The effect of temperature on the gill morphology and ion transporter distribution in the branchial epithelium of a freshwater teleost, the Koi carp (Cyprinus carpio, L.) was examined. Three different water temperatures were used to detect changes in expression level of transporter proteins in the gill epithelial cells. With increasing temperature, the expression level of all three ion transporters studied declined, and the gill lamellae protruded out of the cell mass, thus increasing the surface area of the branchial epithelium. A hypothetical organization of the transporter proteins within the ionocytes is proposed.

Massively parallel whole transcriptome sequencing and its ability to generate full transcriptome data at the single transcript level provides a powerful tool with multiple interrelated applications, including transcriptome reconstruction, gene/isoform expression estimation, also known as transcriptome quantification. As a result, whole transcriptome sequencing has become the technology of choice for performing transcriptome analysis, rapidly replacing array-based technologies. The most commonly used transcriptome sequencing protocol, referred to as RNA-Seq, generates short (single or paired) sequencing tags from the ends of randomly generated cDNA fragments. RNA-Seq protocol reduces the sequencing cost and significantly increases data throughput, but is computationally challenging to reconstruct full-length transcripts and accurately estimate their abundances across all cell types. We focus on two main problems in transcriptome data analysis, namely, transcriptome reconstruction and quantification. Transcriptome reconstruction, also referred to as novel isoform discovery, is the problem of reconstructing the transcript sequences from the sequencing data. Reconstruction can be done de novo or it can be assisted by existing genome and transcriptome annotations. Transcriptome quantification refers to the problem of estimating the expression level of each transcript. We present a genome-guided and annotation-guided transcriptome reconstruction methods as well as methods for transcript and gene expression level estimation. Empirical results on both synthetic and real RNA-seq datasets show that the proposed methods improve transcriptome quantification and reconstruction accuracy compared to previous methods.

Antisense RNA (asRNA) strategies are identified as an effective and specific method for gene down-regulation at the post-transcriptional level. In this study, the major purpose is to find a correlation between the expression level and minimum free energy to enable the design of specific asRNA fragments. The thermodynamics of asRNA and mRNA hybridization were computed based on the fluorescent protein reporter genes. Three different fluorescent proteins (i) green fluorescent protein (GFP), (ii) cyan fluorescent protein (CFP) and (iii) yellow fluorescent protein (YFP) were used as reporters. Each fluorescent protein was cloned into the common pUC19 vector. The asRNA fragments were randomly amplified and the resulted antisense DNA fragments were inserted into the constructed plasmid under the control of an additional inducible plac promoter and terminator. The expression levels of fluorescent reporter protein were determined in real time by plate reader. Different results have been observed according to the fluorescent protein and the antisense fragment sequence. The CFP expression level was decreased by 50 to 78% compared to the control. However, with the GFP, the down-regulation did not exceed 30% for the different constructs used. For certain constructs, the effect was the opposite of expected and the expression level was increased. In addition, the YFP showed a weak signal compared to growth media, therefore the expression level was hard to be defined. Based on these results, a thermodynamic model to describe the relationship between the particular asRNA used and the observed expression level of the fluorescent reporter was developed. The minimum free energy and binding percentage of asRNA-mRNA complex were computed by NUPACK software. The expression level was drawn as a function of the minimum free energy. The results showed a weak correlation, but linear trends were observed for low energy values and low expression levels the CFP gene. The linear aspect is not verified for higher energy values. These findings suggest that the lower the energy is, the more stable is the complex asRNA-mRNA and therefore more reduction of the expression is obtained. Meanwhile, the non-linearity involves that there are other parameters to be investigated to improve the mathematical correlation. This model is expected to offer the chance to "fine-tune" asRNA effectiveness and subsequently modulate gene expression and redirect metabolic pathways toward the desired component. In addition, the investigation of the localization of antisense binding indicates that there are some regions that favors the hybridization and promote hence the down-regulation mechanisms. / Master of Science

