A carcinogênese oral é um processo multifásico, onde componentes genéticos levam a desregulação de vias de sinalização celular que controlam funções celulares básicas, como divisão, diferenciação e morte celular. Uma das maneiras de compreender a natureza biológica dos cânceres, além do curso clínico, é através do entendimento do processo de progressão e metástase destas neoplasias. Este estudo teve como objetivo avaliar a participação da proteína Twist no desenvolvimento e progressão dos carcinomas epidermóides orais. Com tal proposta, também foi avaliada a participação das proteínas caderina-E e p-Akt, e sua possível interação com Twist no processo de carcinogênese oral. O trabalho em questão analisou a expressão imuno-histoquímica destas proteínas em 30 espécimes de displasia oral, 20 de carcinoma epidermóide oral e 10 de mucosa oral normal, e avaliou também a possível inter-relação dessas proteínas em linhagens derivadas de carcinoma epidermóide de cabeça e pescoço por meio dos ensaios de Western Blotting e imunofluorescência. Os resultados deste estudo demonstraram uma relação inversamente proporcional entre Twist e caderina-E desde os estágios mais precoces da carcinogênese oral. Tal afirmação baseou-se na presença de diferenças significantes entre a expressão imuno-histoquímica de Twist e Caderina-E na amostras de epitélio oral, epitélio displásico e nos espécimes de carcinoma epidermóide oral. Adicionalmente, foi observada a relação inversa entre Twist e a Caderina-E nas linhagens de carcinoma epidermóide de cabeça e pescoço, sendo este evento constatado pelo decréscimo nos níveis protéicos da Caderina-E frente a uma elevação de Twist. Estes resultados sugerem um importante papel de Twist na progressão do carcinoma epidermóide oral, e juntamente com a Caderina-E, pode representar um relevante marcador biológico do câncer oral. / The oral carcinogenesis is a multi-stage process, where genetic components leads to deregulation of cell signaling pathways that control basic cellular functions such as division, differentiation and cell death. One way to understand the biological nature of cancers, besides the clinical course, is through understanding the process of progression and metastasis of these neoplasms. This study aimed to evaluate the role of Twist protein in the development and progression of oral squamous cell carcinomas. With this proposal, was also evaluated the involvement of E-cadherin and p-Akt proteins, and its possible interaction with Twist in the process of oral carcinogenesis. The work in question examined the immunohistochemical expression of these proteins in 30 specimens of oral dysplasia, 20 oral squamous cell carcinoma and 10 normal oral mucosa, and also evaluated the possible interrelationship of these proteins in lines derived from squamous cell carcinoma of head and neck by means of Western blotting assays and immunofluorescence. The results of this study showed an inverse relationship between Twist and E-cadherin since the earliest stages of oral carcinogenesis. These results were based on the presence of significant differences between the immunohistochemical expression of Twist and ECadherin in samples of oral epithelium, dysplastic epithelium and in specimens of oral squamous cell carcinoma. In addition, we observed the inverse relationship between Twist and E-Cadherin in the lines of squamous cell carcinoma of head and neck; this event was evidenced by the decrease in protein levels of E-Cadherin forward to a high of Twist. These results suggest an important role of Twist in the progression of oral squamous cell carcinoma, and along with E-cadherin may represent a relevant biomarker of oral cancer.

