The genus Aspergillus possesses broad functionality and occupation of ecological niches. Underpinning this are changes in the transcriptome of these species. Transcriptional changes are clinically relevant with respect to understanding triazole resistant isolates of Aspergillus fumigatus. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a highly specific means of measuring changes in gene expression. The most common method of which requires normalization to experimentally validated, stably expressed reference genes. Ideal reference genes are unaffected by differences in the experimental conditions or strains/isolates and are expressed at levels near the target gene(s). The first study reviewed current practices for reference gene selection and validation for RT-qPCR gene expression analysis of the genus, Aspergillus. Information on the species examined, experimental conditions, sample type, normalization strategy, reference gene(s) and their state of validation was obtained from 90 primary studies. Twenty reference genes were used, with the most popular reference genes used encoding beta-tubulin, actin, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase. Seventeen of the 90 studies experimentally validated the expression stability of the reference genes used, out of which eight used more than one reference gene. The results of three studies conflicted with others described in the literature, with no experimental validation of the reference genes available to aid in interpreting the conflicting findings. In the Genome-Wide Association Study, genes noted to increase in expression in response to itraconazole and/or voriconazole treatment of A. fumigatus were extracted from published RNA-sequencing or RT-qPCR studies. Ten ATP-binding cassette transporters, four major facilitator superfamily transporters and 16 transcription factors were identified. Collectively, the findings of this thesis show a large disparity in experimentally validated reference genes as well as future targets of gene expression analysis in triazole resistant isolates of A. fumigatus. / Thesis / Master of Science (MSc) / Aspergillus is a globally distributed genus of fungi, with some species threatening opportunistic human infection. To combat infection with the opportunistic species, Aspergillus fumigatus, antifungal drugs including: itraconazole, voriconazole and amphotericin B are used. Recent years have seen a rise in antifungal resistance in A. fumigatus. To understand this and other mechanisms in Aspergillus, changes in gene expression must be examined. My thesis aimed to determine how reference genes are selected for reverse-transcription quantitative polymerase chain reaction, a method applied to measure gene expression changes in Aspergillus. It was discovered that very few studies between the years 2001 and 2020 experimentally validated that the reference genes used were stably expressed, with only 17 out of 90 studies providing validation. In part two of my thesis, genes overexpressed in A. fumigatus when exposed to antifungal drugs, from formerly published articles, were summarized to better understand the role of gene expression in antifungal drug resistance.

Celiac disease (CD) is a common heritable immune related disorder where chronic inflammationof the small intestine is induced by the ingestion of gluten. The immune response leads to theinflammation and flattening of intestinal mucosa due to the damaged villi and thus results indefects in the absorption of nutrients. This defect can affect any organ or body system and exposeto the risk of lifelong complications such as cancer, autoimmune diseases and other complexdiseases. Now a day, celiac disease is becoming one of the well-studied models of complexdisorders.The PI3K- FOX signaling pathway is activated by many regulators and growth factors and playsa key role in cell cycle. Two components of this pathway, INSR and FOX, play crucial roles indiverse aspects of embryogenesis from the initial tissue genesis up to organ formation. INSR andFOX take part in development, differentiation, proliferation, apoptosis and stress resistance aswell as metabolism. SNP´s could affect the expression of neighboring genes. These SNP´s areshown to be as eQTLs, genomic loci that regulate the expression of genes. The aim of this studywas to detect and quantitate the expression of INSR and certain FOX genes in celiac disease.Quantitative real time PCR (QPCR) was used to analyze the expression of INSR, FOXO1,FOXO4 and FOXD3 genes in 38 celiac cases and 50 control samples. Three reference genesACTB, EPCAM and PGK1 were tested for their expression stability and their average was used inthe normalization procedure. Gene expression results were analyzed using the ΔCt method. Theexpression of INSR, FOXO1, FOXO4 and FOXD3 were described as their fold change in CDcompared to normal non-celiac mucosa. Our results indicated that FOXO4 and INSR wereexpressed less by 0.60 fold and FOXO1 was expressed less by 0.23 fold in CD samples. Theresults are preliminary and further studies will be needed to confirm if these findings are a resultof the intestinal inflammation in CD or if these genes are partly driving the disease itself.