Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-05-04T17:52:11Z No. of bitstreams: 2 Dissertação - Lanusse Andrade Fernandes - 2015.pdf: 1698917 bytes, checksum: b237e0d0c80dfe44acee928225ef0aa4 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-05T12:45:38Z (GMT) No. of bitstreams: 2 Dissertação - Lanusse Andrade Fernandes - 2015.pdf: 1698917 bytes, checksum: b237e0d0c80dfe44acee928225ef0aa4 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-05-05T12:45:38Z (GMT). No. of bitstreams: 2 Dissertação - Lanusse Andrade Fernandes - 2015.pdf: 1698917 bytes, checksum: b237e0d0c80dfe44acee928225ef0aa4 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Handroanthus impetiginosus, known as ipê roxo, is a species present in several Brazilian biomes, with ecological and economic importance. The species has suffered strong exploration thanks to the quality of its wood. Despite of its ecological importance, economic value and medicinal potential, there is virtually no genomic study published with this species. This work aimed to take advantage of the large sequencing capacity of next generation platforms to generate, assemble and functional annotate the sequences of the entire transcriptome of H. impetiginosus. Seeds were collected from two trees on the Campus II of the Universidade Federal de Goiás. The seeds were germinated, their aerial tissues collected and the RNA extracted and sent to the University of Illinois to be sequenced on the HiSeq 2500 platform (Illumina). A total of 148,359,530 sequencing reads were generated. The adapters and low-quality sequences were removed. Contaminating sequences were detected and eliminated, and the remaining sequences were normalized. Sequences were then assembled, annotated and polymorphisms were detected. A total of 190,885 scaffolds were assembled from 23,002 genes. Mimulus guttatus was the species with the most Blast best hit against the sequences of H. impetiginosus. Of all the Gene Ontology (GO) annotated sequences, the majority had categories for Biological Process (50.94%), followed by Cellular Component (28.34%) and Molecular Function (20.72%). The most frequent GO categories related to Biological Process were metabolic and cellular processes, while in Cell Components the most frequent were cell and organelles. Catalytic activity and binding of ions and molecules were the most frequent annotated categories of Molecular Function. As the sequences were generated from a pool of RNA from four plants, 25,169 InDels and 278,121 SNPs were identified. As expected, InDels are depleted in coding regions (CDS) when compared to SNPs. These results will be important for future studies with an interest in gene expression of the species. Furthermore, the possible SNPs identified can facilitate genetic studies and genomic H. impetiginosus populations. / Handroanthus impetiginosus, conhecida como ipê-roxo, é uma espécie presente em vários biomas brasileiros, tendo importância ecológica e econômica. O ipê-roxo vem sofrendo forte exploração graças à qualidade de sua madeira. Apesar de sua importância ecológica, seu valor econômico e de seu potencial medicinal, inexistem estudos genômicos publicados com esta espécie. Assim, este trabalho teve como objetivo principal aproveitar a grande capacidade de sequenciamento das plataformas de nova geração para gerar, montar e fazer anotação funcional da sequência do transcritoma de Handroanthus impetiginosus. Foram coletadas sementes de duas árvores matrizes de H. impetiginosus dispersas pelo Campus II da Universidade Federal de Goiás. As sementes foram germinadas, seus tecidos aéreos coletados e o RNA foi extraído e enviado à Universidade de Illinois para ser sequenciado na plataforma HiSeq 2500 da Illumina. Foi gerado um total de 148.359.530 sequências. Os adaptadores e as sequências de baixa qualidade foram removidos. Em seguida, possíveis sequências contaminantes foram detectadas e eliminadas, e as sequências restantes foram normalizadas. Ao final da normalização, as sequências foram montadas, anotadas e os polimorfismos detectados. Foram montados 190.885 transcritos (scaffolds), totalizando 23.002 genes. Mimulus guttatus, popularmente conhecida como flor de macaco, foi a espécie com maior número de sequências mais similares às de Handroanthus impetiginosus. De todas as sequências anotadas com categorias GO (Gene Ontology) a maioria teve anotações para Processo Biológico (50.94%), seguido de Componente Celular (28.34%) e Função Molecular (20.72%). As sequências mais frequentes relacionadas à Processo biológico estão ligadas a processos metabólicos e celulares, em Componentes Celulares as sequências mais frequentes estão ligadas a célula e a organelas. Já em Função Molecular as sequências mais frequentes estão relacionadas a atividade catalítica e ligação de íons e moléculas. Como as sequências foram geradas a partir de amostras de RNA de quatro plantas, 25.169 InDels e 278.121 SNPs foram identificados. Como esperado, os InDels são significativamente mais deplecionados em regiões codantes (CDS) que os SNPs.Estes resultados serão importantes para futuros estudos com interesse na expressão gênica da espécie. Além disso, os possíveis SNPs identificados poderão facilitar estudos de genética e genômica de populações para H. impetiginosus.