As mutações no gene PROP1 são a causa genética mais comum da deficiência combinada de hormônios hipofisários. Até o momento, diversas mutações missense e pequenas deleções foram descritas sendo a mutação 301-302 delAG a mais freqüente. Nosso objetivo foi estudar as mutações em DNA de pacientes com hipopituitarismo e padronizar a extração de DNA de células de swab oral, usando um método com NaCl e comparar com um kit comercial (Purigene, Minneapolis, EUA). Amplificamos os 3 exons do gene PROP1 do DNA obtido de células orais e de sangue periférico. Identificamos a mutação 301-302delAG em 6 pacientes, 4 em homozigose (33%) e 2 em heterozigose (16%) e a mutação G51A em heterozigose em um único paciente. Em dois irmãos, filhos de pais consangüíneos, não foi possível amplificar os 3 exons do gene PROP1 enquanto que os os genes LHX3 e LHX4 foram amplificados com sucesso. Para confirmar a hipótese de deleção do PROP1, o Southern blotting foi realizado usando como sonda o produto de PCR do exon 2 do gene PROP1 e um fragmento do gene CYP21A2 como sonda controle. A banda referente ao CYP21A2 estava presente nos pacientes e nos controles enquanto a banda referente ao PROP1 estava ausente nos irmãos e presente na mãe e nos controles. Para definir a extensão da deleção usamos um mapa de STS próximos ao gene e o STS GDB:314805 localizado a 6,0 kb a montante do PROP1 não foi amplificado nos pacientes. Entretanto, o gene Q8N6H0 a 18 kb a juzante e o STS WI-16216 a 59 kb a montante do PROP1 foram amplificados com sucesso nos pacientes e controles indicando que a deleção está localizada dentro de 81 Kb. Para determinar os limites da deleção, várias reações de PCR foram realizadas com primers desenhados progressivamente distantes de gene PROP1, cobrindo toda a região. Isto nos permitiu determinar a região deletada de 9,6 kb a juzante e 11 kb a montante do gene PROP1, com o tamanho máximo deletado de 18,4 kb. Por ambos os métodos de extração obtivemos um DNA de boa qualidade, permitindo o amplificação dos 3 exons do gene PROP1. A extração com NaCl foi mais rápida e mais barata resultando em maior quantidade de DNA quando comparada com o kit comercial. Em conclusão, descrevemos a deleção completa do gene PROP1 em dois irmãos com o fenótipo clássico de hipopituitarismo associado à hipófise hipoplásica ou aumentada e padronizamos a extração de DNA de células de mucosa oral com NaCl, que apresentou custo mais baixo e resultado mais rápido quando comparado a extração por um kit comercial, indicando que o swab oral é uma fonte prática de obtenção de DNA para estudos genéticos. / PROP1 gene mutations are the most common cause of genetic combined pituitary hormone deficiency. To date, several missense mutations and small deletions have been described and the 301-302 del AG is the most frequent. Our objective was to study PROP1 mutations in patients with hypopituitarism and standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit (Purigene, Minneapolis, USA). We amplified the 3 exons of PROP1 gene in DNA obtained from oral cells and peripheral blood cells. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In two siblings, a boy and a girl, born to consanguineous parents we failed to amplify PROP1 gene by PCR whereas LHX3 and LHX4 genes were successfully amplified. To confirm the hypothesis of PROP1 gene deletion, Southern blotting was performed using PROP1 exon 2 gene PCR product as a probe and a fragment of CYP21A2 gene as a control probe. The CYP21A2 band was present in patients and controls whereas PROP1 band was absent in both siblings and present in their mother and in controls. To define the extension of this deletion we used STS mapping approach and no amplification for a STS GDB:314805 6.0 kb downstream of PROP1 was found. However, Q8N6H0 gene located 18 kb upstream and the STS WI-16216 located 59 kb downstream of PROP1 were successfully amplified indicating that the deletion is placed within 81 Kb. To determine the limits of the deletion a number of PCR covering this region were then carried out with primers located progressively distant from PROP1. This allowed us determine the deleted region from 9.6 kb upstream to 11 kb downstream of PROP with a maximum deletion size of 18.4 kb. Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared with the commercial kit. In conclusion, we describe a complete deletion of PROP1 gene in two siblings with classical hypopituitarism phenotype associated with hypoplastic or enlarged pituitary gland and standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that oral swabs are a reliable DNA source for genetic studies.

B and T lymphocytes are critical to the adaptive immune response against invading microorganisms. B and T cells develop in the bone marrow and thymus, respectively, and initiate a series of proliferative responses once they encounter their cognate antigen in the peripheral lymphoid organs. These developmental and functional processes are controlled by different networks of transcriptional regulators that repress and activate gene expression. Identifying proteins that activate or repress specific genes and integrating these proteins into their transcriptional networks is critical to understanding lymphocyte development and function. The study of B lymphopoiesis and CD4+ T cell functional responses has greatly increased our understanding of how transcriptional regulators and other proteins cooperate to specify cell fates and responses. While many of the key components of these protein networks have been defined, several factors have yet to be described. Chemically induced random mutagenesis is a powerful tool for identifying genes that have critical biological functions. Justy mutant mice were generated by injecting wild-type mice with of N-Ethyl-N-Nitrosourea (ENU), a mutagen, which generated a unique point mutation in the mouse Gon4-like (Gon4l) gene. This mutation was found to specifically blunt B cell development and impair the functional responses of CD4+ T cells. Given that the Gon4l protein contains domains implicated in transcriptional regulation and B lymphopoiesis and T cell responses are regulated transcriptionally, the aim of this project was to characterize T and B lymphocyte populations from Justy mice and provide insights into the mechanisms underlying the regulation of gene expression during these biological processes. The work presented in this dissertation demonstrates that the protein encoded by Gon4l is essential for B lymphopoiesis, likely through the repression of alternate lineage genes. This work also shows that in CD4+ T cells, decreased Gon4l protein expression results in reduced levels of proliferation in response to exogenous IL-2 or T cell receptor (TCR) engagement. Additionally, Justy mutant CD4+ T cells display a reduced ability to generate IFNγ-producing cells in response to Th1 polarization in vitro. Collectively, these defects correlate with elevated levels of genes known to specifically inhibit the above developmental and functional processes. Thus, this dissertation proposes that Gon4l acts as a transcriptional repressor within the protein networks controlling B lymphopoiesis and CD4+ T cell responses.

