Analysis of the regulation of Nodal function by SPC-mediated cleavage during early mammalian development

RÉSUMÉ Après implantation dans l'utérus, le foetus de mammifère est composé de trois populations différentes de cellules: l'epiblast, l'ectoderme extraembryonnaire et l'endoderme viscéral. Pendant la gastrulation, les cellules de l'epiblast donnent naissance aux trois lignées germinales: l'ectoderme, le mésoderme et l'endodermes. Les lignées germinales produisent par la suite les différents tissus et organes du corps embryonnaire et adulte. Les cellules de l'ectoderme extraembryonnaire donnent par la suite le composant foetal du placenta qui est essentiel à la survie de l'embryon dans l'utérus. L'épiblast et l'ectoderme extraembryonnaire sont entourés par l'endoderme viscéral et forment une structure connue sous le nom de bouton embryonnaire. L'endoderme viscéral joue un rôle important dans l'embryogenèse car il comporte une sous-population de cellules appelées l'endoderme viscéral antérieur dont les signaux influencent l'épiblast adjacent et déterminent le futur axe antéro-postérieur de l'embryon. La protéine de signalisation Nodal de la famille des TGFß est essentielle dans l'épiblast pour spécifier le mésendoderme, l'endoderme viscéral antérieur, ainsi que pour maintenir les cellules souche de l'ectoderme extraembryonnaire. Ainsi, dans les embryons mutants pour Nodal, aucun axe antéro-postérieur n'est établi, les lignées germinales ne sont pas spécifiés et le placenta ne se développe pas. Au niveau moléculaire, comme pour les protéines de la famille des TGFß, Nodal est initialement synthétisée sous forme de précurseur avant d'être clivée de façon endoproteolytique par des protéanes sécrétées, les proprotéines convertases du type subtilisin (SPC), qui suppriment la partie inhibitrice N-terminale du pro peptide. Dans ce contexte, le projet de ma thèse a été d'analyser l'influence des SPC sur la fonction de Nodal en employant une combinaison d'approches génétiques et biochimiques. Premièrement, nous avons constaté que le clivage du précurseur par les protéases active Nodal, mais en même temps augmente son turn-over et diminue la portée de son action. Deuxièmement, dans l'embryon, il apparaît que Nodal est activé par l'action combinée de Furin et de PACE4, deux protéases sécrétées qui sont spécifiquement exprimées dans les cellules de l'ectoderme extraembryonnaire, donc adjacentes au domaine d'expression de Nodal. De manière similaire aux mutants de Nodal, les embryons mutants pour les deux protéases ne forment pas d'endoderme viscéral antérieur et ne gastrulent pas. Cependant, certains gènes cible de Nodal restent exprimés, suggérant que toutes les activités de Nodal ne sont pas dépendent du clivage par les SPCs. En effet, la génération et l'analyse de mutants portant un allèle knock-in qui code pour une forme mutante de Nodal résistante aux SPC, ont montré que ces mutants ont les caractères phénotypique des mutants de Nodal seulement de façon partielle. La formation de mésoderme est partiellement induite, et de façon remarquable, la forme de Nodal résistante aux SPC est capable d'agir à une distance de sa source, maintenant l'expression de ses propres protéases et d'autres gènes essentiels pour la spécification de l'ectoderme extraembryonnaire. Ensemble, ces résultats prouvent que par leur action directe les protéases extraembryonnaire modulent la signalisation de Nodal pendant le développement mammifère précoce. SUMMARY : Early after implantation in the uterus, the mammalian conceptus is composed of three different cell populations: the epiblast, the extraembryonic ectoderm and the visceral endoderm. During gastrulation, epiblast cells give rise to the three embryonic germ layers: the ectoderm, the mesoderm and the endoderm. These germ layers then generate the different tissues and organs of the embryonic and adult bodies. In parallel, extraembryonic ectoderm cells give rise to the fetal component of the placenta, which is essential for the survival of the embryo in the uterus. Both the epiblast and extraembryonic ectoderm are surrounded by the visceral endoderm to form a structure known as the egg cylinder. The visceral endoderm plays an important role as it harbours a subpopulation of cells called the anterior visceral endoderm, from which signals influence the adjacent epiblast and determine the future antero-posterior embryonic axis. The TGFß-related signalling protein Nodal is required within the epiblast to specify the mesoderm, the endoderm,the anterior visceral endoderm and is also essential to maintain stem cells in the extraembryonic ectoderm. Thus, in Nodal null conceptuses, no antero-posterior axis is established, the germ layers are not specified and the placenta does not develop. At the molecular level, Nodal, like related proteins of the TGFß family, is initially synthesized as a precursor and undergoes endoproteolytic cleavage by secreted proteases of the subtilisin-like proprotein convertases (SPC) to remove an inhibitory N-terminal pro peptide. In the embryo, Nodal is activated by the combined action of Furin and PACE4, two secreted SPCs that are specifically expressed in cells of the extraembryonic ectoderm, thus adjacent to the Nodal expression domain. Similar to Nodal null .embryos, mutant embryos lacking both these proteases fail to specify the anterior visceral endoderm and to undergo gastrulation. However, these mutants still express a subset of Nodal target genes, suggesting that part of Nodal activity is independent on cleavage by SPCs. Indeed, by generating and analyzing mutants with a knock-in allele that encodes an SPC-resistant mutant form of Nodal, I could show that they retain a subset of Nodal activities. Mesoderm formation is partially induced, but most remarkably, SPC-resistant Nodal form is able to act at a distance from its source, maintaining the expression of its proteases and of other genes essential for maintenance of the extraembryonic ectoderm.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Meta-analysis of studies analyzing the role of human papillomavirus in the development of bladder carcinoma

