O gene, uma seqüência de nucleotídeos necessária para a síntese de moléculas funcionais, é transcrito e regulado por um conjunto de processos e fatores extremamente complexos. Entender o momento e o tecido em que os genes são expressos, as isoformas funcionais, as regiões controladoras e os fatores envolvidos na regulação da expressão de cada gene é um dos grandes desafios da biologia molecular moderna. Hoje, com a enorme quantidade de informações de seqüências genômicas e de transcriptomas, aliado ao desenvolvimento de métodos computacionais para agrupar e analisar estes dados em larga escala, o estudo dos fenômenos relacionados à transcrição e regulação gênica está passando por uma revolução. Por exemplo, é possível medir, concomitantemente, a expressão gênica de milhares de genes em diferentes tecidos, assim como identificar diversos fenômenos que atuam nestes genes. Neste trabalho nós desenvolvemos e aplicamos métodos computacionais no estudo de quatro temas envolvendo aspectos chave da transcrição e regulação gênica. No primeiro trabalho, nós abordamos a expressão gênica tecido-específica através do estudo dos genes expressos no cérebro e em dez regiões cerebrais de camundongo. No segundo trabalho, nós identificamos seqüências potencialmente envolvidas no controle da transcrição gênica através do estudo de motivos sobre representados na região promotora dos genes de receptores olfativos. No terceiro trabalho, analisamos o transcriptoma humano quanto a presença de eventos de retenção de intron, um tipo de splicing alternativo. No quarto trabalho, nós abordamos a complexidade do transcriptoma e a regulação da expressão gênica através do estudo de pares de genes senso-antisenso em humanos e camundongos. Em todos os trabalhos, obtivemos resultados que nos permitiram tirar conclusões específicas sobre cada fenômeno estudado e nos mostraram a importância de estudá-los através de uma abordagem em larga escala. Adicionalmente, verificamos que os nossos métodos computacionais foram eficientes e adequados para o estudo da transcrição e regulação gênica em Homo sapiens e Mus musculus. / Genes, nucleotide sequences necessary for the synthesis of functional molecules, are transcribed and regulated by extremely complex cellular and molecular processes. To understand when and in which tissues the genes are expressed, their functional isoforms, control regions and the factors involved in gene regulation is one of major challenges of modern molecular biology. Today, the availability of complete genome sequences and transcriptomes, together with the development of new computational methods allows the study of phenomena related to the transcription and gene regulation in a large scale. For example, it is possible to quantify, concomitantly, gene expression of thousands of genes in different tissues and analyze different aspects of their regulation. In this work we developed and applied computational methods to the study of four key aspects of gene transcription and regulation. In the first study, we addressed tissue specific gene expression through the study of genes that are preferentially expressed in the brain and ten different mouse brain regions. In the second study, we identified sequences that are potentially involved in the control of gene transcription through the study of motifs that are over represented in the promoter region of olfactory receptor genes. In the third study, we browsed the human for the presence of intron retention, a type of alternative splicing. In the fourth study, we addressed the transcriptoma complexity and gene expression regulation through the study of pair of sense-antisense genes in human and mouse. In all studies, our results allowed us to make specific conclusions about each phenomenon analyzed which showed us the importance of a large scale approach. In addition, we verified that our computational methods can be efficiently applied to the study of transcription and gene regulation in Homo sapiens and Mus musculus.

Les éléments LINE-1 (L1) sont une classe abondante de rétrotransposons représentant 17% du génome humain. La région 5’UTR des sous-familles les plus récentes (L1PA1 à 6) contient un promoteur bidirectionnel contenant non seulement un promoteur sens interne mais aussi un promoteur antisens, nommé ASP. Dans les cellules normales, l’un des mécanismes impliqués dans la régulation du promoteur de L1 est la méthylation ADN. Dans les tumeurs, une hypométhylation globale affectant notamment les L1 est observée. Il a été mis en évidence que cette hypométhylation pouvait induire la transcription, à partir de l’ASP, de transcrits chimères ou LCT (L1 Chimeric Transcript). Ces LCT sont composés en 5’ de la séquence L1 et se poursuivent dans la région génomique adjacente. Afin d’étudier l’impact pangénomique de cette dérégulation et son implication dans les processus tumoraux, un outil bio-informatique dédié, nommé CLIFinder, a été développé pour identifier dans des données de RNA-seq paired-end orientés des LCT putatifs. Les RNA-seq de 13 gliomes, qui sont les cancers du cerveau les plus fréquents chez l’adulte, et de 3 tissus cérébraux contrôles ont été étudiés. CLIFinder identifie 2675 chimères dans les gliomes dont 84% impliquent des L1 récents (PA1 à 7) pleine taille supposés posséder un ASP fonctionnel et 50% sont détectées spécifiquement dans les échantillons tumoraux. 78 chimères correspondent à des LCT déjà décrits dans la littérature. De même, l’étude de RNA-seq d’autres types tumoraux (lignée MCF7 et métastases ovariennes) par CLIFinder identifie des chimères en commun suggérant une récurrence de certaines d’entre elles. L’étude d’un groupe de chimères par marche en 5’ par RT-PCR valide que 89% (56/63) des chimères impliquant des L1 récents (L1PA1 à PA7) sont initiées dans la région de l’ASP et correspondent à des LCT alors que toutes les chimères testées impliquant des L1PA8 sont initiées en amont de cette région. Des études de RT-qPCR sur une cohorte plus large de 51 gliomes montrent que les 56 LCT testés, incluant des LCT spécifiques de tumeurs, sont exprimés non seulement dans les tumeurs mais aussi dans les contrôles. Par contre, 70% des LCT spécifiques de tumeurs, montrent alors une surexpression tumorale significative. Ces résultats suggèrent donc une transcription basale provenant de l’ASP dans les tissus normaux et que la dérégulation transcriptionnelle liées aux LCT dans les gliomes passe par une surexpression. Par ailleurs, afin de déterminer le ou les mécanismes sous-jacents impliqués dans l’augmentation de l’activité transcriptionnelle de l’ASP, deux hypothèses ont été testées. La première implique l’hypométhylation du promoteur de L1. Toutefois mes résultats tendent à réfuter cette hypothèse puisqu’aucune diminution de la méthylation ADN n’est retrouvée au niveau de la région promotrice des L1 impliqués dans la transcription de LCT surexprimés. Par contre, les gènes associés à des LCT dont l’expression est dérégulée en contexte tumoral présentent une dérégulation dans le même sens que celle du LCT. De plus, les variations d’expression de gènes corrèlent systématiquement avec celle des LCT correspondants. Ceci suggère qu’une augmentation d’activité transcriptionnelle aux loci des LCT serait responsable de la surexpression de ceux-ci. Enfin 2 LCT candidats surexprimés et ayant un potentiel de biomarqueur prédictif de la survie des patients, pourraient jouer un rôle fonctionnel dans l’initiation, la progression et/ou l’agressivité tumorale. En conclusion, mes travaux ont validé que CLIFinder se positionne comme un outil pertinent permettant d’identifier, de façon pangénomique, les LCT exprimés dans différents types tumoraux à partir de données de RNA-seq paired-end orientées. L’observation d’une récurrence ainsi que d’une surexpression tumorale de certains LCT suggère qu’ils pourraient jouer un rôle fonctionnel dans les processus de tumorigenèse. / LINE-1 (L1) is the most abundant class of retrotransposons which represents 17% of the human genome. The 5’ region of the youngest L1 sub-families (L1PA1 to 6) contains a bidirectional promoter consisting, in addition to the internal sense promoter, of an antisense promoter, called ASP. In normal cells, the main defense mechanism, developed to counteract the deleterious effect of L1 activity, consists in L1 promoter DNA methylation. A hallmark of cancer genomes consists in a global DNA hypomethylation which affects especially L1 promoters. In tumors, evidences suggest that this hypomethylation could result in transcription from ASP of aberrant L1-Chimeric Transcripts (LCTs) composed of L1 5’end and its adjacent sequence. To investigate the pangenomic extent of this transcriptional deregulation and its impact in tumoral processes, a dedicated bioinformatic tool, CLIFinder, was designed to select putative LCTs among RNA-seq oriented paired-end reads. RNA-seq analyses of 13 gliomas, which are the most common brain cancer in adults, and 3 control brains were performed.CLIFinder identifies 2675 chimeras in gliomas, among which 84% involves recent L1 (PA1 to 7) full size, supposed to possess a functional ASP, and 50% are detected specifically in tumors samples. 78 chimeras correspond to LCT already described in literature. In addition, study of additional RNA-seq data from other tumor types (MCF7 and ovarian metastasis) by CLIFinder identifies common chimeras suggesting that some of them can be recurrent. The analysis of a group of chimeras by 5’ walk RT-PCR validate that 89% (56/63) of chimeras implying recent L1 (L1PA1 to 7) are initiated at the ASP region and therefore correspond to LCT; whereas all tested chimeras implying an L1PA8 element are transcribed from an upstream region. RT-qPCR studies on a larger cohort of 51 gliomas show that all 56 tested LCT, even identified by CLIFinder as “tumor specific”, are not only expressed in tumors but also in controls. Nevertheless, 70% of the “tumor specific” LCTs are significantly overexpressed in tumors. My results suggest that, even L1 5’ UTR methylation, some ASP are active in normal tissues and lead to a basal LCT expression in normal tissues. Moreover, a transcriptional deregulation linked to LCTs in tumors exists and implies a LCTs’ overexpression.In order to determine the underlying mechanisms involved in the increase of transcriptional activity of ASP, two hypothesis were tested. The first one implies L1 promoter hypomethylation. My results tend to refute this hypothesis because no decrease of the DNA methylation is found at the promoter region of L1 linked to overexpressed LCTs. On the other hand, the genes associated to LCT presenting an expression deregulation in tumors demonstrate a deregulation in the same way. Moreover, gene expression variations correlates systematically with the one corresponding LCTs. This suggests that an increase of transcriptional activity at the LCTs loci would be responsible of their overexpression. Finally, 2 candidate LCT overexpressed and presenting as potential predictive biomarkers for patient’s survival, could play a functional role in initiation, progression and/or the tumoral aggressiveness.In conclusion, my work has validated CLIFinder as a useful tool to identify, at pangenomic level, LCTs expressed in different tumor types from paired-end stranded RNA-seq data. The observation of the recurrence and tumoral overexpression for some LCTs suggests that they may play a functional role in tumoral processes.

Les métastases osseuses sont des complications fréquentes de nombreux cancers tels que le cancer du poumon, de la prostate et du sein. Chez la femme, 70% des patientes présentant un cancer du sein avancé développent des métastases qualifiées d’ostéolytiques. Le développement de ce type de métastases entraine la destruction massive de l’os causant chez les patientes des fractures pathologiques. Les traitements actuellement dispensés en clinique tels que les bisphosphonates et le dénosumab (anti-RANKL) ne sont pas curatifs mais seulement palliatifs. Pour cette raison, notre laboratoire s’intéresse particulièrement aux évènements moléculaires et cellulaires précoces aboutissant à l’émergence de métastases osseuses. Les récepteurs Roundabout (ROBO) ont été initialement décrits comme des régulateurs cruciaux de la migration neuronale et vasculaire lors du développement. ROBO4 est le dernier récepteur décrit et diverge des autres récepteurs ROBO, notamment par son expression spécifique et limitée aux cellules endothéliales et hématopoïétiques. Toutefois, lors d’une analyse transcriptomique comparative le récepteur ROBO4 a été retrouvé surexprimé dans la lignée tumorale ostéotropique MDA-B02. Les récentes expériences, réalisées par notre équipe, visant à inhiber l’activité du récepteur ROBO4, nous ont permis de considérer le récepteur ROBO4 comme un médiateur de l’ancrage à l’os des cellules tumorales MDA-B02. En effet, L’utilisation d’un anticorps anti-ROBO4 réduit considérablement l'adhérence des cellules MDA-B02 sur les cellules ostéoblastiques MC3T3-E1 in vitro et l’ancrage à l’os in vivo. La surexpression de ROBO4 dans un modèle cellulaire de cancer du sein murin nous a conduit à mettre en évidence la présence de deux formes de la protéine ROBO4 aux propriétés antagonistes : (i) une forme glycosylée détectée exclusivement dans les cellules endothéliales et (ii) une forme non glycosylée, issue d’un transcrit alternatif et présente en forte quantité dans les cellules tumorales MDA-B02. L’identification d’un variant protéique du récepteur ROBO4 aux propriétés fonctionnelles et structurales différentes nous a permis d’envisager de nouvelles pistes dans le décryptage des fonctions de ROBO4 dans le processus tumoral et pourrait conduire à la mise au point de thérapies innovantes pour empêcher la formation de métastases dans la moelle osseuse / Bone metastases are frequent complications of many cancers such as lung, prostate and breast cancer. In women, 70% of patients with advanced breast cancer develop metastases known as osteolytic. The development of this type of metastasis leads to the mass destruction of bone causing pathological fractures in patients. Current clinical treatments such as bisphosphonates and denosumab (anti-RANKL) only slow the progression of the disease. For this reason, our laboratory is particularly interested in early molecular and cellular events leading to the emergence of bone metastases. Roundabout receptors (ROBO) were initially described as crucial regulators of neuronal and vascular migration during development. ROBO4 is the last receptor described and diverges from other ROBO receptors, in particular by its specific expression and limited to endothelial and hematopoietic cells. However, in a comparative transcriptomic analysis, the ROBO4 receptor was found to be overexpressed in the MDA-B02 osteotropic tumor line. Recent experiments carried out by our team, aiming at inhibiting the activity of the ROBO4 receptor, allowed us to consider the ROBO4 receptor as a mediator of MDA-B02 tumor cells anchorage to the bone. Indeed, the use of an anti-ROBO4 antibody considerably reduces the adhesion of the MDA-B02 cells to the MC3T3-E1 osteoblastic cells in vitro and the anchoring to the bone in vivo. Overexpression of ROBO4 in a murine breast cancer cell model led us to demonstrate the presence of two forms of the ROBO4 protein with antagonistic properties: (i) a glycosylated form detected exclusively in endothelial cells and (ii) one unglycosylated form derived from an alternative transcript and present in high amounts in MDA-B02 tumor cells. The identification of a protein variant of the ROBO4 receptor with different functional and structural properties has allowed us to consider new opportunities in decoding the functions of ROBO4 in the tumor process and could lead to the development of innovative therapies to prevent the formation of metastases in the bone marrow

Orientador: Michel Eduardo Beleza Yamagishi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T17:21:19Z (GMT). No. of bitstreams: 1 Herai_RobertoHirochi_D.pdf: 3625854 bytes, checksum: 3f19d10a9b0bb7f77091197cd302f66e (MD5) Previous issue date: 2010 / Resumo: A grande quantidade de dados biológicos gerados recentemente permitiu verificar que os genomas são repletos de seqüências repetitivas (SR), como microsatélites e elementos genéticos móveis, altamente improváveis de ocorrer estatisticamente se os genomas fossem gerados a partir de uma distribuição aleatória de nucleotídeos. Tal comprovação motivou a classificação de tais seqüências e também a construção de diversas ferramentas de bioinformática, além de mecanismos de armazenamento baseados em sistemas de gerenciamento de bancos de dados (SGBD) para permitir localizá-las e armazená-las para posterior estudo. Entretanto, foi com a comprovação biológica da importância das SR, como no mecanismo de interferência por RNAi (SR reversa complementar), que as SR despertaram maior interesse por parte da comunidade científica. Atualmente, já há fortes evidências que associam as SR com fenômenos biológicos bastante interessantes, como o processamento de RNA por cis-splicing e a formação de transcritos quiméricos, freqüentes em organismos inferiores e muito raro em organismos superiores. Tais tipos de transcritos podem ser gerados a partir de trans-splicing ou, como conjecturamos nesse trabalho, pela transposição de elementos genéticos móveis (como por exemplo transposons ou retrotransposons). Em virtude disso, este projeto propõe a construção de metodologias de Bioinformática, disponibilizadas na WEB, para detectar transcritos quiméricos em genomas de organismos, tanto em versões draft ou em alta qualidade, e também estudar as SR que ocorrem no locus gênico dos transcritos envolvidos na formação de uma seqüência quimérica. As ferramentas propostas permitiram identificar, a partir de bibliotecas de transcritos de full-length cDNA, tanto de humanos quanto de bovinos, novos transcritos quiméricos provenientes de células de tecidos normais, e que não seguem splice-sites canônicos na região de fusão dos transcritos envolvidos. Além disso, as seqüências encontradas apresentam uma elevada taxa de concentração de pares de SR do tipo reverso complementar no locus gênico dos dois transcritos que formam a seqüência quimérica. As ferramentas propostas podem ser utilizadas para outros organismos e direcionar trabalhos experimentais para tentar comprovar em bancada novos transcritos quiméricos, tanto em organismos inferiores quanto em superiores / Abstract: The recent availability of a huge amount of biological data allowed to know about the high concentration of repetitive sequences (SR) like microsatellites and genetic mobile elements in different genomes. Repetitive sequences are improbable to occur statistically if genome data were generated by a random distribution of nucleotides. Such observation motivated the classification of repetitive sequences, and the construction of several bioinformatics tools. Furthermore, several mechanisms to store repetitive sequences, which are based on data base management systems (DBMS) were proposed and created. They can be used to search for specific sequences to make a posteriori study. However, it was with the biological confirmation of the importance of repetitive sequences, like by the RNA interference (reverse complement, or inverted repeat) mechanism, that the scientific community gained more interest by such sequences. Actually, there is strong evidence that associates the repetitive sequences with some interesting biological phenomena, like in RNA processing by cis-splicing, and in chimeric transcript formation mechanism. This last one is very frequently in inferior organism, but rare in superior organisms. Such types of transcripts can be generated by trans-splicing, or like conjectured in this work, by the retrotransposition of mobile genetic elements (like transposons or retrotransposons). In this way, this work proposed the construction of several Bioinformatics methodologies, available in the WEB, to detect new evidences of chimeric transcripts in genomes of different organisms, both in draft genome and in high quality genome assemblage. We also studied repetitive sequences in gene loci of the involved transcripts in a chimeric sequence formation. The proposed tools allowed us to identify, using a full-length cDNA databank, new chimeric transcript candidates in human and in bovine genome. They are from cells of normal tissues, and do not follow canonical splice-sites in the fusion region of the involved transcripts. Moreover, it was possible to show that the detected sequences have high concentration pairs of reverse complement type of repetitive sequences in gene loci of the two involved transcripts, which originated a new chimeric transcript candidate. The created bioinformatics tools can be used in other organisms in addition to the one used in this work, leading to the proposition of new experimental work to try to prove in vivo new chimeric transcripts, both in superior organism and in inferior organism / Doutorado / Bioinformatica / Doutor em Genetica e Biologia Molecular

O gene, uma seqüência de nucleotídeos necessária para a síntese de moléculas funcionais, é transcrito e regulado por um conjunto de processos e fatores extremamente complexos. Entender o momento e o tecido em que os genes são expressos, as isoformas funcionais, as regiões controladoras e os fatores envolvidos na regulação da expressão de cada gene é um dos grandes desafios da biologia molecular moderna. Hoje, com a enorme quantidade de informações de seqüências genômicas e de transcriptomas, aliado ao desenvolvimento de métodos computacionais para agrupar e analisar estes dados em larga escala, o estudo dos fenômenos relacionados à transcrição e regulação gênica está passando por uma revolução. Por exemplo, é possível medir, concomitantemente, a expressão gênica de milhares de genes em diferentes tecidos, assim como identificar diversos fenômenos que atuam nestes genes. Neste trabalho nós desenvolvemos e aplicamos métodos computacionais no estudo de quatro temas envolvendo aspectos chave da transcrição e regulação gênica. No primeiro trabalho, nós abordamos a expressão gênica tecido-específica através do estudo dos genes expressos no cérebro e em dez regiões cerebrais de camundongo. No segundo trabalho, nós identificamos seqüências potencialmente envolvidas no controle da transcrição gênica através do estudo de motivos sobre representados na região promotora dos genes de receptores olfativos. No terceiro trabalho, analisamos o transcriptoma humano quanto a presença de eventos de retenção de intron, um tipo de splicing alternativo. No quarto trabalho, nós abordamos a complexidade do transcriptoma e a regulação da expressão gênica através do estudo de pares de genes senso-antisenso em humanos e camundongos. Em todos os trabalhos, obtivemos resultados que nos permitiram tirar conclusões específicas sobre cada fenômeno estudado e nos mostraram a importância de estudá-los através de uma abordagem em larga escala. Adicionalmente, verificamos que os nossos métodos computacionais foram eficientes e adequados para o estudo da transcrição e regulação gênica em Homo sapiens e Mus musculus. / Genes, nucleotide sequences necessary for the synthesis of functional molecules, are transcribed and regulated by extremely complex cellular and molecular processes. To understand when and in which tissues the genes are expressed, their functional isoforms, control regions and the factors involved in gene regulation is one of major challenges of modern molecular biology. Today, the availability of complete genome sequences and transcriptomes, together with the development of new computational methods allows the study of phenomena related to the transcription and gene regulation in a large scale. For example, it is possible to quantify, concomitantly, gene expression of thousands of genes in different tissues and analyze different aspects of their regulation. In this work we developed and applied computational methods to the study of four key aspects of gene transcription and regulation. In the first study, we addressed tissue specific gene expression through the study of genes that are preferentially expressed in the brain and ten different mouse brain regions. In the second study, we identified sequences that are potentially involved in the control of gene transcription through the study of motifs that are over represented in the promoter region of olfactory receptor genes. In the third study, we browsed the human for the presence of intron retention, a type of alternative splicing. In the fourth study, we addressed the transcriptoma complexity and gene expression regulation through the study of pair of sense-antisense genes in human and mouse. In all studies, our results allowed us to make specific conclusions about each phenomenon analyzed which showed us the importance of a large scale approach. In addition, we verified that our computational methods can be efficiently applied to the study of transcription and gene regulation in Homo sapiens and Mus musculus.