Les Lymphocytes T CD4 (LT CD4) sont des cellules du système immunitaire adaptatif extrêmement plastiques qui, en fonction des signaux présents dans le microenvironnement cellulaire, ont la capacité de se différencier en différentes sous-populations de LT CD4 possédant des fonctions distinctes. Ce processus est hautement régulé par l'expression de facteurs de transcription (FT) clés tels que T-Bet, GATA-3, RORgammaT et Foxp3, nécessaires à la mise en place des lignages Th1, Th2, Th17 et Treg respectivement. Néanmoins, ces protéines n'agissent pas seules, et d'autres facteurs de transcription sont nécessaires pour amplifier, soutenir et maintenir ces différents lignages. Chaque lignage permet de lutter efficacement face à différents types de pathogènes ; toutefois, si la réponse immune n'est pas adaptée, ils peuvent également être responsables du développement de maladies auto-immunes. Afin de mettre en évidence les voies de signalisation et les facteurs de transcription impliqués dans la différenciation des LT CD4 pathogènes, nous avons utilisé le modèle de l'Encéphalomyélite Auto-immune Expérimentale (EAE), un modèle murin de Sclérose En Plaques (SEP). Dans ce modèle, nous avons mis en évidence le rôle clef de deux facteurs de transcription, Foxo3 et Eomes, dans la différenciation des LT CD4. En effet, les souris déficientes en Foxo3 développent une EAE moins sévère que les souris WT, et cette moindre sévérité de la maladie est associée à une proportion réduite de cellules productrices d'IFN-gamma et de GM-CSF in vivo, suite à l'immunisation. L'analyse du transcriptôme des souris Foxo3KO et WT a révélé que la déficience en Foxo3 a pour conséquence une diminution drastique de l'expression du FT Eomes. Bien que cette protéine soit nécessaire à la mise en place des réponses cytotoxiques dans les LT CD8 et les NK, son rôle précis dans les LT CD4 reste peu connu. D'un point de vue moléculaire, nous avons pu prouver, par des techniques d'Immuno-Précipitation de la Chromatine (ChIP) et des analyses de gènes rapporteurs, que le FT Eomes est un gène cible direct de Foxo3 dans les LT CD4.[...] / CD4 T cells are extremely plastic, and depending on the cytokines that are present within the microenvironment, they have the ability to differentiate into several subpopulations. This process is finely regulated by the expression of Master Regulator of each lineage such as T-Bet, GATA-3, RORgammaT and Foxp3, that are mandatory for the differentiation of Th1, Th2, Th17 and Treg cells respectively. However, they do not act alone, and several other transcription factors are required to stabilize, amplify and lock CD4 T cell lineages. Each subpopulation of CD4 T cells is highly specialized in the elimination of particular types of pathogen; however, in case of dysregulation of the immune response, they can also be involved in the development of autoimmune diseases. In order to determine how such properties are acquired by pathogenic CD4 T cells, we used the Experimental Autoimmune Encephalomyelitis (EAE) model which mimic Multiple Sclerosis pathology. In this model, we identified two transcription factors, Foxo3 and Eomes, that are critical for the differentiation of a particular and highly pathogenic subset of CD4 T cell. Indeed, Foxo3-deficient mice develop a less severe disease as compared to WT littermate and this decreased disease severity is associated with a decreased proportion of IFN-gamma and GM-CSF producing cells. Transcriptomic analysis of Foxo3KO versus WT CD4 T cells revealed that the most downregulated gene within Foxo3KO CD4 T cells is Eomes, which is essential for/to the acquisition of cytotoxic functions and production of IFN-gamma by NK and CD8 T cells. At the molecular level, using Chromatin Immuno-Precipitation experiments and Luciferase assays, we showed that Eomes is a direct target gene of Foxo3 in CD4 T cells. Then, in order to determine which of the downregulated gene is responsible for the decreased production of IFN-gamma and GM-CSF, we decided to overexpress Eomes in Foxo3KO CD4 T cells. Eomes overexpression restored IFN-gamma and, to a lesser extent, GM-CSF production by CD4 T cells, thus indicating that Eomes is involved in IFN-gamma and GM-CSF regulation in CD4 T cells.[...]