La voie de l’AMPc est une voie impliquée dans la physiopathologie de nombreuses tumeurs endocrines. Les phosphodiestérases sont des enzymes clés de la voie de l’AMPc dans la mesure où elles permettent de réguler finement les niveaux intra-cellulaires d’AMPc en hydrolysant l’AMPc en son catabolite inactif, le 5’AMP. L’hyperplasie macronodulaire bilatérale des surrénales est une étiologie rare de syndrome de Cushing ACTH-indépendant responsable d’un syndrome de Cushing souvent modéré contrastant avec la taille des nodules surrénaliens. Sa physiopathologie est mal connue.Nous avons tout d’abord pu montrer dans ce travail que les variants faux sens de la phosphodiestérase 11A pouvaient participer au développement des tumeurs corticosurrénales bilatérales sécrétrices de cortisol. Ces variants sont, en effet, non seulement retrouvés de façon plus fréquente chez les patients porteurs d’une hyperplasie nodulaire comparés à des sujets contrôles, mais ils ont également des conséquences fonctionnelles in vitro.Nous avons, dans une seconde partie, montré grâce à une étude de transcriptome ayant porté sur un groupe d’adénomes cortisoliques comparés à un groupe d’adénomes non-sécrétant que plusieurs gènes codant pour la voie de l’AMPc étaient impliqués dans la sécrétion cortisolique et que la phosphodiestérase 8 B semblait avoir un rôle prépondérant. Ces résultats ont ensuite été confirmés au niveau protéique. / Pas de résumé en anglais

Doctor of Philosophy / Department of Plant Pathology / Dorith Rotenberg / Frankliniella occidentalis, the western flower thrips, transmits the plant-pathogenic virus, Tomato spotted wilt virus (TSWV), through a circulative-propagative transmission strategy. The virus infects and replicates in the insect, traversing membrane barriers as it moves from the midgut to the salivary glands for subsequent inoculation of a plant host. Based on well-characterized virus-vector systems, many molecular interactions occur as the virus completes an infection cycle in the vector, and knowledge of transcriptome-wide response of thrips to TSWV has been limited. My research goals were to gain insight into i) the molecular responses that occur in thrips vectors of orthotospoviruses, ii) the role of antiviral defense in viruliferous thrips, and iii) plant transgenic-based strategies for studying thrips gene function and crop-pest control. To this end, my specific research objectives were to: 1) generate, assemble, and annotate a RNA-Seq-derived transcriptome for F. occidentalis using the thrips genome, and to quantify global gene expression in response to TSWV activity in larval, pre-pupal, and adult developmental stages, 2) conduct a time-course experiment to determine the effect(s) of challenging TSWV-exposed and non-exposed thrips with dsRNAs of F. occidentalis Dcr-2 or AGO2 by hemocoel injection, and 3) construct transgenic plants expressing a thrips-gene specific dsRNA hairpin to target a vital gene. My research has catalogued insect response to TSWV activity in thrips during development and provides candidate sequences for functional analysis of genes involved in insect development and defense. Successful silencing of the antiviral RNAi pathway in thrips revealed increased mortality and decreased offspring production in both virus-exposed and non-exposed insects. Arabidopsis plants were developed to express dsRNA of vacuolar ATP synthase (V-ATPase) and preliminary feeding bioassays to explore the effect of these transgenics on thrips fitness indicate a need for further description of thrips dsRNA uptake. In total, my research contributes new basic knowledge underpinning the complex and dynamic relationship between thrips vectors and the plant viruses they transmit.

