Prenatal mortality in the swine ranges from 30%~40%. Little is known about genes that involve in the preovulation events that the initiation of swine oocyte development. The main objective of this study was to utilize suppression subtractive hybridization¡]SSH¡^to delineate the differential gene expression between in vitro culture mature and antral-follicle oocytes of swine development. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryos. Porcine ovaries obtained from the slaughterhouse were used to collect oocytes from follicle with a diameter ≥ 3 mm. After in vitro mature for two days, oocytes with first polar body were subjected to as the testers and were lysed for mRNA extraction. Pools of 26 denuded oocytes without culture were submitted to suppressive subtraction hybridization (SSH) as the drivers. Forward and reverse subtractions were performed to identify candidate genes differentially expressed between in vitro culture mature and primordial-follicle oocytes. A total of one hundred and thirty-five differential expressed plasmid clones were sequenced, and each was analyzed by BLAST programs. Of these transcripts, 40 clones were subjected to differential screening by a dot blot cDNA array. We identified three genes like zona pellucida glycoprotein (ZP1), 1-aminocyclopropane-1-carboxylate synthase (ACS), and death associated protein 5¡]DAP 5¡^while other numerous clones remain novel. The in vivo functions of the genes remain further investigation.

RNA sequencing is a powerful new approach to analyzing differential expression of transcripts between treatments. Many statistical methods are now available to test for differential expression, each one reports results differently. This thesis presents a workflow of five popular methods and discusses the results. A pipeline was built in the R language to analyze four of these packages using a real RNA-seq dataset. At present, researchers must prepare RNA-seq data prior to analysis to achieve reliable results. Filtering is a necessary preparatory step in which transcripts exhibiting low levels of genetic expression are removed from further analysis. Yet, little research is available to guide researchers on how best to choose this threshold. This thesis introduces a study designed to determine if the choice of filter threshold has a significant effect on individual package performance. Increasing the filtering threshold was shown to decrease the sensitivity and increase the specificity of the four statistical methods studied. / Graduate