Au cours de la gestation précoce, la régulation de la physiologie endométriale est cruciale au bon déroulement de l’implantation. Chez les mammifères, une famille de facteurs de transcription est fortement impliquée dans la régulation de la physiologie endométriale, les facteurs STAT. Chez la vache, des analyses haut-débit ont révélé que l’expression endométriale de STAT1 est régulée au cours de la période préimplantatoire. Le but de cette thèse est donc d’apporter de nouvelles données sur l’expression et la régulation endométriales de STAT1 mais également sur ses fonctions biologiques au cours de la gestation précoce chez la vache.Grâce à différents modèles physiologiques et expérimentaux, l’impact de la progestérone, de l’IFNT (signal majeur de reconnaissance maternelle de la gestation chez les ruminants) et de la gestation sur l’expression et la régulation de STAT1 (y compris sa phosphorylation) a été analysé dans l’endomètre bovin et sur des cultures primaires de cellules endométriales. Ainsi, l’expression de STAT1 (transcrit et protéine) ainsi que sa phosphorylation sont augmentés en présence du conceptus et de l’IFNT, indépendamment du taux circulant de progestérone à l’implantation chez la vache. Pour avoir une meilleure connaissance des rôles de STAT1, l’identification de ses gènes cibles a été entreprise : d’abord avec une approche gènes candidats (avec la famille des gènes SOCS), puis par une approche exploratoire.Les facteurs SOCS sont connus pour être des régulateurs négatifs de la voie de signalisation des cytokines. L’utilisation des différents modèles physiologiques et expérimentaux évoqués plus haut a permis l’analyse de l’expression et de la régulation des huit membres de la famille des gènes SOCS au cours de la gestation précoce chez la vache. L’application d’un protocole d’immunoprécipitation de la chromatine sur des cultures primaires de cellules stromales bovines montre le recrutement rapide de STAT1 par l’IFNT sur les promoteurs des gènes SOCS IFNT-dépendants. D’autre part, l’identification systématique des gènes cibles de STAT1 a été entreprise via l’élaboration d’un protocole d’immunoprécipitation de la chromatine suivit de séquençage haut-débit, appliqué à des échantillons d’endomètre bovin. L’ensemble de ces travaux suggèrent l’implication de STAT1 dans la signalisation endométriale de l’IFNT, dans la régulation du système immunitaire maternel et également dans le contôle des phénomènes d’apposition et d’adhérence, fonctions cruciales à l’implantation chez la vache. / During the early stage of the pregnancy, the regulation of the endometrial physiology is crucial to the right establishment of the implantation. In mammals, a transcription factor family is highly involved in the regulation of endometrial physiology, the STAT family. In cattle, high-throughput analyses light up the regulation of endometrial STAT1 expression during the pre-implantation period. Thus, the aim of this work is to bring new insights about endometrial STAT1 expression and regulation but also on its biological functions during the early pregnancy in cattle. Using physiological and experimental models, the impact of progesterone, IFNT (major signal of the maternal recognition of pregnancy in ruminants) and pregnancy on the expression and the regulation of STAT1 transcript and protein (including its phosphorylation status) have been analyzed in the bovine endometrium and endometrial cells. Thus, STAT1 (transcript, protein and phosphorylation) is up-regulated by the presence of the conceptus and by IFNT but independent of progesterone level at implantation in cattle. To better understand endometrial STAT1 functions, the identification of STAT1 target genes has been initiated: first, on a candidate genes family, SOCS genes, and secondly, with an explorative approach.The proteins SOCS are known to be negative regulator of cytokine signalling pathway. Using physiological and experimental models previously quoted, the eight members of SOCS genes expression and regulation were analyzed during the early pregnancy in cattle. Chromatin immunoprecipitation protocol applied on stromal cells show the recruitment of STAT1 on SOCS promoters by a rapid treatment of IFNT. Moreover, the exhaustive identification of STAT1 target gene has been initiated, using a chromatin immunoprecipitation followed by high-throughput sequencing on bovine endometrium samples. Collectively, this data suggests the involvement of STAT1 in IFNT signalling pathway but also in the regulation of maternal immune system and the apposition/adhesion process, all that being crucial for the implantation in cattle.

