HOX11 is a homeodomain transcription factor that has been implicated in leukaemic transformation associated with T-cell acute lymphoblastic leukaemia (T-ALL). Its role in leukaemogenesis remains enigmatic, nevertheless, in vitro and in vivo studies have provided additional evidence supporting the role of HOX11 as an oncogene. The mechanism by which HOX11 transforms cells is yet to be elucidated, however, HOX11 has been postulated to function by binding regulatory elements within the promoter regions of specific target genes in order to control gene transcription. The identification of transcriptional targets is thus thought to be critical to our understanding of the pathways controlled by this master gene regulator. To date, only three candidate HOX11 target genes have been reported and given that HOX11 overexpression can have a profound impact on cell behaviour, it is likely that many more exist. In this study, we sought to further understand the role of HOX11 in tumorigenesis by: 1) The identification of novel putative HOX11 target genes by profiling gene expression in response to HOX11 in a number of cell lines using a combination of RDA, cDNA microarray and GeneChip approaches and 2) confirming target gene status by assessing whether the proximal promoters of the leading candidates identified are transcriptionally regulated by HOX11. To identify genes whose expression was altered by HOX11, three techniques were employed, namely representational difference analysis, cDNA microarray and Affymetrix GeneChip array. Because of the relative novelty of these technologies, all three methods were employed in a complementary manner. While representational difference analysis did not require dedicated equipment and enabled the identification of novel genes, the technique was labour-intensive and also exhibited a number of problems including high levels of background. Emphasis was therefore placed on the more systematic microarray approaches that enabled a global investigation of expression patterns and thus the identification of a range of candidate target genes. Initially, this involved cDNA microarray experiments, however, during the course of this work Affymetrix GeneChip technology became available. The latter was identified as the most appropriate technology for the identification of candidate target genes because of its relative ease of use, as well as its employment of multiple independent probe pairs which greatly improved background noise, increased the range and accuracy of detection, minimized the effects of cross hybridization and drastically reduced the rate of false positives and miscalls. Using these combined approaches, several genes of interest were identified which were differentially regulated in the presence of HOX11 and thus may represent oncogenically or physiologically relevant target genes. These included OSTEOPONTIN, PAG, GUANOSINE DIPHOSPHATE DISSOCIATION INHIBITOR 3, SUR8, GAS3, C-KIT, VEGFC, NOR1 and SMARCD3. In order to confirm their role as target genes, four candidates (C-KIT, VEGFC, NOR1 and SMARCD3) were characterized in terms of the ability of their proximal promoters to be transcriptionally regulated by HOX11 using luciferase reporter assays. Significant repression of the proximal promoters of C-KIT and VEGFC by HOX11 was observed, which provided further evidence for their status as target genes. This repression was, however, in stark contrast to the transcriptional activation seen when the C-KIT and VEGFC proximal promoters were co-transfected with a HOX11 mutant lacking the third helix of the DNA-binding homeodomain. This unexpected finding suggested that the transcriptional activity of HOX11 is complex and highly context-dependent, and in particular, highlighted the importance of an intact homeodomain for HOX11 function. C-KIT and VEGFC are both involved in tyrosine kinase signal transduction pathways, as a receptor tyrosine kinase and tyrosine kinase ligand, respectively. C-KIT plays an important role in the survival and self-renewal of haematopoietic cells. It is a previously identified and relatively well characterized oncogene known to be regulated by other transcription factors (SCL/TAL1 and LMO) implicated in the pathogenesis of T-ALL. VEGFC is a member of the vascular endothelial growth factor family that functions in angiogenesis and lymphangiogenesis. A paracrine loop involving VEGFC and its receptor VEGFR-3 has previously been implicated in leukaemic cell survival. While further work is required in order to confirm the status of VEGFC and C-KIT as oncogenically-relevant HOX11 target genes and to characterize their exact mode of regulation, these findings implicate receptor tyrosine kinases in HOX11-mediated tumorigenesis and underscore their potential importance as therapeutic targets in haematological malignancies.