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O esgotamento das reservas de energia nÃo-renovÃvel, como petrÃleo, carvÃo e gÃs natural, tÃm estimulado a busca por fontes alternativas geradoras de energia. Neste contexto, o pinhÃo manso (Jatropha curcas L.) tem recebido especial atenÃÃo por parte dos pesquisadores, devido à presenÃa de grande quantidade de Ãleo em suas sementes, que pode ser convertida em biodiesel. O objetivo deste trabalho foi investigar o comportamento de genes relacionados ao metabolismo de lipÃdios durante os processos de desenvolvimento e germinaÃÃo da semente de pinhÃo manso. Para quantificar com exatidÃo os nÃveis de expressÃo gÃnica por RT-qPCR foi feita uma seleÃÃo entre nove candidatos a genes de referÃncia. Nossos resultados mostraram que na anÃlise de sementes em desenvolvimento, os genes GAPDH, UCP, ACT11, PP2A2 e CICLOF foram os mais estÃveis. Para as sementes em germinaÃÃo, os genes considerados mais estÃveis foram EF1-α, PP2A2, GAPDH, PUB3 e ACT11. Para validar nossos resultados com genes de referÃncia, foi utilizado o padrÃo de expressÃo do gene que codifica a proteÃna oleosina no qual foi observado que o mesmo foi similar aos observados em artigos cientÃficos pesquisados, indicando que os genes de referÃncia estavam apropriados para normalizaÃÃo dos dados de RT-qPCR. ApÃs obtenÃÃo desses dados, efetuou-se um estudo da expressÃo por RT-qPCR de 20 genes envolvidos com o metabolismo de lipÃdios. Nossos resultados revelaram que os genes oleosina, β-cetoacil-ACP Sintase I e II, tioesterase A e triacilglicerol lipase I, bem como outros genes envolvidos na biossÃntese de lipÃdios, alcanÃaram altos nÃveis de expressÃo no desenvolvimento da semente. Os genes acil-CoA sintetase, tiolase e triacilglicerol lipase II, relacionados com a degradaÃÃo de lipÃdios apresentaram altos nÃveis de transcritos na germinaÃÃo da semente. Os dados obtidos neste trabalho contribuem para o entendimento das vias metabÃlicas estudadas, fornecendo subsÃdios para a produÃÃo, via engenharia genÃtica, de variedades melhoradas do pinhÃo manso. / The depletion of non-renewable energy such as oil, coal and natural gas, has stimulated the search for alternative sources of energy generation. In this context, physic nut (Jatropha curcas L.) has received special attention from researchers due to the presence of large amounts of oil in its seeds that can be converted into biodiesel. The objective of this study was to investigate the behavior of genes related to lipid metabolism during the development and germination of physic nut seeds. To accurately quantify the levels of gene expression by RT-qPCR, a selection of nine candidates for reference genes was performed. Our results showed that the GAPDH, UCP, ACT11, PP2A2 and CICLOF were the most stable genes during the development of the seeds. In germinating seeds, EF1-α, PP2A2, GAPDH, PUB3 and ACT11 were considered the most stable genes. To validate our findings with reference genes, we used the expression profile of the gene encoding the oleosin protein in which that was similar to those observed in the literature evaluated, indicating that they were suitable reference genes for data normalization by RT-qPCR. After obtaining these data, we performed a gene expression study by RT-qPCR of 20 genes involved in lipid metabolism. Our results revealed that the oleosin, β-ketoacyl-ACP Synthase I and II, thioesterase A and triacylglycerol lipase I genes, as well as other genes involved in lipid biosynthesis, achieved high expression levels in developing seeds. Acyl-CoA synthetase, thiolase and triacylglycerol lipase II, genes related to the degradation of lipids, showed high transcript levels in germinating seeds. The data obtained in this study contribute to the understanding of the metabolic pathways studied, providing subsidies for production of improved varieties of physic nut via genetic engineering.