Erythropoiesis, the process of producing mature erythrocytes from the haematopoietic stem cells (HSCs) that reside in the bone marrow, is tightly regulated at both the cell and molecular level by a well defined set of extracellular cytokine signals and intracellular transcription factors. Diseases affecting erythropoiesis are among the most commonly inherited conditions and result from disturbances to the cellular and molecular events that normally regulate this process. Erythroid Kruppel-like factor (EKLF/KLF1) is a transcription factor that is essential for erythropoiesis. EKLF is the founding member of the Kruppel-like factor family of transcription factors that bind to GC rich CACC-Box elements within gene promoters and activate transcription. The β-like globin genes are critical targets of EKLF through its binding at sites within the proximal promoters and the upstream locus control region (LCR) enhancer. Mice lacking EKLF die prior to birth by E16 with a phenotype that closely resembles the human disease thalassaemia. Thalassaemia is due to mutations in the α or β-globin genes, leading to globin chain imbalance, red cell destruction and ineffective erythropoiesis. However, restoration of expression of γ-globin (a β-like gene) failed to prevent embryonic lethality in EKLF knockout mice and suggested that additional target genes were critical to erythropoiesis. This thesis describes investigation into the transcriptional network of EKLF and an in depth analysis of previously uncharacterised phenotypes present in the EKLF knockout mouse. I have identified a suite of target genes for EKLF that include critical components of the cells cycle. I have also tested the hypothesis that EKLF is able to function in vivo as a tumour suppressor gene. Additionally, I report a role for EKLF in the determination of cell fate within the haematopoietic system and describe the development of a new approach to globally understanding erythroid transcription factor function. A previously performed microarray transcriptional profiling study provided a set of potential target genes for EKLF. I have expanded on this study by identifying that the cell cycle genes p18INK4c, and E2f2 are direct transcriptional targets of EKLF, where binding of EKLF occurs at the promoter and a novel intronic enhancer region, respectively. I have also described a previously undiscovered cell cycle phenotype of aberrant entry into S-phase in EKLF -/- erythroid cells that is directly related to abrogated expression of E2f2. The Kruppel-like factor family of genes have been implicated as players in the tumour process. By constructing a model for the loss of EKLF within HSCs in vivo, I have tested whether EKLF is functional as a tumour suppressor. The loss of EKLF in vivo was found to be insufficient to generate erythroleukaemia, however did result in erythroid hyperplasia, extramedullary haematopoieis and a mild macrocytic anaemia. In addition to regulation of erythropoiesis, EKLF performs a critical role in the lineage choice for a megakaryocyte-erythroid progenitor (MEP) between the megakaryocytic and erythroid lineages. This thesis describes that in the absence of EKLF, MEPs fail to commit properly to either lineage and proceed along a promiscuous pathway sharing the hallmarks of both megakaryocytes and erythroid cells. A detailed molecular mechanism for this phenotype remains undetermined, but is likely to involve interactions with the megakaryocyte transcription factor Fli1 and other members of the Kruppel-like factor family, such as BKLF (KLF3). While the transcriptional mechanisms that drive erythropoiesis have been slowly discovered, the development of chromatin immunoprecipitation (ChIP) assays and next generation DNA sequencing technology has presented the potential to rapidly enhance the progression of these studies. In this thesis I describe the development of ChIP-seq using Applied Biosystems SOLiD technology, an approach to rapidly identify binding sites for erythroid transcription factors in an unbiased genome wide approach. The work described in this thesis has expanded the transcriptional network of EKLF to include critical components of the cell cycle and has suggested many additional target genes from ChIP-seq requiring validation. As one of the major transcription factor players during erythropoiesis, EKLF performs many critical functions that include the regulation of the cell cycle, lineage selection and erythroid development. I suggest that current and future studies of EKLF function will influence our understanding of erythropoiesis and refine our understanding of human conditions such as thalassaemia and erythroleukaemia.