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Submitted by ANA PAULA SBARDELLA null (paulasbardella@gmail.com) on 2016-08-30T12:48:56Z No. of bitstreams: 1 ANAPAULASBARDELLA_GENÉTICA_E_MELHORAMENTO_ANIMAL_MESTRADO_VERSÃO_FINAL.pdf: 2368521 bytes, checksum: c62c877ae578820d87db27a37651a178 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-31T12:58:56Z (GMT) No. of bitstreams: 1 sbardella_ap_me_jabo.pdf: 2368521 bytes, checksum: c62c877ae578820d87db27a37651a178 (MD5) / Made available in DSpace on 2016-08-31T12:58:56Z (GMT). No. of bitstreams: 1 sbardella_ap_me_jabo.pdf: 2368521 bytes, checksum: c62c877ae578820d87db27a37651a178 (MD5) Previous issue date: 2016-07-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A mastite é responsável por grandes perdas econômicas na bovinocultura leiteira e necessita de maior compreensão de suas bases genéticas para que métodos de melhoramento genético possam ser desenvolvidos. Neste sentido, estudos de expressão gênica têm sido empregados. Portanto, os objetivos deste trabalho foram: 1. Detectar, por meio de três métodos computacionais distintos, a expressão gênica diferencial de dados obtidos por sequenciamento de RNA extraído de glândula mamária mantida em um sistema extracorpóreo de bovinos de leite mestiços Holandês-Zebu e infectada experimentalmente com Streptococcus agalactiae; 2. Estudar o perfil de expressão do total de genes identificados e aqueles comuns aos três métodos relacionados ao desenvolvimento da mastite, a fim de contribuir para melhor entendimento de processos biológicos, de componente celular e função molecular e vias biológicas envolvidas com a expressão desta característica. Quatro vacas foram abatidas e os úberes foram colhidos, perfundidos e inoculados com S. agalactiae. Para cada úbere, dois quartos foram inoculados (anterior esquerdo - AE; e posterior esquerdo - PE) e dois quartos foram utilizados como controle (anterior direito - AD; e posterior direito - PD). Biópsias foram feitas do tecido alveolar nos tempos 0 e 3 horas após a perfusão do tecido. O RNA das amostras foi extraído e sequenciado com a plataforma HiSeq2000 (Illumina). A partir das leituras obtidas os transcritos foram montados utilizando como referência a anotação do genoma bovino UMD 3.1 e então foram avaliadas quanto à sua expressão diferencial e funcionalidade. Métodos que assumem distribuição binomial negativa executados pelos métodos computacionais edgeR e baySeq e o método que assume distribuição beta binomial negativa executado pelo Cuffdiff, foram utilizados para análise de expressão gênica diferencial. Foram identificados 5.158 genes provenientes da união dos resultados obtidos com os três métodos computacionais. Esses genes foram investigados pela plataforma DAVID revelando o enriquecimento de termos de ontologia gênica, dentre os quais destacaram-se os processos de resposta a estímulos, sistema imune e processos apoptóticos. O perfil de genes diferencialmente expressos em amostras afetadas foi associado com a diferenciação, proliferação, migração e apoptose celular, desempenhando importante papel no local da inflamação por células do sistema imune, além de ativar vias de sinalização relacionadas ao sistema imune que são importantes nas primeiras três horas de infecção. Foram identificados apenas 122 genes com expressão diferencial em comum pelos três métodos utilizados. A restrição causada pelo uso da intersecção dos resultados obtidos com os três métodos não se mostrou adequada para realizar o enriquecimento funcional pois muitos genes de interesse foram desconsiderados e perdeu-se suporte estatístico nas análises de enriquecimento funcional. Assim, sugerimos que para esse tipo de estudo é mais adequado a utilização dos resultados de um dos métodos ou a união dos resultados dos três métodos. Este estudo permitiu maior entendimento das bases genéticas do desenvolvimento da mastite em úbere extracorpóreo de bovinos leiteiros mestiços holandês-Zebu, infectado experimentalmente por S. agalactiae, durante as primeiras horas de infecção. / Mastitis is responsible for huge economic losses in dairy cattle production which requires a better understanding of its genetic bases in order that genetic breeding methods could be developed. Therefore, some gene expression studies have been developed. The aims of this study were: 1. To detect, through three different computational methods, the differential gene expression data obtained by RNA sequencing from mammary glands of Holstein-Zebu crossbred dairy cattle, kept in an extracorporeal system and experimentally infected with Streptococcus agaclatiae; 2. To study the total gene expression profile and those genes common to the three methods, related with mastitis, in order to contribute to a better understanding of biological processes, cellular components, molecular functions and metabolic pathways related with the expression of this trait. Four cows were slaughtered and its udders were perfused and inoculated with S. agalactiae. For each udder, 2/4 were inoculated (front left - AE, and rear left - PE) and 2/4 were used as control (front right - AD, and rear right - PD). Biopsies were obtained from the alveolar tissue at 0 and 3 hours after the tissue perfusion. The RNA sample was extracted and sequenced with HiSeq 2000 platform (Illumina). From the reads obtained transcripts were assembled using as the reference the bovine genome annotation UMD 3.1, and then were evaluated for differential expression and functionality. Methods that assumes negative binomial distribution performed by edgeR and baySeq and the method that assumes beta negative binomial distribution performed by Cuffdiff were used for differential gene expression analysis. From the union of the results obtained with all computational methods, a total of 5,158 genes were identified. These genes were investigated by DAVID platform showing the enrichment of gene ontology terms, which were highlighted the response processes stimuli, immune response and apoptotic processes. The profile of differentially expressed genes identified in the affected samples was associated with differentiation, proliferation, migration and apoptosis, playing a major role in the inflammation site by immune system cells and in the activation of signaling pathways related to the immune system, which are important in the early three hours of infection. Only 122 differentially expressed genes were identified in common by the three methods. The restriction caused by the use of the intersection of the results obtained with the three methods was not interesting to perform functional enrichment analysis because there is loss of many interesting genes and the functional enrichment analysis has no statistical support. Thus, for this kind of study, we suggest that could be most appropriate the use of the results obtained with one method or the union of the results obtained with more than one method. This study allowed a better understanding of the genetic basis of mastitis developing in extracorporeal udder of Holstein-Zebu crossbred dairy cattle, during the early hours of experimentally infection by S. agalactiae.