Yes / Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2 . We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2 . In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

COSTA, Washington Luiz Gomes. Análise espacial e temporal da expressão de genes relacionados ao metabolismo de lipídios em sementes de pinhão manso (Jatropha curcas L.). 2013. 87 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2013. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-19T12:29:04Z No. of bitstreams: 1 2013_dis_wlgcosta.pdf: 2152820 bytes, checksum: afc16b51f6d293f4d04a7abbf8fdca67 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:28:32Z (GMT) No. of bitstreams: 1 2013_dis_wlgcosta.pdf: 2152820 bytes, checksum: afc16b51f6d293f4d04a7abbf8fdca67 (MD5) / Made available in DSpace on 2016-08-02T20:28:32Z (GMT). No. of bitstreams: 1 2013_dis_wlgcosta.pdf: 2152820 bytes, checksum: afc16b51f6d293f4d04a7abbf8fdca67 (MD5) Previous issue date: 2013 / The depletion of non-renewable energy such as oil, coal and natural gas, has stimulated the search for alternative sources of energy generation. In this context, physic nut (Jatropha curcas L.) has received special attention from researchers due to the presence of large amounts of oil in its seeds that can be converted into biodiesel. The objective of this study was to investigate the behavior of genes related to lipid metabolism during the development and germination of physic nut seeds. To accurately quantify the levels of gene expression by RT-qPCR, a selection of nine candidates for reference genes was performed. Our results showed that the GAPDH, UCP, ACT11, PP2A2 and CICLOF were the most stable genes during the development of the seeds. In germinating seeds, EF1-α, PP2A2, GAPDH, PUB3 and ACT11 were considered the most stable genes. To validate our findings with reference genes, we used the expression profile of the gene encoding the oleosin protein in which that was similar to those observed in the literature evaluated, indicating that they were suitable reference genes for data normalization by RT-qPCR. After obtaining these data, we performed a gene expression study by RT-qPCR of 20 genes involved in lipid metabolism. Our results revealed that the oleosin, β-ketoacyl-ACP Synthase I and II, thioesterase A and triacylglycerol lipase I genes, as well as other genes involved in lipid biosynthesis, achieved high expression levels in developing seeds. Acyl-CoA synthetase, thiolase and triacylglycerol lipase II, genes related to the degradation of lipids, showed high transcript levels in germinating seeds. The data obtained in this study contribute to the understanding of the metabolic pathways studied, providing subsidies for production of improved varieties of physic nut via genetic engineering. / O esgotamento das reservas de energia não-renovável, como petróleo, carvão e gás natural, têm estimulado a busca por fontes alternativas geradoras de energia. Neste contexto, o pinhão manso (Jatropha curcas L.) tem recebido especial atenção por parte dos pesquisadores, devido à presença de grande quantidade de óleo em suas sementes, que pode ser convertida em biodiesel. O objetivo deste trabalho foi investigar o comportamento de genes relacionados ao metabolismo de lipídios durante os processos de desenvolvimento e germinação da semente de pinhão manso. Para quantificar com exatidão os níveis de expressão gênica por RT-qPCR foi feita uma seleção entre nove candidatos a genes de referência. Nossos resultados mostraram que na análise de sementes em desenvolvimento, os genes GAPDH, UCP, ACT11, PP2A2 e CICLOF foram os mais estáveis. Para as sementes em germinação, os genes considerados mais estáveis foram EF1-α, PP2A2, GAPDH, PUB3 e ACT11. Para validar nossos resultados com genes de referência, foi utilizado o padrão de expressão do gene que codifica a proteína oleosina no qual foi observado que o mesmo foi similar aos observados em artigos científicos pesquisados, indicando que os genes de referência estavam apropriados para normalização dos dados de RT-qPCR. Após obtenção desses dados, efetuou-se um estudo da expressão por RT-qPCR de 20 genes envolvidos com o metabolismo de lipídios. Nossos resultados revelaram que os genes oleosina, β-cetoacil-ACP Sintase I e II, tioesterase A e triacilglicerol lipase I, bem como outros genes envolvidos na biossíntese de lipídios, alcançaram altos níveis de expressão no desenvolvimento da semente. Os genes acil-CoA sintetase, tiolase e triacilglicerol lipase II, relacionados com a degradação de lipídios apresentaram altos níveis de transcritos na germinação da semente. Os dados obtidos neste trabalho contribuem para o entendimento das vias metabólicas estudadas, fornecendo subsídios para a produção, via engenharia genética, de variedades melhoradas do pinhão manso.