We have studied the effect of interferon α (IFN-α) on MHC class II expression by human buccal epithelial cells (BEC), and mRNA expression by BEC and mucosal-associated mononuclear cells (MAMC). In 6 experiments, freshly collected BEC were suspended at a concentration of 1.0 x 105/ml in RPMI 1640 and incubated in the presence of 0-10000 IU/ml of human lymphoblastoid IFN-α (HuIFN-α). Zero and six hour samples were analyzed by single color flow cytometry using FITC-labeled murine IgG1 monoclonal antibody to HLA-DR. Preparations were also analyzed for expression of cytokine transcripts (IL-2 IL-4, IL-5, IL-6, IL-8, IFN-γ, GM-CSF) by reverse transcriptase polymerase chain reaction (RT-PCR). Increasing concentrations of IFN-α resulted in proportionate increases in the percentage of HLA-DR + BEC (r = 0.7897, p = 0.0627) and in the percentage of HLA-DR + staining at higher intensities (101 to 102 log fluorescence intensity) (LFI) (r = 0.40l0, p = 0.0424). The percentage of HLA-DR + BEC rose from a mean of 1.5% with no IFN-α to 7% with 10000 IU/ml IFN-α (p < 0.05). The percentage of HLA-DR + BEC staining at 101 to 102 LFI rose from a mean of 8.3% with no added IFN-α to 19.2% with 10000 IU/ml IFN-α (p <0.05). Unstimulated BEC constitutively expressed IL-8 and GM-CSF. IFN-α stimulated preparations also expressed IFN-γ, possibly due to the presence of MAMC, which comprised 2-9% of the total cell population. These data indicated that HuIFN-α upregulates MHC class II expression by human BEC, possibly by enhancing IFN-γ production by MAMC present in the culture preparations.

No description available.

The presence of the translocation t(9;22)(q34;q11), leading to the BCR::ABL1 fusion transcript, is the hallmark of chronic myeloid leukemia (CML). Nevertheless, atypical presentation at diagnosis can be challenging. However, although most patients with CML are diagnosed with the e13a2 or e14a2 BCR:: ABL1 fusion transcripts, about 5% of them carry rare BCR::ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3, and e6a2. In particular, the e6a2 fusion transcript has been associated with clinically aggressive disease frequently presenting in accelerated or blast crisis phases. To date, there is limited evidence on the efficacy of front-line second-generation tyrosine kinase inhibitors for this genotype. Here, we report two patients, in whom the diagnosis of CML was challenging. The use of primers recognizing more distant exons from the common BCR::ABL1 breakpoint region correctly identified the atypical BCR::ABL1 e6a2 fusion transcript. Treatment with the second-generation tyrosine kinase inhibitor nilotinib was effective in our patient expressing the atypical e6a2 BCR::ABL1 fusion transcript.

Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack. In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools. Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods. Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.

The regulation of glucagon-like peptide-1 and peptide YY hormone levels are regulated based on different influential factors, but primarily levels are dependent upon ingested food content. As meals today become more fat-enriched, there is greater requirement for evaluation of these hormones that regulate insulin and satiety levels within the body. We have shown that the gene expression transcript production of glucagon-like peptide-1 and peptide YY are modulated by different concentrations, and times of short-chain fatty acids and long-chain fatty acids. Although the peptide hormone levels have the influential physiological role on effector tissue, the regulation of these hormones begins at the transcript levels. Recent research indicates that glucagon-like peptide-1 and peptide YY hormones are altered in response to different free-fatty acids. The present investigation generally demonstrated an overall decrease in both hormones after chronic exposure to fatty acids. Intestinal secretin tumor cell line (STC-1 cells) was used as a representative for intestinal L-cells. Quantitative real-time PCR analysis was used to determine the changes in RNA transcripts. Overall, there was a decrease in the 3-hour timeline, which continued to decrease in the 16-hour and 24-hour timelines for glucagon-like peptide-1. Peptide YY transcript expression in 3-hours increased significantly after exposure to propionate, a significant decrease after exposure to acetate, and no significant increase or decrease after exposure to butyrate. However, there was a significant decrease in peptide YY once reaching 24-hour exposure. It was determined there is a threshold for different concentrations of free-fatty acids to influence glucagon-like peptide-1 and peptide YY production, which was present in the different concentrations of butyrate. Lastly, exposure to both concentrations of linolenic acid caused a significant decrease in glucagon-like peptide-1 and peptide YY.