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape and wine quality by influencing the colour and mouthfeel of red wine. These compounds are synthesized in different parts of the berry and during different stages of berry development. In addition, environmental and viticultural factors such as light exposure can also alter the flavonoid composition of grapes. An understanding of how synthesis of these compounds is coordinated, their relationship to wine quality and the influence of bunch light exposure on the flavonoid composition of grapes, could be used to improve fruit quality by enhanced viticultural management. The first part of this study sought to investigate the relationship between the different products of the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions (warm and cool) and determine their role in grape and wine quality. In collaboration with a major winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of 80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin, seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive comparison of the two methods is discussed. In both years, the grapes from warm and cool climates formed two distinct data sets based on flavonoid composition. There was a correlation between anthocyanins and flavonols for both the warm and cool climate samples in both years, however those from the warm region had lower anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two regions clearly separated based on yield and despite the weak correlations in both regions, the levels of total anthocyanins were inversely related to yield. In addition, there was no relationship with any of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for quality using these flavonoid compounds. The second part of the study was to investigate the effect of bunch light exposure on flavonol synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering, pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the boxes were removed and then every second day (light induced), along with exposed controls for one week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by HPLC and Real Time-PCR (RT-PCR) respectively. As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering, pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1 expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes. These results show that flavonols are able to be induced by bunch light exposure at different times during berry development, including times when flavonols are not normally being synthesised. This suggests bunch light exposure can override the developmental control of flavonol accumulation. To further investigate the light induced expression of VvFLS1 in grapevines the molecular mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive element (MRE) and several light responsive elements (LRE) were identified in the promoter region of these genes. To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay suspension cells. Cells were bombarded with constructs containing potential transcription factors and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a grapevine transcription factor specific for VvFLS1 was sought. Two techniques were employed to identify potential transcription factor regulators of the VvFLS1 promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST) database with AtMYB12 and the second involved using DNA microarray technology to identify candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and characterisation. These results have made significant progress in unravelling the molecular mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required to unravel the transcriptional control of flavonol biosynthesis. This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An understanding of the synthesis of flavonoids and how they may be coordinated, particularly in response to light, could be used to improve fruit quality by enhanced viticultural management. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326767 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

<p>The reproductive organs of conifers and angiosperms differ in morphology in several fundamental respects. The conifer Norway spruce <i>(Picea abies)</i> form pollen and seed cones from separate meristems whereas angiosperms bear bipartite flowers with sepals and petals surrounding two inner whorls of stamens and carpels. Despite these differences in morphology this thesis present data to suggest that reproductive development in conifers and angiosperms is regulated by a similar molecular mechanism. This implies an evolutionary conservation of the major mechanism for reproductive development since the origin of seed plants. </p><p>Flower organ identity in angiosperms is determined by regulatory genes belonging to the MADS-box gene family of transcription factors. This thesis presents the cloning and characterisation of four novel MADS-box genes from Norway spruce<i>.</i> Three of these genes <i>DAL11</i>, <i>DAL12</i> and <i>DAL13 </i>are most closely related to angiosperm B function genes <i>i.e.</i> genes required for petal and stamen development. <i>DAL11</i>, <i>12</i> and <i>13</i> all are specifically active in developing pollen cones, with different temporal and spatial expression pattern. Functional analysis in transgenic Arabidopsis and yeast suggest that the reproductive aspect of the B-function is conserved between conifers and angiosperms. The results also suggest that the B-function in conifers is separated into one shoot identity and one organ identity determinant. </p><p>A fourth gene presented;<i> DAL10,</i> is specifically expressed in vegetative parts of pollen- and seed cones. Phylogenetically <i>DAL10</i> is not closely related to any of the known angiosperm clades, but rather forms a separate clade with other gymnosperm genes, suggesting a gymnosperm specific function. We suggest that the <i>DAL10</i> activity reflects a function in the determination of the reproductive shoot.</p>