Mouse PEA3 is the founding member of the PEA3 subfamily of ETS transcription factors that includes ERM and ER81. Numerous studies implicate PEA3 subfamily members in a diversity of human cancers, especially breast cancer. Dominant-negative PEA3 (L1NPEA3En) effectively represses activated transcription by all three PEA3 subfamily members. When expressed under control of the MMTV promoter, L1NPEA3En significantly delays the appearance of mammary tumors and reduces their number and size in mouse models of HER2 mediated breast cancer. In addition, L1NPEA3En is not expressed in the mammary tumors that do develop in these mice. These findings strongly suggest a required role for PEA3 subfamily members or other ETS proteins with similar DNA binding specificity in HER2-mediated oncogenesis. The primary objective of this research was to identify the PEA3 subfamily target genes that could play a role in the initiation and progression of tumors, specifically in the breast. To achieve this, a recombinant adenovirus carrying L1NPEA3En was constructed to express L1NPEA3En in three human mammary tumor cell lines: MDA-MB-468, BT-549 and MDA-MB-361. Gene expression analysis using Affymetrix® GeneChip® technology identified a common set of 39 downregulated and 2 upregulated genes in cells expressing L1NPEA3En compared to control cells in all three tumor cell lines. Differentially expressed genes included some that have been shown to play key roles in tumorigenesis such as activating transcriptionfactor 3, heat shock 70kD protein lA and interleukin-8. In addition one colon carcinoma cell line, SW620, was used for gene expression analysis and 7 genes identified in the mammary tumor cell lines were also identified in the colon carcinoma cell line. The results suggest a role for PEA3 subfamily genes in a multiple human cancers mediated through a small subset of common target genes. The genes identified as being differentially expressed by ~NPEA3En hold potential value not only as targets for therapeautic drug discovery, but also as diagnostic or prognostic markers for human cancers, specifically breast cancer. / Thesis / Master of Biological Science (MBioSci)

No description available.

Safe and effective oocyte cryopreservation will have a considerable impact on clinical practice in assisted reproduction. Great improvements have been made in recent years to oocyte vitrification procedures, although further controlled trials are necessary to ensure safety, and it is necessary to know more about pregnancy and live birth outcomes. This study aims to validate various methods of oocyte vitrification as assessed by comparative target gene analysis, hence contributing to information available to clinicians advising women about fertility preservation options before cancer treatment. Target genes investigated were: the maternal effect genes Deleted in Azoospermia Like (DAZL), Maternal Antigen That Embryos Require (MATER/NLRP5) and Zygote Arrest 1 (ZAR1); three genes involved in cell cycle progression and cell death, tumour suppressor protein 53 (p53), B-cell lymphoma 2 (BCL2), BCL2-Associated X Protein (BAX); three genes known to affect spindle and chromatin structure, oocyte-specific histone 1 (H1FOO), kinesin family member 11 (KIF11) and mitotic arrest deficient 2 (MAD2); together with Factor In the GermLine, Alpha (FIGLα) which regulates zona pellucida proteins, octamer-binding transcription factor 4 (OCT4/POU5F1) which is associated with pluripotency and oocyte developmental competence, and superoxide dismutase 2, mitochondrial (SOD2) which responds to oxidative stress in the mitochondria. These genes may be useful indicators of oocyte quality following vitrification. Lysis, complementary DNA (cDNA) amplification, polyadenylic acid polymerase chain reaction (polyA PCR) and quantitative polymerase chain reaction (QPCR) were used to investigate gene expression patterns in failed-to-fertilize non-vitrified, vitrified and slow frozen human MII oocytes. Comparative gene analyses included oocytes vitrified using closed and open carriers, and two different media. Results indicate that the impact of vitrification varies by gene and oocyte variability, highlighting the importance of studies based on single oocytes and the need for caution in interpretation of generalised findings. OCT4 and also β-actin were significantly affected by all methods investigated, while FIGLα, MAD2, ZAR1 and DAZL were affected by some methods. Oocyte survival rate after thawing and the number of genes expressed by individual oocytes were higher with media incorporating dimethyl sulfoxide (DMSO) and Dextran Serum Supplement (DSS) and first-step warming in a larger volume. All methods led to altered expression of target genes, most noticeably when the second media was used. Further quantitative studies of the impact of OCT4, FIGLα and β-actin should be conducted, together with clinical comparisons between media and a longitudinal multi-centre study regarding outcomes arising from different vitrification methods.