FAPEAL - Fundação de Amparo à Pesquisa do Estado de Alagoas / Estima-se que 50% dos genes codificadores de proteínas possuem oscilação rítmica em diferentes tecidos de mamíferos. Curiosamente, metade das proteínas desses genes possuem seus RNAm correspondentes com expressão constitutiva (arrítmica), ressaltando a relevância de eventos pós-transcricionais para a oscilação de proteínas. Os “High throughput Assays” (HTA) circadianos são extremamente importantes, pois fornecem informações acerca da expressão de milhares de transcritos e de proteínas, uma rica coleção cronobiológica que pode ajudar na resolução de diferentes problemas científicos, não apenas os abordados nas pesquisas originais. Embora altamente reprodutivos e informativos, ensaios de biologia molecular circadiana apresentam algumas divergências em seus resultados, ressaltando a necessidade do aprimoramento de métodos de normalização e análise dos diferentes ensaios de expressão. Até o momento, não se conhecem os impactos da normalização do RNA em ensaios de expressão de pequenos RNAs, como miRNAs. Este estudo objetivou: (1) analisar a co-expressão de miRNAs e RNAm e proteínas de genes alvos; (2) identificar e validar miRNAs candidatos ao sistema molecular circadiano; (3) analisar o impacto da normalização do RNA total em estudos circadianos de miRNAs. Através da sistematização de diferentes dados circadianos de HTA (RNA-seq, small RNA-seq, Chip-seq e proteoma), de bioinformática e de interações miRNA:RNAm validadas, identificamos uma lista de 152 microRNAs (miRNAs) candidatos ao controle pós-transcricional dos ritmos moleculares. Desses, os dois mais relevantes, miR29b-3p e miR-23b-3p, foram experimentalmente validados como importantes para a manutenção do período dos ritmos das células U2OS PER2:LUC. Análises de HTA também permitiram a identificação de diferenças nas fases de expressão de miRNAs 3p e 5p, com genes alvos divergentes. Interessantemente, a identificação de padrões de expressão de RNAm e proteínas de genes alvos de miRNAs, permite sugerir um mecanismo de ajuste das amplitudes dos ritmos das proteínas dependente da fase do RNAm. Por fim, através do controle de etapas experimentais, demonstramos oscilação circadiana na concentração do RNA total de diferentes regiões de cérebros de camundongos. A normalização desse ritmo, durante a síntese de cDNA, afeta o perfil de expressão de transcritos, incluindo os dos genes relógio Per1-2 e Bmal1. Ademais, através de análises com RNA exógeno, ou spike-ins, demonstramos que o ajuste da concentração do RNA compromete a análise de miRNAs, possivelmente por interferências nos rendimentos de extração e nas reações de síntese de cDNA. Este estudo apresenta dois novos miRNAs importantes para a manutenção da ritmicidade circadiana e uma nova estratégia de análise de qPCR para estudos cronobiológicos de miRNAs.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-04-05T19:37:55Z No. of bitstreams: 2 Dissertação - Fernanda Raquel Martins Abreu - 2014.pdf: 2480777 bytes, checksum: f8c50ff359945dffab56d7874ad25086 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-06T11:34:39Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda Raquel Martins Abreu - 2014.pdf: 2480777 bytes, checksum: f8c50ff359945dffab56d7874ad25086 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-04-06T11:34:39Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda Raquel Martins Abreu - 2014.pdf: 2480777 bytes, checksum: f8c50ff359945dffab56d7874ad25086 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-04-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Drought, a major problem concerning a sustainable rice production in Brazil and worldwide, is responsible for a series of plant responses, including modification in gene expression, accumulation of metabolites and protein synthesis. In order to verify the correspondence between five Arabidopsis genes (PLDα1, LEW2, GluR2, Lsi1 e EIN2), previously related to drought tolerance, and their respective orthologs in rice, the present study analyzed two contrasting rice genotypes for drought, Douradão, the tolerant genotype, and Primavera, the susceptible one. The genotypes were submitted to drought stress and subsequently evaluated for gene expression by quantitative real-time Polymerase Chain Reaction (qPCR). The comparison of gene expression, between leaf and root tissues, showed a greater expression in roots, within their vegetative stage, and leaves, within their reproductive stage. Differential expression were observed mainly among the genes whose orthologs in Arabidopsis encode phospholipase Dα1 (PLDα1) and ethylene-insensitive protein (EIN2); these proteins are directly related to abscisic acid (ABA), a phytohormone that when identified in higher concentration in cells triggers the expression of drought stress-responsive genes, besides it is also responsible for the regulating the water loss (by transpiration) by controlling of stomatal movement. The results suggested that orthologs genes were in fact drought stress-responsive genes in rice, and emphasized the feasibility of PLDα1 and EIN2 overexpression in rice plants, supporting plant breeding programs in the development of drought tolerant genotypes. / A seca, um dos principais problemas para a sustentabilidade do cultivo de arroz no Brasil e no mundo, é responsável por uma série de respostas em plantas, incluindo mudanças na expressão gênica, acúmulo de metabólitos e síntese de