The fundamental goal of proteomics is to gain an understanding of the expression and function of the proteome on the level of individual proteins, on the level of defined cell types and on the level of the entire organism. In this thesis, the human proteome is explored using membrane protein topology prediction methods to define the human membrane proteome and by global protein expression profiling, which relies on a complex study of the location and expression levels of proteins in tissues and cells. A whole-proteome analysis was performed based on the predicted protein-coding genes of humans using a selection of membrane protein topology prediction methods. The study used a majority decision-based method, which estimated that approximately 26% of the human genes encode for a membrane protein. The prediction results are displayed in a visualization tool to facilitate the selection of antigens to be used for antibody generation. Global protein expression profiles in a large number of cells and tissues in the human body were analyzed for more than 4000 protein targets, based on data from the antibody-based immunohistochemistry and immunofluorescence methods within the framework of the Human Protein Atlas project. The results revealed few cell-type specific proteins and a high fraction of human proteins expressed in most cells, suggesting that cell and tissue specificity is attained by a fine-tuned regulation of protein levels. The expression profiles were also used to analyze the relationship between 45 cell lines by hierarchical clustering and principal component analysis. The global protein expression patterns overall reflected the tumor origin of the cells, and also allowed for identification of proteins of importance for distinguishing different categories of cell lines, as defined by phenotype of progenitor cell. In addition, the protein distribution in 16 subcellular compartments in three of the human cell lines was mapped. A large fraction of proteins were localized in two or more compartments and, in line with previous results, a majority of proteins were detected in all three cell lines. Finally, mass spectrometry-based protein expression levels were compared to RNA-seq-based transcript expression levels in three cell lines. Highly ubiquitous mRNA expression was found and the changes of expression levels between the cell lines showed high correlations between proteins and transcripts. Large general differences in abundance of proteins from various functional classes were observed. A comparison between categories based on expression levels revealed that, in general, genes with varying expression levels between the cell lines or only expressed in one cell line were highly enriched for cell-surface proteins. These studies show a path for a systematic analysis to characterize the proteome in human cells, tissues and organs. / QC 20110317 / The Human Protein Atlas project

Submitted by Kian Eghrari Moraes null (kianem@gmail.com) on 2017-03-14T19:44:37Z No. of bitstreams: 1 Dissertação_Kian_Eghrari_Moraes.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-21T19:54:29Z (GMT) No. of bitstreams: 1 moraes_ke_me_jabo.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) / Made available in DSpace on 2017-03-21T19:54:29Z (GMT). No. of bitstreams: 1 moraes_ke_me_jabo.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os híbridos de milho transgênicos, em geral, apresentam o locus transgênico em hemizigose. O objetivo do trabalho foi avaliar o efeito do número de alelos transgênicos em híbridos de milho em relação à expressão de Cry1F nas folhas, ataque de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) e a produtividade de grãos, utilizando cinco híbridos isogênicos nas versões transgênicas homozigota e hemizigota, além da versão convencional de um dos híbridos. O nível de expressão da proteína Cry1F (PRYF) foi quantificado pela técnica de ELISA. Nos experimentos de campo, conduzidos na primeira e segunda safras do ano agrícola 2015/2016, avaliou-se o ataque de S. frugiperda em campo (ALC), por infestação natural, e a produtividade de grãos (PG). Dois bioensaios foram realizados em laboratório para avaliar a sobrevivência larval de S. frugiperda de 1º instar (SL) alimentadas com as folhas dos híbridos. Os híbridos transgênicos, homozigotos e hemizigotos, não apresentaram silenciamento gênico. Os híbridos homozigotos apresentaram maior concentração de proteína Cry1F. Quando houve elevado ALC, na primeira safra, os híbridos transgênicos foram superiores à testemunha convencional na PG, entretanto, não houve diferença entre os híbridos homozigotos e hemizigotos. Os híbridos transgênicos também foram superiores à testemunha convencional nos bioensaios, sendo que os homozigotos apresentaram as menores médias de SL. A presença de um alelo transgênico a mais nos híbridos homozigotos propiciou comportamento genético aditivo para a expressão de Cry1F e controle de S. frugiperda, diminuindo ALC e SL, sem diminuir a capacidade produtiva das plantas. Diante do exposto, não há limitações para a utilização de híbridos de milho homozigotos para o transgene, pois apresentaram melhor controle de S. frugiperda em comparação com os híbridos hemizigotos. / Genetically modified (GM) maize hybrids, in general, possess the transgenic locus in a hemizygous state. The aim of the study was to assess the effect of the number of transgenic alleles in maize hybrids regarding the Cry1F leaf expression, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) attack and grain yield, through the evaluation of five isogenic hybrids in the homozygous and hemizygous transgenic versions and a non-GM hybrid. Cry1F protein expression levels (PRYF) were quantified by ELISA. In field experiments conducted during the summer and autumn seasons of 2015/2016, we assessed the leaf-feeding injury of S. frugiperda in the field (LFI) by natural infestation and grain yield (GY). Two bioassays were carried out in the laboratory to evaluate the survival of first-instar S. frugiperda larvae (SL) fed with the maize hybrids. Transgenic hybrids did not present gene silencing. Homozygous hybrids presented higher Cry1F expression levels than hemizygous hybrids. With high LFI during the summer season, transgenic hybrids were superior to the non-GM for GY, however, there was no difference between homozygous and hemizygous hybrids. The transgenic hybrids were superior to the non-GM hybrid in the bioassays, and the homozygotes caused the highest mortality of S. frugiperda. The addition of one transgenic allele in the homozygous hybrids provided an additive genetic effect, increasing PRYF and S. frugiperda control, whereas GY was not affected. In conclusion, there are no limitations to the use of transgenic homozygous maize hybrids, which presented better S. frugiperda control comparing to hemizygous hybrids.