Orientadores: Marcos Antonio Machado, Alessandra Alves de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T04:53:23Z (GMT). No. of bitstreams: 1 Coqueiro_DanilaSouzaOliveira_D.pdf: 3034667 bytes, checksum: 7ce135c75da10c6b26032ec285344dc5 (MD5) Previous issue date: 2013 / Resumo: Avaliou-se as alterações transcricionais em laranja 'Pera' (Citrus sinensis L. Osb.) promovidas por quitosana (CHI) e ácido salicílico (SA), utilizando RNA-seq, e o efeito destes compostos no controle do cancro cítrico (Xanthomonas citri subsp. citri) e da clorose variegada dos citros (CVC - Xylella fastidiosa). As plantas foram tratadas com CHI ou SA e após 48h e 24h, respectivamente, foram coletadas amostras foliares para avaliar seus transcriptomas. Para a avaliação dos eliciadores sobre o cancro cítrico e a CVC, as plantas foram tratadas com CHI ou SA e após 48h e 24h, respectivamente, inoculadas com as duas bactérias separadamente. A partir de 24h da inoculação, foram coletadas amostras foliares para avaliar a curva de crescimento de ambas as bactérias, a redução da severidade e/ou incidência das doenças e respostas de defesa da planta por RT-qPCR. Com os resultados do transcriptoma, observou-se que mais genes foram induzidos pelo tratamento com SA do que com CHI. O tratamento com SA aumentou a expressão de genes que participam da via de sinalização do SA na planta (WRKY50, PR2 e PR-9) e genes da biossíntese do etileno e ácido jasmônico (ACS 12, fator de transcrição contendo domínio AP2 e OPR3). Além disso, promoveu a indução de genes relacionados ao metabolismo secundário, processos redox e estresse biótico. No tratamento com CHI, foi observada maior indução de genes relacionados ao metabolismo secundário. Para ambos os tratamentos, a via da auxina foi reprimida. No experimento para controle do cancro cítrico, observou-se que ambos os eliciadores promoveram reduções na severidade e incidência da doença. Entretanto, a CHI pareceu não interferir diretamente na formação do biofilme pela bactéria, mas pode ter dificultado a multiplicação de X. citri na planta. O SA retardou a entrada da bactéria na planta e, aparentemente, inibiu mais a formação do biofilme bacteriano do que a CHI. Comparações da expressão gênica entre os eliciadores reforçam a ideia de que a CHI tem maior potencial de induzir resistência ao cancro cítrico do que SA. No experimento para o controle da CVC, observou-se que a CHI induziu importantes genes da via do SA (NPR1, TGA, EDS1) e etileno (EIN-3, PR-4) 24h após a inoculação. Aplicações exógenas de SA potencializaram o seu efeito endógeno na planta, pois houve indução de NPR1, TGA e PRs. Entretanto, não foi possível estabelecer uma relação clara entre a multiplicação de X. fastidiosa, a incidência da doença e o uso da CHI e SA em laranja 'Pera', já que na maioria das avaliações não houve redução da população bacteriana em amostras foliares e não houve redução da incidência em plantas tratadas. Com base nos resultados, observou-se que CHI e SA induziram diversos genes envolvidos em respostas de defesa em laranja 'Pera'. Entretanto, essas respostas podem ser moduladas diferencialmente a depender do patógeno que afeta a planta, pois os eliciadores foram eficientes no controle da X. citri, um patógeno que coloniza o mesófilo da planta, entretanto não foram efetivos no controle da X. fastidiosa, um patógeno que coloniza o xilema da planta, embora respostas de defesa tenham sido expressas nos momentos iniciais (24h) após a inoculação com X. fastidiosa / Abstract: This study was carried out to evaluate transcriptional modification in sweet orange 'Pera' (Citrus sinensis L. Osb.), promoted by chitosan (CHI) and salicylic acid (SA), using RNA-seq, and the effect of these compounds on citrus canker (Xanthomonas citri subsp. citri) and citrus variegated chlorosis (CVC - Xylella fastidiosa). Plants were treated with CHI or SA and after 48h and 24h, respectively, leaf samples were collected to assess the transcriptome. In the experiments for disease assessment, the plants were treated with CHI or SA and after 48h and 24h, respectively, inoculated. Starting from 24h after inoculation, leaf samples were collected to evaluate the multiplication of the pathogens (X. citri and X. fastidiosa), reduction of the severity and / or incidence and plant defense responses by RT-qPCR. Based upon the transcriptome results, it was observed that more genes were induced by SA than by CHI. SA treatment increased the expression of genes that participate in the SA signaling pathway in the plant (WRKY50, PR2 and-PR9), and genes involved in the biosynthesis of ethylene and jasmonic acid (ACS 12, transcription factor containing AP2 and OPR3 domain). Besides these, SA promoted induction of genes of secondary metabolism, redox processes and biotic stress. The treatment with CHI exhibited higher induction of genes related to secondary metabolism. For both treatments, the auxin pathway was suppressed. In the experiment for the control of citrus canker, it was observed that both elicitors reduced the severity and incidence of the disease. However, CHI seems not to interfere directly in biofilm formation, but may have hindered the multiplication of