Seven genes involved in metabolism were tested as direct targets of the Notch signalling pathway. For each gene the occupancy of its enhancers by Su(H), its transcriptional response to Notch pathway and its biological functionality was verified in vitro and in vivo.

Previous studies in our laboratory demonstrated the expression of WT1 in a significant number of glioma cells and established its role in promoting tumor cell proliferation. Here, we noted the effect(s) of manipulating WT1 levels on the expression levels of genes that were previously shown to be regulated by WT1. We found no correlation between the expression levels of WT1 and PDGF-A, Snai1 and E-cadherin and a consistent inverse correlation between WT1 and IGF-1R expression in U251-MG cells. To ascertain whether the increased IGF-1R levels resulting from WT1 silencing could account for decreased cellular proliferation, we utilized siRNA mediated knockdown of IGF-1R and found a modest decrease in cellular proliferation. Gene expression profiling in U251-MG cells was then used to identify candidate target genes for WT1. Several genes whose levels directly correlated with WT1 were observed to have putative or established oncogenic role(s) in glioma cells or other malignancies. Among the genes correlated inversely, meanwhile, a tumor-suppressor role was attributed to some. Real time RT-PCR helped to substantiate these microarray findings in U251-MG cells. We also characterized the expression and function of WT1 in U1242-MG and GBM6 cells. Interestingly, in these cells WT1 facilitated cell invasiveness but had no discernible influence on cellular proliferation. The expressions of the candidate WT1 target genes were studied also determined in these 2 cell lines. At least 3 genes were consistently down-regulated with WT1 silencing in the three cell lines- INPP5A, CD97, and TYMS. To determine whether CD97 assisted WT1 in facilitating cellular invasion, we silenced CD97 expression using siRNA and noted a significant decrease in the cells’ ability to invade through Matrigel-coated filters. We propose that WT1 profoundly impacts the glioma cells’ invasive ability, and this function is mediated by CD97 alone or in conjunction with other pro-invasive molecules. Our findings argue for the oncogenic role of WT1 in the specific context of glioma cells. They also point to a novel pro-invasive protein- CD97- in glioma cells. Further studies are necessary to confirm the mechanism by which CD97 promotes invasion as well as to explore its potential as a diagnostic and/or therapeutic target.

Xanthomonas axonopodis pv. manihotis (Xam) est une bactérie à gram négatif causant le Cassava Bacterial Blight (CBB) sur Manihot esculenta Crantz. Le manioc représente une des sources les plus importantes de carbohydrates pour près d'un milliard de personnes sur terre et une source importante d'énergie du fait de sa forte concentration en amidon. Le CBB constitue une limitation importante à la production massive de manioc et nos connaissances sur cette maladie sont encore insuffisantes. La pathogénie de nombreuses phytobactéries dépend de l'injection d'effecteurs de type III via un système de sécrétion de type III dans la cellule eucaryote hôte Parmi tous les effecteurs référencés aujourd'hui, les effecteurs de type TAL pour Transcription Activator-Like sont particulièrement intéressant. Une fois injectés dans la cellule végétale, les effecteurs TAL sont importés au noyau et y modulent l'expression de gènes cibles au bénéfice de la bactérie. Chez Xam, TALE1Xam est le seul gène de cette famille qui a été étudié au niveau fonctionnel. Cette étude a pour objectif majeur d'identifier les gènes de manioc dont l'expression est modifiée en présence de TALE1Xam. Le transcriptome de plantes de manioc inoculées avec XamΔTALE1Xam vs. XamΔTALE1Xam (TALE1Xam) a été analysé par RNAseq. Les données obtenues confrontées à la recherche bioinformatique de promoteurs de gènes potentiellement directement activés par TALE1Xam ont permis d'établir une liste de gènes ciblés par TALE1Xam candidats. Un candidat majeur ressort de cette analyse comme étant particulièrement intéressant, il s'agit d'un gène codant un facteur de transcription de type B3 régulant l'activité de protéines de type "Heat Shock". L'analyse fonctionnelle de ce candidat permettra de valider sa fonction en tant que gène de sensibilité du manioc à Xam. / Xanthomonas axonopodis pv. manihotis (Xam) is a gram negative bacteria causing the Cassava Bacterial Blight (CBB) in Manihot esculenta Crantz . Cassava represents one of the most important sources of carbohydrates for around one billion people around the world as well as a source of energy due to its high starch levels content. The CBB disease represents an important limitation for cassava massive production and little is known about this pathosystem. Bacterial pathogenicity often relies on the injection in eucaryotic host cells of effector proteins via a type III secretion system (TTSS). Between all the type III effectors described up to now, Transcription Activator-Like Type III effectors (TALE) appear as particularly interesting. Once injected into the plant cell, TAL effectors go into the nucleus cell and modulate the expression of target host genes to the benefit of the invading bacteria by interacting directly with plant DNA. In Xam, only one gene belonging to this family has been functionally studied so far. It consists on TALE1xam. This work aim to identify cassava genes whose expression will be modified upon the presence of TALE1xam. By means of cassava plants challenged with Xam Δ TALE1xam vs. Xam + TALE1xam together with the TAL effectors code, statistical analyses between RNAseq experiments and a microarray containing 5700 cassava genes, we seek out direct TALE1xam target genes. Hence, through transcriptomic, functional qRT validation and specific artificial TALEs design we proposed that TALE1xam is potentially interacting with a Heat Shock Transcription Factor B3. Moreover we argue that this gene is responsible of the susceptibility during Xam infection. Furthermore this work represents the first complete transcriptomic approach done in the cassava/Xam interaction and open enormous possibilities to understand and study CBB.