proteínas. No intuito de verificar a correspondência entre cinco genes de Arabidopsis (PLDα1, LEW2, GluR2, Lsi1 e EIN2), previamente relacionados com a tolerância à seca, e seus respectivos genes ortólogos em arroz, o presente estudo analisou dois genótipos de arroz contrastantes no nível de tolerância à seca, Douradão, genótipo tolerante, e Primavera, genótipo susceptível. Os genótipos foram submetidos a experimento de seca e posterior avaliação de expressão gênica via PCR quantitativa em tempo real, qPCR (real time quantitative-Polimerase Chain Reaction). Ao se comparar os níveis de expressão entre os tecidos foliar e radicular, desses genótipos, foi observado, de uma forma geral, que houve maior expressão em raízes no estádio vegetativo e em folhas no estádio reprodutivo. Níveis diferenciais de expressão entre os genótipos foram observados principalmente para os genes ortólogos responsáveis por produzir fosfolipase Dα1 (PLDα1) e proteína etileno insensitiva (EIN2); estes produtos estão diretamente relacionados ao ácido abscísico (ABA), um fito hormônio induzido por estresse de seca que, quando em alta concentração nas células, além de acionar a expressão de muitos genes, desempenha um papel vital na regulação da perda de água por transpiração ao controlar os movimentos estomáticos. Os resultados apresentados nesta pesquisa sugeriram que os genes ortólogos estariam de fato atuando em resposta à tolerância à seca em arroz, e reforçaram a hipótese de que a superexpressão desses genes, em especial, PLDα1 e EIN2, pode auxiliar programas de melhoramento genético de arroz no desenvolvimento de cultivares mais tolerantes à seca.

La dysfonction diastolique du ventricule gauche (DDVG) réfère à une rigidité ainsi qu’à des troubles de relaxation au niveau de ce ventricule pendant la phase de la diastole. Nos connaissances sur les mécanismes moléculaires sous-jacents de cette pathologie demeurent limités. Les analyses géniques sont indispensables afin de bien identifier les voies par lesquelles cette maladie progresse. Plusieurs techniques de quantification de l’expression génique sont disponibles, par contre la RT-qPCR demeure la méthode la plus populaire vu sa haute sensibilité et de ses coûts modérés. Puisque la normalisation occupe un aspect très important dans les expériences de RT-qPCR, nous avons décidé de sélectionner des gènes montrant une haute stabilité d’expression dans un modèle de DDVG de lapin. Nous avons alors exposé 18 lapins blancs soit à une diète normale (n=7) ou bien à une diète hypercholestérolémiante additionnée de vitamine D2 (n=11). La DDVG a été évaluée par des mesures échocardiographiques. L’expression de l’ARNm de dix gènes communément utilisés dans la littérature comme normalisateur (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, et G6pd) a été mesurée par RT-qPCR. L’évaluation de leur stabilité a été vérifiée par les algorithmes de geNorm et Normfinder. Sdha et Gapdh ont obtenu les meilleurs scores de stabilité (M<0.2) et ont été suggérés par le geNorm, comme meilleure combinaison. Par contre, l’utilisation de Normfinder mène à la sélection d’Hprt1 et Rpl5 comme meilleure combinaison de gènes de normalisation (0.042). En normalisant par ces deux combinaisons de gènes, l’expression de l’ARNm des peptides natriurétiques de type A et B (Anp et Bnp), de la protéine chimiotactique des monocytes-1 (Mcp-1) et de la sous unité Nox-2 de la NADPH oxydase ont montré des augmentations similaires chez le groupe hypercholestérolémique comparé au groupe contrôle (p<0.05). Cette augmentation d’expressions a été corrélée avec plusieurs paramètres échocardiographiques de DDVG. À notre connaissance, c’est la première étude par laquelle une sélection de gènes de référence a été réalisée dans un modèle de lapin développant une DDVG. / Left ventricular diastolic dysfunction (LVDD) is characterized by the diminution of ventricle’s performance, its incapacity to relax normally and an increase of blood filling pressure. LVDD is considered as a main cause of heart failure in approximately 50% of patients suffering from this disease. However, the molecular mechanisms underlying LVDD remain unclear. Real-time quantitative PCR (RT-qPCR) is widely used in gene-expression studies. In this study we attempt to establish normalization genes for gene expression analysis in a rabbit model of LVDD. Eighteen New-Zealand white rabbits were exposed to normal (n=7) or 0.5% hypercholesterolemic (n=11) diet supplied by vitamin D2; an LVDD animal model previously characterized in our Laboratory. LVDD was assessed by echocardiography. RT-qPCR was performed using cDNA from left ventricle samples and measuring the stability of 10 candidate genes as normalisers (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). We found that Sdha and Gapdh are the most stable genes using geNorm analysis with very high stability average M <0.2. By contrast, Hprt1 and Rpl5 were found to be the best combination for normalization with a stability value of 0.042 when using Normfinder. Comparison of both normalization strategies highlighted an increase of Anp, Bnp, Mcp-1 and Nox-2 mRNA expression in the hypercholesterolemic rabbits (p<0.05) compared to normal controls. This increase correlates with different DD parameters and validates the development of the disease in our model. To our knowledge, this is the first study highlighting stable reference genes for RT-qPCR normalization in a validated rabbit model of LVDD.

No description available.

Advanced maternal age has been shown to influence follicular and luteal dynamics in bovine ovary resulting in reduced fertility. The overall objective of the four studies presented in this thesis is to identify the maternal age-associated transcriptional changes in granulosa cells of the dominant follicles during follicle development. In the first study, mRNA expression levels of housekeeping genes were measured by real–time quantitative PCR (RT-qPCR) in granulosa cells of dominant follicles and FSH-stimulated follicles to select and validate suitable reference genes for relative gene expression analyses during maternal and follicular aging. Stability of six reference genes (GAPDH, ACTB, EIF2B2, UBE2D2, SF3A1 and RNF20) was analyzed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined based on these programs. Geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) was more appropriate reference control than individual genes for the comparison of relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies. In the second study, maternal age-associated changes in the transcriptome of granulosa cells recovered at the time of selection of the dominant follicle from aged (n=3) and young cows (n=3) were determined by EmbryoGENE bovine oligo-microarrays (EMBV3, Agilent Technology). The mRNA expression of five transcripts (CYP19A1, PCNA, GJA1, TPM2, and VNN1) was confirmed in a different set of granulosa cell samples by RT-qPCR to validate microarray data. A total of 169 genes/isoforms were differentially expressed (≥ 2-fold-change; P ≤ 0.05) in aged cows vs. young cows. These transcripts revealed inefficient 1) control of gonadotropins, and gonadotropin-induced changes in the cytoskeleton and extracellular matrix, 2) lipid metabolism and steroidogenesis 3) cell proliferation, cell cycle control and intercellular communication, and 4) higher oxidative stress responses in aged cows vs. young cows. In the third study, changes in the transcriptome of granulosa cells of the preovulatory follicle 24 h after LH treatment from aged (n= 3) and young (n=3) were determined. A total of 1340 genes were expressed differentially (≥ 2-fold change; P ≤ 0.05) in aged cows vs. young cows. The mRNA expression of five transcripts (RGS2, PTGS2, TNFAIP6, VNN1, NR5A2 and GADD45B) was confirmed in a different set of granulosa cell samples to validate microarray data. These transcripts were related to delayed 1) response to LH treatment 2) cellular differentiation and luteinization and 3) progesterone synthesis. Intra-follicle levels of progesterone were lower (P < 0.05) in aged cows compared to young and mid-aged cows. The fourth study compared the aged-associated changes in the transcriptome of granulosa cells during follicle development from the time of dominant follicle selection to preovulatory stage (24 h after LH). In comparison to young cows, aged cows expressed fewer differentially expressed genes/isoforms (1206 vs. 2260, respectively) at ≥ 2-fold-change (P ≤ 0.05) in the granulosa cells of the preovulatory (24 h after LH treatment) vs. the dominant follicle at selection. These transcripts in aged cows were related to late and inefficient 1) organization of cytoskeleton and cytoplasm, 2) differentiation, 3) lipid and cholesterol metabolism, 4) proliferation and 5) higher response to oxidative stress and free radical scavenging in the preovulatory follicles vs. the dominant follicle at selection. In conclusion, maternal age-alters the gene expression of granulosa cells of the dominant follicles during follicle development and results in a compromised follicular environment.