X. citri in the plant. The SA slowed down the entry of the bacteria into the plant and, apparently, inhibited the formation of biofilm more efficiently than the CHI. Comparisons of gene expression between elicitors reinforce the idea that CHI has higher potential to induce resistance to citrus canker than SA. In the experiment for the control of CVC, it was observed that the CHI induced important genes of the SA (NPR1, TGA, EDS1) and ethylene (EIN-3, PR-4) pathways 24h after inoculation. Exogenous applications of SA potentiated its endogenous effect in the plant, since there was induction of EDS-1, NPR1, TGA and PRs. However, it was not possible to establish a clear relationship between the multiplication of X. fastidiosa, the incidence of the disease and the use of CHI and SA in 'Pera' sweet orange, since most of the assessments did not show reduction of bacterial populations in leaf samples and there was no reduction of the incidence in treated plants. Based upon the results of this study, it was observed that CHI and SA induced several genes involved in defense responses in 'Pera' sweet orange. However, these responses can be modulated differentially depending on the pathogen that affects the plant. This fact was demonstrated in this study, as elicitors were effective in controlling X. citri, a pathogen that colonizes the mesophyll of the plant, but were not effective in controlling X. fastidiosa, a pathogen that colonizes the xylem of the plant, although defense responses were expressed in the early stages (24 h) after inoculation with X. fastidiosa / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Diversos estudos elucidaram mecanismos envolvidos com a organogênese in vitro, porém pouco é conhecido a respeito da fase de aquisição de competência, fundamental para que a regeneração ocorra. Alguns genes já foram identificados por interferirem na fase de aquisição de competência em tomateiro (Solanum lycopersicum), mas ainda existem diversas lacunas a serem esclarecidas. Para investigar a expressão de genes e o controle hormonal na fase de aquisição de competência, foram utilizados os mutantes de tomateiro, sob o background genético da cultivar Micro-Tom (MT), MT-Rg1 e MT-pro (procera), os quais afetam positiva ou negativamente a organogênese in vitro, respectivamente. Embora a resposta constitutiva a giberelina no mutante MT-pro seja conhecida, a identidade molecular do gene RG1 permanece indefinida. O mutante MT-Rg1 apresenta aumento tanto na formação de gemas caulinares quanto de raízes e reduz o tempo necessário para a indução desses órgãos, devido à diminuição do período para a aquisição de competência. A partir do estabelecimento das fases de aquisição de competência e indução da organogênese in vitro para MT e MT-Rg1, foram identificados genes diferencialmente expressos entre estes genótipos. Entre estes genes, CELL DIVISION CYCLE ASSOCIATED 7 e LACCASE 1A estão regulados positivamente em MT-Rg1 e todos estão fortemente relacionados à fase de aquisição de competência, e a alterações na proliferação de células do protoxilema durante o início da organogênese. Por outro lado, a resposta constitutiva à giberelina no mutante MT-pro reduz a formação de gemas caulinares e raízes e aumenta a formação de calos in vitro, sem afetar o tempo requerido para a indução de gemas caulinares e raízes. De forma oposta a MT-Rg1, o gene CDCA7 apresenta expressão reduzida durante a fase de aquisição de competência em MT-pro, diminuindo o número de células do protoxilema em divisão. Outro fator importante para a divisão celular no mutante MT-pro é o aumento da expressão do gene WUS, causando um aumento da proliferação das stem cells, que são células indiferenciadas relacionadas à formação de novos órgãos. Esta proliferação celular inadequada, somada a uma alteração desfavorável na homeostase das citocininas, justifica o efeito negativo do alelo pro na formação de gemas caulinares, o que permitiu a criação de um novo modelo para organogênese in vitro / Several studies have enabled the discovery of mechanisms to achieve in vitro organogenesis; however, little is known about the phase of acquisition of competence, essential for regeneration. A few genes have been identified to interfere in the acquisition of the competence phase in tomato (Solanum lycopersicum), but there are still many gaps to be filled. We have used the mutants, under the genetic background of the Micro-Tom cultivar, MT-Rg1 and MT-pro (procera), which positively or negatively affect in vitro organogenesis, respectively, to investigate gene expression and the hormonal control in the phase of acquisition of competence. Despite the fact that the constitutive gibberellin response in the procera mutant is well-established, the molecular identity of RG1 gene remains unknown. The MT-Rg1 mutant presents an increase in the formation of both shoot and roots and a reduced period for the induction of these organs, because of the reduced time required for acquisition of competence.We searched for the identity of differentially expressed genes between MT and MT-Rg1 after the establishment of the competence acquisition phase and organogenesis induction stages. Among those