MiRNAs são pequenas moléculas de RNAs endógenos não codificantes com aproximadamente 23nt que atuam na regulação da expressão gênica. A sua função é inibir a tradução de genes transcritos através de um mecanismo que viabiliza a ligação do miRNA com o mRNA alvo levando à inibição da tradução ou a degradação do RNA mensageiro. Estudos evidenciam a relação dos miRNAs com diversos processos biológicos como proliferação celular, diferenciação, desenvolvimento e doenças. Uma vez que estão envolvidos na regulação gênica, também alteram as vias metabólicas. Atualmente, as ferramentas computacionais disponíveis para o estudo dos miRNAs são o miRBase, microCosm, o miRGen e o miRNAmap. Elas possuem informações sobre as sequências dos miRNAs, genes alvos e sobre elementos que estão próximos à região dos miRNAs. Embora o avanço até o momento, não existia que relacionasse os miRNAs com as vias metabólicas, para isso foi construída a plataforma miRNApath que auxilia no estudo da função dos miRNAs por meio da análise do seus alvos dentro vias metabólicas. De modo semelhante, também não existia uma abordagem que relacione dados de expressão miRNAs e seus alvos dentro de um mesmo experimento. Para tanto, neste trabalho foi feita uma abordagem utilizando bibliotecas de SAGE (Serial Analysis of Gene Expression) que será incorporada no miRNApath. O miRNApath encontra-se disponível em http://lgmb.fmrp.usp.br/mirnapath. / MiRNAs are small molecules of endogenous non-coding RNAs with approximately 23nt in length that acts over gene expression regulation. Its function is inhibit the translation of gene transcripts through a mechanism that links the miRNA with its mRNA target leading to a translational repression or degradation. Studies show the relation of RNAs in many biological processes like cell proliferation, dierentiation and development of diseases. Since they are involved in gene regulation, they also change the metabolic pathways. Currently, the available computational tools for the study of miRNAs are miRBase, microCosm, miRGen and miRNAmap. They have information about miRNAs sequences, targets and features. Despite the the advances, until now, there is no tool that correlates the miRNAs with metabolic pathways, therefore we developed the miRNApath platform that helps in the analysis of miRNAs function through the study of its targets that are into the metabolic pathway. In the same way, there is no approach that put together information of expression of miRNAs and its targets in the same experiment. In this work we develop an approach with SAGE (Serial Analysis of Gene Expression ) libraries that will be integrated to miRNApath. The plataform is avaible at http://lgmb.fmrp.usp.br/mirnapath.