<p>Horizontal cells are retinal interneurons that modulate the output from photoreceptors. Two horizontal cell (HC) subtypes are commonly identified in the vertebrate retina: axon-bearing and axon-less HCs. In this work, we have identified Isl1 as a novel HC marker and demonstrated that Lim1 and Isl1 distinguish axon-bearing and axon-less HCs, respectively. In the chick retina, axon-less HCs are furthermore split into two different subtypes based on the expression of GABA and TrkA.</p><p>We have demonstrated that during early chick retinogenesis, HCs expressing either Lim1 or Isl1 are generated consecutively as two equally large sub-groups at different time points. Moreover, these newborn HCs undertake an unexpected bi-directional migration before settling in their final laminar position. Different HC subtypes complete this migration at different times.</p><p>We investigated the role of activin signaling during HC subtype generation. Activin or its inhibitor follistatin was administrated during the main phase of HC generation and analyzed when HCs had completed migration. Activin caused a significant decrease in both HC subtypes and decreased the proliferation of retinal precursor cells. Follistatin increased the number of late born (Isl1+) HCs, which migrated to the HC-layer during a prolonged migration period. Both treatments affected retinal histology, but only activin influenced the generation of retinal populations other than HCs. These effects were most likely mediated by altered proliferation in certain retinal precursor cells.</p><p>The data on HC subtype ratios, birth-dates, migration, apoptosis and extrinsic activin modulation favor a scenario where the mature proportions of HC subtypes are generated sequentially from a specific HC-precursor cell lineage early in development and remain stable thereafter. These proportions are not adjusted by apoptosis, but rather by the combined actions of transcription factors and extrinsic signaling. Our studies on HC subtypes and their development promises to facilitate future studies on HC development, evolution and function.</p>

In order to function properly, organisms have a complex control mechanism, in which a given gene is expressed at a particular time and place. One way to achieve this control is to regulate the initiation of transcription. This step requires the assembly of several components, i.e., a basal/general machinery common to all expressed genes, and a specific/regulatory machinery, which differs among genes and is the responsible for proper gene expression in response to environmental or developmental signals. This specific machinery is composed of transcription factors (TFs), which can be grouped into evolutionarily related gene families that possess characteristic protein domains. In this work we have exploited the presence of protein domains to create rules that serve for the identification and classification of TFs. We have modelled such rules as a bipartite graph, where families and protein domains are represented as nodes. Connections between nodes represent that a protein domain should (required rule) or should not (forbidden rule) be present in a protein to be assigned into a TF family. Following this approach we have identified putative complete sets of TFs in plant species, whose genome is completely sequenced: Cyanidioschyzon merolae (red algae), Chlamydomonas reinhardtii (green alga), Ostreococcus tauri (green alga), Physcomitrella patens (moss), Arabidopsis thaliana (thale cress), Populus trichocarpa (black cottonwood) and Oryza sativa (rice). The identification of the complete sets of TFs in the above-mentioned species, as well as additional information and reference literature are available at http://plntfdb.bio.uni-potsdam.de/. The availability of such sets allowed us performing detailed evolutionary studies at different levels, from a single family to all TF families in different organisms in a comparative genomics context. Notably, we uncovered preferential expansions in different lineages, paving the way to discover the specific biological roles of these proteins under different conditions. For the basic leucine zipper (bZIP) family of TFs we were able to infer that in the most recent common ancestor (MRCA) of all green plants there were at least four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments. Currently, following the approach described above, up to 57 TF and 11 TR families can be identified, which are among the most numerous transcription regulatory families in plants. Three families of putative TFs predate the split between rhodophyta (red algae) and chlorophyta (green algae), i.e., G2-like, PLATZ, and RWPRK, and may have been of particular importance for the evolution of eukaryotic photosynthetic organisms. Nine additional families, i.e., ABI3/VP1, AP2-EREBP, ARR-B, C2C2-CO-like, C2C2-Dof, PBF-2-like/Whirly, Pseudo ARR-B, SBP, and WRKY, predate the split between green algae and streptophytes. The identification of putative complete list of TFs has also allowed the delineation of lineage-specific regulatory families. The families SBP, bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP and FHA significantly differ in size between algae and land plants. The SBP family of TFs is significantly larger in C. reinhardtii, compared to land plants, and appears to have been lost in the prasinophyte O. tauri. The families bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP and FHA preferentially expanded with the colonisation of land, and might have played an important role in this great moment in evolution. Later, after the split of bryophytes and tracheophytes, the families MADS, AP2-EREBP, NAC, AUX/IAA, PHD and HRT have significantly larger numbers in the lineage leading to seed plants. We identified 23 families that are restricted to land plants and that might have played an important role in the colonization of this new habitat. Based on the list of TFs in different species we have started to develop high-throughput experimental platforms (in rice and C. reinhardtii) to monitor gene expression changes of TF genes under different genetic, developmental or environmental conditions. In this work we present the monitoring of Arabidopsis thaliana TFs during the onset of senescence, a process that leads to cell and tissue disintegration in order to redistribute nutrients (e.g. nitrogen) from leaves to reproductive organs. We show that the expression of 185 TF genes changes when leaves develop from half to fully expanded leaves and finally enter partial senescence. 76% of these TFs are down-regulated during senescence, the remaining are up-regulated. The identification of TFs in plants in a comparative genomics setup has proven fruitful for the understanding of evolutionary processes and contributes to the elucidation of complex developmental programs. / Organismen weisen einen komplexen Steuerungsmechanismus auf, bei dem die Aktivität eines Gens räumlich und zeitlich reguliert wird. Eine Möglichkeit der Kontrolle der Genaktivität ist Regulation der Initiation der Transkription. Eine Voraussetzung für die Transkriptionsinitiation ist die Zusammenlagerung verschiedener Komponenten: eine allgemeine Maschinerie, die für alle exprimierten Gene gleich ist und eine spezifische Maschinerie, die sich von Gen zu Gen unterscheidet und die für die korrekte Genexpression in Abhängigkeit der Entwicklung und von Umweltsignalen verantwortlich ist. Diese spezifische Maschinerie besteht aus Transkriptionsfaktoren (TFs), welche in evolutionär verwandte Genefamilien eingeteilt werden können, die charakteristische Proteindomänen aufweisen. In dieser Arbeit habe ich die Proteindomänen genutzt, um Regeln aufzustellen, die die Identifizierung und Klassifizierung von TFs erlauben. Solche Regeln wurden als Graphen modelliert, in denen die Familien und Proteindomänen als Knoten repräsentiert wurden. Verbindungen zwischen den Knoten bedeuten, dass eine Proteindomäne in einem Protein entweder vorhanden sein sollte oder nicht vorhanden sein darf, damit das Protein einer TF-Familie zugeordnet wird. Mit Hilfe dieses Ansatzes wurden vermutlich vollständige Datensätze von TFs in Pflanzenspezies generiert, deren Genom komplett sequenziert wurde: C. merolae, C. reinhardtii, O. tauri, P. patens, A. thaliana, P. trichocarpa and O. sativa. Diese kompletten TF-Sätze sowie weitergehende Informationen und Literaturhinweise wurden unter der Internetadresse http://plntfdb.bio.uni-potsdam.de/ öffentlich zugänglich gemacht. Die Datensätze erlaubten es, detailliertere evolutionäre Studien mit unterschiedlichen Schwerpunkten durchzuführen. Diese reichten von der Analyse einzelner Familien bis hin zum genomweiten Vergleich aller TF-Familien in verschiedenen Organismen. Als Resultat besonders erwähnenswert ist, dass bevorzugt einige bestimmte TF-Familien in verschiedenen Spezies expandierten. Diese Studien ebnen den Weg, um die spezifische biologische Rolle dieser Proteine unter verschiedenen Bedingungen zu ergründen. Für die wichtige TF-Familie bZIP konnte gezeigt werden, dass der letzte gemeinsame Vorfahr aller Grünpflanzen mindestens vier bZIP Gene hatte, die funktionell in die Antwort auf oxidativen Stress eingebunden waren. Aus den vier Gründergene entstand durch Genverdopplung und –differenzierung eine große Familie, die Eigenschaften hervorbrachte, die die Besiedelung neuer Lebensräume ermöglichten. Mit Hilfe des oben beschriebenen Ansatzes können derzeit aus der Vielzahl der Transkriptionsregulatorfamilien in Pflanzen bis zu 57 TF und 11 TR Familien identifiziert werden. Drei Familien mutmaßlicher TFs markieren die Trennung zwischen Rhodophyta (Rotalgen) und Chlorophyta (Grünalgen): G2-like, PLATZ und RWPRK. Diese könnten eine besondere Rolle bei der Evolution eukaryotischer photosynthetisch aktiver Organismen gespielt haben. Neun zusätzliche Familien (ABI3/VP1, AP2-EREBP, ARR-B, C2C2-CO-like, C2C2-Dof, PBF-2-like/Whirly, Pseudo ARR-B, SBP und WRKY) kennzeichnen die Trennung zwischen Grünalgen und Streptophyten. Die Identifizierung putativer kompletter Listen an TFs erlaubte auch die Identifizierung abtammungsspezifischer regulatorischer Familien. Die Familien SBP, bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP und FHA unterscheiden sich signifikant in ihrer Größe zwischen Algen und Landpflanzen. Die SBP Familie ist in C. reinhardtii signifikant größer als in Landpflanzen. In der Parasinophyte O. tauri scheint diese Familie verloren gegangen zu sein. Die Familien bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP und FHA expandierten präferenziell mit der Kolonialisation an Land. Sie könnten eine wichte Rolle während dieses einschneidenden Ereignisses der Evolution gespielt haben. Später, nach der Trennung von Bryophyten und Tracheophyten sind die Familien MADS, AP2-EREBP, NAC, AUX/IAA, PHD und HRT stärker in den Linien, die zu Samenpflanzen führten, gewachsen. 23 TF-Familien wurden identifiziert, die es nur in Landpflanzen gibt. Sie könnten eine besondere Rolle bei der Besiedelung des neuen Lebensraum gespielt haben. Aufbauend auf die Transkriptionsfaktordatensätze, die in dieser Arbeit erstellt wurden, wurde mittlerweile damit begonnen, experimentelle Hochdurchsatz-Plattformen zu entwickeln (für Reis und für C. reinhardtii), um Änderungen in der Genaktivität der TF-Gene unter verschiedenen genetischen, Entwicklungs- oder Umweltbedingungen zu untersuchen. In dieser Arbeit wird die Analyse von TFs aus A. thaliana im Verlauf der Seneszenz vorgestellt. Seneszenz ist ein Prozess, der zur Zell- und Gewebeauflösung führt, um Nährstoffe aus den Blättern für den Transport in reproduktive Organe freizusetzen. Es wird gezeigt, dass sich die Expression von 187 TF Gene verändert, wenn sich die Blätter voll entfalten und schließlich teilweise in den Prozess der Seneszenz eintreten. 76% der TFs waren runterreguliert, die übrigen waren hochreguliert.