genes, CDCA7 and LAC1A are upregulated in MT-Rg1 and these genes appear to be strongly related with the acquisition of competence phase and changes in proliferation of protoxylem cells during early organogenesis. The constitutive response to gibberellin in the MT-pro mutant decreases the formation of shoot and roots and increase in vitro calli formation, without reducing the induction phase of shoots and roots. Unlike MT-Rg1, MT-pro reduces the CDCA7L expression during the acquisition of competence phase, causing a reduction of the protoxylem dividing cells. Another important factor for cell division in MT-pro mutant is the increased expression of the WUS gene, leading to an abnormal proliferation of stem cells. Thereby, this abnormal cell proliferation, in addition to an unfavorable change in the cytokinin homeostasis, justify the negative effect of the pro allele in the shoot formation, which enabled the proposal of a new model for in vitro organogenesis

Orientador: Paulo Eduardo Martins Ribolla / Resumo: O Aedes aegypti é o mosquito mais sinantrópico e antropofílico da família Culicidae. Esta espécie sempre coabita com os seres humanos e é extremamente oportunista. A dispersão vetorial está diretamente relacionada à capacidade das fêmeas de encontrar um parceiro com sucesso em um cenário urbano geralmente irregular. No presente trabalho, investigamos alterações transcricionais em fêmeas de Ae. aegypti durante o processo de cortejo e após o acasalamento. Observamos uma alteração substancial na expressão gênica desencadeada logo após o contato com machos de Ae. aegypti, que por sua vez não estavam totalmente correlacionados com as alterações desencadeadas pelo contato. Após analisar genes compartilhados significativamente e diferencialmente regulados entre contato específico e inseminação, a maior parte da mudança transcriptômica observada desencadeada por contato é revertida após o acasalamento, indicando uma situação intermediária entre as condições virgens e de cortejo que supomos ser crucial para o sucesso do acasalamento. Após o contato, vários genes quimiossensoriais relacionados são reprimidos, especialmente proteínas de ligação a odorante. A maioria desses genes retorna a taxas de expressão mais altas após o acasalamento. Nenhum desses genes é significativamente regulado pelo encontro de uma espécie diferente, em nosso estudo, Aedes albopictus. Os resultados apresentados aqui podem ser aplicados a uma abordagem inovadora de controle, focada nos sistemas semioquímicos de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aedes aegypti is the most synanthropic and anthropophilic mosquito of Culicidae. This species always cohabits with humans and is extremely opportunistic. Vector dispersal is directly related to the ability of the females on successfully finding a mate in a generally patchy urban scenario. In the present work, we investigate transcriptional changes in Ae. aegypti females during the courtship process and after mating. We observe a substantial alteration in gene expression triggered just upon contact with Ae. aegypti males, which in turn was not fully correlated to the changes triggered by the contact. After analysing shared significant differentially regulated genes between conspecific contact and insemination, the major part of the observed transcriptomic change triggered by contact is reversed after mating, indicating an intermediary situation between naive and mating conditions that we hypothesize to be crucial for mating success. Upon contact, several chemosensory related genes are repressed, especially odorant binding proteins. Most of these genes return to higher expression rates after mating. None of these genes are significantly regulated by the encounter of a different species, Aedes albopictus. The results presented here might be applied to an innovative control approach focusing on the semiochemical systems of mosquitoes in an effort to disrupt undesirable host–insect interaction to reduce the risk of pathogen transmission to humans. / Mestre

abstract: Cancer researchers have traditionally used a handful of markers to understand the origin of tumors and to predict therapeutic response. Additionally, performing machine learning activities on disparate data sources of varying quality is fraught with inherent bias. The Caris Life Sciences Molecular Database (CMD) is an immense resource for discovery as it contains over 215,000 molecular profiles of tumors with consistently gathered clinical grade molecular data along with immense amounts of clinical outcomes data. This resource was leveraged to generate two artificial intelligence algorithms aiding in diagnosis and one for therapy selection. The Molecular Disease Classifier (MDC) was trained on 34,352 cases and tested on 15,473 unambiguously diagnosed cases. The MDC predicted the correct tumor type out of thirteen possibilities in the labeled data set with sensitivity, specificity, PPV, and NPV of 90.5%, 99.2%, 90.5% and 99.2% respectively when considering up to 5 predictions for a case. The availability of whole transcriptome data in the CMD prompted its inclusion into a new platform called MI GPSai (MI Genomic Prevalence Score). The