L’implantation est caractérisée par les premiers contacts cellulaires permanents entre l’endomètre, tapissant l’utérus, et le conceptus (disque embryonnaire et tissus extra-embryonnaires). Cette étape se trouve être l’un des plus importants points de contrôle de la gestation nécessitant un dialogue finement régulé entre ces deux entités. Concernant les ruminants, un déclin de la fertilité a été observé notamment chez les vaches laitières hautes productrices. La moitié des gestations s’arrête pendant la période pré-implantatoire due à des mortalités embryonnaires précoces ainsi qu’à des défauts utérins. Depuis 10 ans, des analyses exploratoires ont été mises en place dans le but d’étudier les profils d’expression de gènes endométriaux sous l’influence du cycle oestral, de la gestation précoce ou encore des stéroïdes ovariens comme la progestérone et les oestrogènes. Ces études sont essentielles pour l’identification des gènes endométriaux clés pour la survie et la croissance du conceptus avant l’implantation. Notre laboratoire a réalisé une analyse transcriptomique à partir d’échantillons endométriaux collectés sur des vaches cycliques et gestantes au 20 ème jour post-oestrus correspondant respectivement à la phase folliculaire et au premier jour d’implantation. Plusieurs familles de facteurs de transcription apparaissent différentiellement exprimées dans cette étude, notamment FOXL2, un membre de la famille des Forkhead Box transcription factor considéré comme le gène clé de la différenciation ovarienne. Ce travail de thèse s’est intéressé à l’implication de FOXL2 dans la physiologie endométriale. FOXL2 est exprimé et régulé pendant le cycle oestral et la gestation précoce dans l’endomètre de ruminants. De plus, la progestérone a été identifiée comme le régulateur majeur de l’expression endométriale de FOXL2 chez la vache et la brebis alors que l’effet des estrogènes n’a pas été démontré. A partir d’une approche gènes candidats, la surexpression de FOXL2 induit la régulation différentielle de onze gènes potentiellement cibles de FOXL2 dans des cultures primaires endométriales de cellules stromales et épithéliales glandulaires. En particulier, PTGS2 qui est un gène impliqué dans la réceptivité utérine apparait inhibé par FOXL2 alors que SCARA5 et RSAD2, tout deux impliqués dans la réponse immunitaire sont stimulés. Enfin, DLX5 apparait différentiellement régulé entre les cellules stromales et épithéliales glandulaires sous l’impact d’une surexpression de FOXL2. Pour conclure, l’expression endométriale de FOXL2 est fortement liée au processus de réceptivité utérine qui se déroule avant l’implantation et peut moduler l’expression de gènes endométriaux essentiels. De nouvelles analyses sont nécessaires pour déterminer si FOXL2 est le gardien de la physiologie reproductive femelle. / Implantation is characterized by the first permanent cellular interactions between the endometrium, lining the uterus, and the conceptus (embryonic disk and extra-embryonic tissues) and appears to be one of the most important checkpoints of successful pregnancy. Regarding ruminant species, and more specifically dairy cows, half of pregnancies abort during the pre-implantation period due to early embryonic death and uterine defects. In the last decade, exploratory approaches have been developed to study endometrial genes expression under the influence of oestrous cycle, early pregnancy, and ovarian steroid hormones in order to identify systematically crucial endometrial genes for conceptus growth and survival leading to a successful implantation in ruminant specifically. A microarray analyse made at the laboratory based on endometrial samples collected from cyclic and pregnant cows at 20 days post-oestrous, corresponding respectively to the follicular phase and the implantation initiation. Several members of the transcription factor families appeared to be differentially expressed in this study including FOXL2, a member of the Forkhead box L sub-class originally considered as a key gene for ovarian differentiation. My PhD thesis focused on the implication of FOXL2 gene in endometrial physiology. FOXL2 gene had been demonstrated to be expressed and regulated during the oestrous cycle and early pregnancy in ruminant endometrium. Moreover, progesterone was identified as a master regulator of FOXL2 endometrial expression in both cattle and sheep whereas estrogens have no impact. Based on candidate genes approach, over-expression of FOXL2 gene induces a regulation of eleven putative FOXL2 target genes in primary endometrial stromal and glandular epithelial cells. In particular, PTGS2 which is a positive regulator gene for uterine receptivity was shown to be inhibited whereas SCARA5 and RSAD2 expressions that were involved in immune response were shown to be stimulated as well as DLX5 expression was differentially regulated between stromal and glandular epithelial cells. Collectively, FOXL2 endometrial expression is strongly linked to the uterine receptivity process prior to the implantation and modulates the expression of essential endometrial genes. Further investigations will be required to investigate whether FOXL2 is the gatekeeper of female reproduction in the vertebrate species.

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.