Microalgae are widely viewed as a potential source of renewable biofuels. Microalgae are highly productive and can be cultured in recycled water on margial or non-agricultural land. Despite their advantages, the industrial scale deployment of microalgae faces numerous challenges including relatively little knowledge of the algae themselves and the comparatively expensive infrastructures required for culture. The green microalga, Botryococcus braunii is particularly interesting because it synthesizes long-chain (C30- C40) hydrocarbons that can be converted to liquid fuel by hydrogenation and catalytic cracking. Moreover, B. braunii is the major fossil present in the Ordovician oil shales and kerogen deposits. Although studied since the 1970s, very little is known regarding critical aspects of B. braunii, notably its molecular biology. In higher plants molecular clocks have been well defined and transcript profiling has revealed a sophisticated network of circadian scheduling of metabolic processes. Characterization of temporal controls over hydrocarbon synthesis is therefore of importance to optimization of biofuel production from B. braunii. In this project B. braunii (Race B, strain Guadeloupe) were cultured in a 12-hour photoperiod and either maintained in that regime or transferred to constant light. Algae were sampled every 4 hours, during a 28-hour time-course and mRNA extracted. mRNA was reverse-transcribed to cDNA and sequenced using a paired-end protocol on an Illumina HiSeq 2000 platform. Over 2 billion sequence reads of 100 bp were generated and assembled de novo, into a complete transcriptome for B. braunii. The transcriptome was comprehensively annotated using global and targeted protocols and differential expression and co-expression analyses were performed. Metabolic pathway analysis confirmed the presence, and photoperiodic regulation of the MEP/DOXP Terpenoid Backbone synthesis pathway. Targeted annotation and expression analysis revealed two predicted B. braunii circadian clock components, which were incorporated into a B. braunii circadian clock model. In non-hierarchical cluster analysis, contigs of the B. braunii transcriptome clustered under four distinct patterns of diel expression. Networks of co- and anti-expressed contigs were elucidated by hierarchical clustering. These results demonstrate the exquisite control over metabolism in B. braunii. Such knowledge is essential for the industrial applications of B. braunii, either directly or through the engineering of selected B. braunii genes or molecular pathways into alternative chassis.

The nucleolus of eukaryotic cells contain a number of the intron-coding small nucleolar RNAs (snoRNAs), which functions are related to covalent modification of pre-rRNAs. The snoRNA that from long, phylogenetically conserved sequence complementarity to 28S, 18S, 5.8S and 5S rRNAs are designated as 28S, 18S, 5.8S and 5S snoRNAs, respectively. In the present study, studying on human 5S snoRNAs had been carried out. The human genome encoding candidate 5S snoRNAs were searched using database mining. The transcripts of 5S snoRNA genes were identified by RT-PCR analyses and DNA sequencing. No appreciable diversities of 5S snoRNA genes were observed as evidenced by single strand conformation polymorphism (SSCP) and high resolution agarose gel. Moreover, sequence conservation of 5S snoRNAs reflects a requirement for maintaining their secondary structure on exerting their function. The results of RT-PCR analyses revealed a tissue-specific transcription of 5S snoRNAs. A 5S snoRNA designated as N117 was identified to be highly expressed in normal brain. On the contrary, its expression markly decreased in brain tumor (meningioma). This seems to be associated with the expression of host gene, which encodes a protein similar to synapsin III protein. Consequently, this may implicate that the use of snoRNA as a potential index for the transcription of its host gene.

The application of ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) technology to identify and quantify metabolomics data is relatively new. An important feature of the FTICR-MS metabolomics data is the high percentage of missing values. In this thesis, missing value analysis showed that the missing value percentages were up to 50% and the control treatment, NaOH.ww, had the highest missing value percentage among the treatments in the aqueous FTICRMS sets. A simulation study was done for the FTICR-MS data to compare selection methods, the Kruskal-Wallis test and the MTP and Limma functions in Bioconductor, an open source project to facilitate the analysis of high-throughput data. The study showed that MTP was sensitive to variations among treatments, while the Kruskal- Wallis test was relatively conservative in detecting variations. As a result, MTP had a much higher false positive rate than Kruskal-Wallis test. The performance of Limma for sensitivity and false positive rate was between the Kruskal-Wallis test and MTP. Data sets with missing values were also simulated to assess the performance of imputation methods. Study showed that variances among treatments diminished or disappeared after imputations, but no new differentially expressed masses were created. This gave us confidence in using imputation methods. Summary of analysis results of some of the frogSCOPE data sets was given in the last chapter as an illustration. / Graduate

>Magister Scientiae - MSc / A large volume of gene expression data exists in public repositories like the NCBI’s Gene Expression Omnibus (GEO) and the EBI’s ArrayExpress and a significant opportunity to re-use data in various combinations for novel in-silico analyses that would otherwise be too costly to perform or for which the equivalent sample numbers would be difficult to collects exists. For example, combining and re-analysing large numbers of data sets from the same cancer type would increase statistical power, while the effects of individual study-specific variability is weakened, which would result in more reliable gene expression signatures. Similarly, as the number of normal control samples associated with various cancer datasets are often limiting, datasets can be combined to establish a reliable baseline for accurate differential expression analysis. However, combining different microarray studies is hampered by the fact that different studies use different analysis techniques, microarray platforms and experimental protocols. We have developed and optimised a method which transforms gene expression measurements from continuous to discrete data points by grouping similarly expressed genes into quantiles on a per-sample basis. After cross mapping each probe on each chip to the gene it represents, thereby enabling us to integrate experiments based on genes they have in common across different platforms. We optimised the quantile discretization method on previously published prostate cancer datasets produced on two different array technologies and then applied it to a larger breast cancer dataset of 411 samples from 8 microarray platforms. Statistical analysis of the breast cancer datasets identified 1371 differentially expressed genes. Cluster, gene set enrichment and pathway analysis identified functional groups that were previously described in breast cancer and we also identified a novel module of genes encoding ribosomal proteins that have not been previously reported, but whose overall functions have been implicated in cancer development and progression. The former indicates that our integration method does not destroy the statistical signal in the original data, while the latter is strong evidence that the increased sample size increases the chances of finding novel gene expression signatures. Such signatures are also robust to inter-population variation, and show promise for translational applications like tumour grading, disease subtype classification, informing treatment selection and molecular prognostics.

Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

The intestinal tract microbiota – microbial community of the gut – is an important field in microbiology not only because of its critical role in the host development, but also increasingly large number of diseases are associated with certain state of the gut microbiota. The community structure and function of the gut microbiota is relatively well studied in humans and related higher vertebrates, but is severely understudied in fish. This is especially true for the coral reef fishes, who constitute the most diverse assemblage of vertebrates spread over a very local scale, and are essential for the resilience of the reefs. In order to bridge this gap in knowledge, this dissertation studied the community structure, interactions and functions of the gut microbial community from the surgeonfishes in the Red Sea – with special focus on the surgeonfish enteric symbiont Epulopiscium spp. Initially, I studied the composition of the gut microbiota of nine surgeonfish and three nonsurgeonfish species from the Red Sea using 454 pyrosequencing. Upon discovering the high abundance of Epulopiscium spp. in herbivorous surgeonfishes, I then proceeded to identify their phylogenetic diversity, distribution, as well as deducing their coevolutionary relationship with the host. Because Epulopiscium spp. undergo substantial changes in the cell size (grow up to ~600μm) and the DNA concentration (from 85 to over 250pg per cell) throughout their diel lifecycle, I also studied the temporal changes in their expression pattern using RNA-seq. Overall, this dissertation shed light on the complex structure, interaction and function of an important family of coral reef fish from the Red Sea through range of molecular techniques.