algorithm trained on genomic data from 34,352 cases and genomic and transcriptomic data from 23,137 cases and was validated on 19,555 cases. MI GPSai can predict the correct tumor type out of 21 possibilities on 93% of cases with 94% accuracy. When considering the top two predictions for a case, the accuracy increases to 97%. Finally, a 67 gene molecular signature predictive of efficacy of oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer was developed - FOLFOXai. The signature was predictive of survival in an independent real-world evidence (RWE) dataset of 412 patients who had received FOLFOX/BV in 1st line and inversely predictive of survival in RWE data from 55 patients who had received 1st line FOLFIRI. Blinded analysis of TRIBE2 samples confirmed that FOLFOXai was predictive of OS in both oxaliplatin-containing arms (FOLFOX HR=0.629, p=0.04 and FOLFOXIRI HR=0.483, p=0.02). / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

Rodent malaria parasites (RMPs) serve as tractable models for experimental genetics, and as valuable tools to study malaria parasite biology and host-parasitevector interactions. Plasmodium vinckei, one of four RMPs adapted to laboratory mice, is the most geographically widespread species and displays considerable phenotypic and genotypic diversity amongst its subspecies and strains. The phenotypes and genotypes of P. vinckei isolates have been relatively less characterized compared to other RMPs, hampering its use as an experimental model for malaria. Here, we have studied the phenotypes and sequenced the genomes and transcriptomes of ten P. vinckei isolates including representatives of all five subspecies, all of which were collected from wild thicket rats (Thamnomys rutilans) in sub-Saharan Central Africa between the late 1940s and mid 1960s. We have generated a comprehensive resource for P. vinckei comprising of five high-quality reference genomes, growth profiles and genotypes of P. vinckei isolates, and expression profiles of genes across the intra-erythrocytic developmental stages of the parasite. We observe significant phenotypic and genotypic diversity among P. vinckei isolates, making them particularly suitable for classical genetics and genomics-driven studies on malaria parasite biology. As part of a proof of concept study, we have shown that experimental genetic crosses can be performed between P. vinckei parasites to potentially identify genotype-phenotype relationships. We have also shown that they are amenable to genetic manipulation in the laboratory.

Lactate, a metabolic end product of glycolysis in mammals, has emerged as an important energy substrate for the brain. In addition to its energetic role, lactate was shown to modulate the excitability of neurons, to have a neuroprotective role and to participate in long-term memory formation. One previous investigation from our group reported that lactate modulates 4 synaptic plasticity-associated genes and potentiates the activity of the N-Methyl-D-aspartic acid (NMDA) receptor, a major receptor type involved in glutamatergic neurotransmission. The current thesis aimed at first to extend these findings by examining genome-wide transcriptional responses to this metabolite in cortical neurons. Using ribonucleic acid(RNA) sequencing to evaluate expression changes in protein-coding genes, we found that lactate modulates robustly after 1h, 20 genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway and in synaptic plasticity in a NMDA receptor activitydependent manner and that nicotinamide adenine dinucleotide, reduced (NADH), but not pyruvate, reproduces the modulatory effects of lactate on 70% of all differentially expressed genes. In a time course experiment, genes modulated after lactate treatment for 6h and 24h were also identified; these are involved in 9 signaling pathways including circadian rhythm, drug addiction, and retrograde endocannabinoid signaling. Bioinformatics analyses indicated CREB1 and CREM as candidate master regulators of gene expression and the modulatory effect of lactate was prevented by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, indicating a role for this kinase in mediating lactate signaling. An examination of changes in dendritic spines’ morphology and density - a morphologicalcorrelate of synaptic plasticity – has shown that lactate modulated spine density changes induced by potassium chloride (KCl) and carbachol. An additional investigation described in this thesis indicated that different gut microbiota manipulations (germ-free, prebiotics, high-fat diet) regulated mRNA expression of genes involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) - a metabolic cooperation mechanism between astrocytes and glutamatergic neurons. Overall, the results of this thesis help to establish a role for lactate as a signaling molecule in the brain, highlight mechanisms implicated in its signaling, and open new avenues for investigation of links between the gut microbiota and brain energy metabolism.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21813号 / 農博第2326号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5185(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM