invasiveness and growth. However, our results indicated that DNA methyltransferase inhibitor 5-Azacytidine (AZA) consistently retarded cell migration and growth. While unexpected, these findings corroborate recent studies that AZA may induce an interferon response in cancer via increased ERV expression. This body of work illustrates the importance of understanding bioinformatics methods used in RNA-seq analysis of common clinical samples. These studies suggest the potential for TEs as biomarkers for disease progression and novel therapeutic approach to investigate in additional model systems.

Since its description in 2008, Chromera velia has drawn profound interest as the closest free-­-living photosynthetic relative of apicomplexan parasites that are significant pathogens, causing enormous health and economic problems. There-­- fore, this newly described species holds a great potential to understand evolu-­- tionary basis of how photosynthetic algae evolved into the fully pathogenic Apicomplexa and how their common ancestors may have lived before they evolved into obligate parasites. Hence, the aim of this work is to understand how C. velia function and respond to different environmental conditions. This study aims to reveal how C. velia is able to respond to environmental perturbations that are applied individually and simultaneously since, studying stress factors in separation fails to elucidate complex responses to multi stress factors and un-­- derstanding the systemic regulation of involved genes. To extract biologically significant information and to identify genes involved in various physiological processes under variety of environmental conditions (i.e. a combination of vary-­- ing temperatures, iron availability, and salinity in the growth medium) we pre-­- pared strand specific RNA-­-seq libraries for 83 samples in diverse environmental conditions. Here, we report the set of significantly differentially expressed genes as a re-­- sponse to the each condition and their combinations. Several interesting up-­- regulated and down-­-regulated genes were found and their functions and in-­- volved pathways were studied. We showed that the profound regulation of HSP20 proteins is significant under stress conditions and hypothesized that the-­- se proteins might be involved in their movements.

Hypoxia has become a subject of interest among the many environmental stressors as its role in biology is complex and diverse. Hypoxia is a significant low oxygen condition that causes many pathologies and adaptive responses in organisms. It can lead to a moderate or dangerous loss of respiration and can be an indication of tumorigenesis as many tumors lack adequate blood supply. Organisms possess adaptive responses to hypoxia that include hypoxia-inducible factors (HIFs) that activate several downstream pathways that are responsible for altering metabolism and maintaining homeostasis. Within aquatic organisms, hypoxia is an important ecological constraint as oxygen availability within bodies of water can vary greatly over time and space. Therefore, adaptation to hypoxia is likely pervasive, especially in genotypes originating from bodies of water that are prone to hypoxia. Here we report the transcriptional response to acute hypoxia in the clonal freshwater crustacean Daphnia magna. Daphnia were subjected to 1mg/O2 for 12 hours. Then, RNA was extracted, reverse transcribed, and sequenced using Oxford Nanopore MinION. We find that severe hypoxia significantly up-regulates key enzymes in the gluconeogenesis pathway. Additionally, we report genotype-by-environment interactions showing that Daphnia clones from habitats that are hypoxia prone survive better in hypoxia.