Despite an increase of biotechnical studies in third world countries, the bioinformatical side is largely lacking. In this paper we attempt to further the bioinformatical capabilities of east Af-rica. The project consisted of two teaching segments for east African doctorates, one as part of an academic workshop at ILRI, Kenya, and one in a small class at SLU, Sweden. The project also included the generation of two simple to use bioinformatical pipelines with the explicit aim to be reused by novice bioinformaticians from the very same region. The viability of the piplines were verified by generating transcriptional expression level differences for Paeni-bacillus polymyxa strain A26 and whole genome annotations for Setaria digitata. Both pipe-lines may have some merit for the collaborative effort between ILRI and SLU to annotate Eleusine coracana, a draught resilient crop, the annotation of which may save lives. The teaching material, source code for the pipelines and overall teaching impression have been included in this paper.

Polyploidization instantly doubles all genome content by combining two genomes that have markedly different methylation and gene expression levels. This process may be accompanied by genetic and epigenetic changes in each genome. Sequencing of the transcriptome (RNA-seq) and the methylome (bisulfite treated libraries whole genome libraries) were used to measure gene expression and methylation levels of genic regions of allopolyploid cotton petals and petals of their diploid relatives. Many differentially expressed genes detected by RNA-seq were consistent with expression levels previously detected by microarrays. RNA-seq results also reconfirmed the presence of general polyploid gene expression trends like expression level dominance and homoeologous expression biases in Gossypium polyploid species. Expression biases between A- and D-genome homoeologs and expression level dominance was characterized for thousands of genes in tetraploids and a diploid F1-hybrid. Unlike the results of microarray study previously done we found a slightly greater number of genes showing A-genome bias vs genes showing D-genome bias. More commonly the overall expression level from homoeologs of polyploid is heterotic i.e the expression level is greater than the average of the expression levels from the two parent genomes. In addition, genome methylation (CG, CHG, and CHH contexts) of each genome was assessed in the diploid and tetraploid samples. The A- and D-genomes had distinct levels of DNA methylation for each context. DNA methylation may be independently regulating homoeologous expression levels of a small number of genes.

Nous avons utilisé la décomposition par séparateurs minimaux complets. Pour décomposer un graphe G, il est nécessaire de trouver les séparateurs minimaux dans le graphe triangulé H correspondant. Dans ce contexte, nos premiers efforts se sont tournés vers la détection de séparateurs minimaux dans un graphe triangulé. Nous avons défini une structure, que nous avons nommée 'atom tree'. Cette dernière est inspirée du 'clique tree' et permet d'obtenir et de représenter les atomes qui sont les produits de la décomposition. Lors de la manipulation de données à l'aide de treillis de Galois, nous avons remarqué que la décomposition par séparateurs minimaux permettait une approche de type `Diviser pour régner' pour les treillis de Galois. La détection des gènes fusionnés, qui est une étape importante pour la compréhension de l'évolution des espèces, nous a permis d'appliquer nos algorithmes de détection de séparateurs minimaux complets, qui nous a permis de détecter et regrouper de manière efficace les gènes fusionnés. Une autre application biologique fut la détection de familles de gènes d'intérêts à partir de données de niveaux d'expression de gènes. La structure de `l'atom tree' nous a permis d'avoir un bon outils de visualisation et de gérer des volumes de données importantes. / We worked on clique minimal separator decomposition. In order to compute this decomposition on a graph G we need to compute the minimal separators of its triangulation H. In this context, the first efforts were on finding a clique minimal separators in a chordal graph. We defined a structure called atom tree inspired from the clique tree to compute and represent the final products of the decomposition, called atoms. The purpose of this thesis was to apply this technique on biological data. While we were manipulating this data using Galois lattices, we noticed that the clique minimal separator decomposition allows a divide and conquer approach on Galois lattices. One biological application of this thesis was the detection of fused genes which are important evolutionary events. Using algorithms we produced in the course of along our work we implemented a program called MosaicFinder that allows an efficient detection of this fusion event and their pooling. Another biological application was the extraction of genes of interest using expression level data. The atom tree structure allowed us to have a good visualization of the data and to be able to compute large datasets.