PURPOSE We aimed to ascertain the degree of association between bladder cancer and human papillomavirus (HPV) infection. MATERIALS AND METHODS We performed a meta-analysis of observational studies with cases and controls with publication dates up to January 2011. The PubMed electronic database was searched by using the key words "bladder cancer and virus." Twenty-one articles were selected that met the required methodological criteria. We implemented an internal quality control system to verify the selected search method. We analyzed the pooled effect of all the studies and also analyzed the techniques used as follows: 1) studies with DNA-based techniques, among which we found studies with polymerase chain reaction (PCR)-based techniques and 2) studies with non-PCR-based techniques, and studies with non-DNA-based techniques. RESULTS Taking into account the 21 studies that were included in the meta-analysis, we obtained a heterogeneity chi-squared value of Q(exp)=26.45 (p=0.383). The pooled odds ratio (OR) was 2.13 (95% confidence interval [CI], 1.54 to 2.95), which points to a significant effect between HPV and bladder cancer. Twenty studies assessed the presence of DNA. The overall effect showed a significant relationship between virus presence and bladder cancer, with a pooled OR of 2.19 (95% CI, 1.40 to 3.43). Of the other six studies, four examined the virus's capsid antigen and two detected antibodies in serum by Western blot. The estimated pooled OR in this group was 2.11 (95% CI, 1.27 to 3.51), which confirmed the relationship between the presence of virus and cancer. CONCLUSIONS The pooled OR value showed a moderate relationship between viral infection and bladder tumors.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Cloud code test IDE

L'objectiu principal d'aquest projecte final de carrera consisteix en desenvolupar una aplicació web que oferisca un entorn simplificat de desenvolupament integrat (IDE) en el llenguatge C/C++, on els estudiants de batxillerat (o secundària en general) puguen iniciar-se en el seu estudi. La finalitat és proveir un entorn agradable a l'alumnat perquè puga seguir correctament les pràctiques que proposa el professor independentment de les circumstàncies pròpies d'aquest (ubicació temporal o permanentde l'alumnat, sistema operatiu que utilitza, dispositiu emprat per a fer les pràctiques, etc..).

Development of the Iowa Drainage Law Manual, March 2005

Although Iowa has some of the most productive agricultural land in the nation, it also maintains a very extensive road network.Consequently, landowners and roadway officials often must deal with drainage issues affecting private lands and public highways. However, many individuals are unfamiliar with legal drainage requirements, practices, and procedures, which can result in misunderstandings concerning maintenance responsibilities for drainage facilities, sometimes leading to litigation. To assist propertyowners, public agencies, and others with interest in better understanding drainage maintenance responsibilities, a reference manual was developed to describe Iowa’s drainage laws and offer interpretations in a clear and concise manner. To develop a comprehensive drainage manual, researchers identified and reviewed current available literature. These resources described pertinent drainage issues and presented explanations of legal responsibilities. The literature review included manuals and guides from Iowa, surrounding states, and federal agencies. Researchers developed a survey to assess the needs and interestsof potential users of an Iowa drainage law manual. Survey responses were used to identify common problems and concerns among individuals who encounter drainage issues on a regular basis. Issues mentioned in the survey responses included interpretation of drainage laws and commonly encountered questions relating to public improvements and private interests. Many individuals, including county engineers, stated interest in specific topics such as maintenance and/or diversion of drainage, landowner issues, and upstream and downstream impacts. Overall, the survey provided researchers with valuable information regarding drainage issues, problems, current policies, and concerns. A comprehensive manual of Iowa drainage law will assist agencies and individuals in interpreting current code requirements and in implementing effective and beneficial solutions when dealing with drainage issues.

Annual Report of Highway Division Highway Research and Development in Iowa for the Fiscal Year ending June 30, 2005

The Highway Division of the Iowa Department of Transportation engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; second, to identify and implement improved engineering and management practices. This report, entitled Highway Division Highway Research and Development in Iowa, is submitted in compliance with Sections 310.36 and 312.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund respectively. It is a report of the status of research and development projects, which were in progress on June 30, 2005; it is also a report on projects completed during the fiscal year beginning July 1, 2004, and ending June 30, 2005. Detailed information on each of the research and development projects mentioned in this report is available in the Research and Technology Bureau in the Highway Division of the Iowa Department of Transportation.

Annual report of the Iowa Highway Research Board Research and Development Activities FY 2006

The Highway Division of the Iowa Department of Transportation engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; and second, to identify and implement improved engineering and management practices. This report is submitted in compliance with Sections 310.36 and 312.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund, respectively. It is a report of the status of research and development projects, which were in progress on June 30, 2006; it is also a report on projects completed during the fiscal year beginning July 1, 2005, and ending June 30, 2006.

Report of Highway Research Board Research and Development Activities FY2007

The Highway Division of the Iowa Department of Transportation (Iowa DOT) engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; second, to identify and implement improved engineering and management practices. This report, entitled “Iowa Highway Research Board Research and Development Activities FY2007” is submitted in compliance with Sections 310.36 and 312.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund respectively. It is a report of the status of research and development projects, which were in progress on June 30, 2007; it is also a report on projects completed during the fiscal year beginning July 1, 2006, and ending June 30, 2007. Detailed information on each of the research and development projects mentioned in this report is available in the Research and Technology Bureau in the Highway Division of the Iowa Department of Transportation.

Annual Report of Iowa Highway Research Board Research and Development Activities FY 08

RESEARCH AND DEVELOPMENT The Highway Division of the Iowa Department of Transportation (Iowa DOT) engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; second, to identify and implement improved engineering and management practices. This report, entitled "Iowa Highway Research Board Research and Development Activities FY2008" is submitted in compliance with Sections 310.36 and 3 I2.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund respectively. It is a report of the status of research and development projects in progress on June 30, 2008; it is also a report on projects completed during the fiscal year beginning July 1, 2007, and ending June 30, 2008. Detailed information on each of the research and development projects mentioned in this report is available in the Research and Technology Bureau in the Highway Division of the Iowa Department of Transportation. IOWA HIGHWAY RESEARCH BOARD In developing a progressive, continuing and coordinated program of research and development, the Highway Division is assisted by the Iowa Highway Research Board. This advisory group was established in 1949 by the Iowa State Highway Commission to respond to the research denoted in Section 310.36 of the Code of Iowa and now is denoted by 312.3A. The Research Board consists of 15 regular members: seven Iowa county engineers, four Iowa DOT engineers, one representative from Iowa State University, one from The University of Iowa, and two engineers employed by Iowa municipalities. Each regular member may have an alternate who will serve at the request of the regular member. The regular members and their alternates are appointed for a three-year term. The membership of the Research Board as of June 30, 2008, is listed in Table I. The Research Board held nine regular meetings during the period ofJuly 1, 2007, to June 30, 2008. Suggestions for research and development were reviewed at these meetings and recommendations were made by the Board.

Construction Code, November 25, 2008

Construction codes establish minimum standards for structural integrity, life safety, accessibility and energy conservation in construction of buildings and facilities intended for human occupancy. These requirements affect all of the major systems of buildings and facilities.

Annual Report of Iowa Highway Research Board Research and Development Activities 2007

RESEARCH AND DEVELOPMENT The Highway Division of the Iowa Department of Transportation (Iowa DOT) engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; second, to identify and implement improved engineering and management practices. This report, entitled "Iowa Highway Research Board Research and Development Activities FY2008" is submitted in compliance with Sections 310.36 and 3 I2.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund respectively. It is a report of the status of research and development projects in progress on June 30, 2008; it is also a report on projects completed during the fiscal year beginning July 1, 2007, and ending June 30, 2008. Detailed information on each of the research and development projects mentioned in this report is available in the Research and Technology Bureau in the Highway Division of the Iowa Department of Transportation. IOWA HIGHWAY RESEARCH BOARD In developing a progressive, continuing and coordinated program of research and development, the Highway Division is assisted by the Iowa Highway Research Board. This advisory group was established in 1949 by the Iowa State Highway Commission to respond to the research denoted in Section 310.36 of the Code of Iowa and now is denoted by 312.3A. The Research Board consists of 15 regular members: seven Iowa county engineers, four Iowa DOT engineers, one representative from Iowa State University, one from The University of Iowa, and two engineers employed by Iowa municipalities. Each regular member may have an alternate who will serve at the request of the regular member. The regular members and their alternates are appointed for a three-year term. The membership of the Research Board as of June 30, 2008, is listed in Table I. The Research Board held nine regular meetings during the period ofJuly 1, 2007, to June 30, 2008. Suggestions for research and development were reviewed at these meetings and recommendations were made by the Board.

Annual Report of the Iowa Highway Research Board Research and Development Activities FY 2009

The Highway Division of the Iowa Department of Transportation (Iowa DOT) engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; and second, to identify and implement improved engineering and management practices. This report, entitled ―Iowa Highway Research Board Research and Development Activities FY2009‖ is submitted in compliance with Sections 310.36 and 312.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund, respectively. It is a report of the status of research and development projects in progress on June 30, 2009. It is also a report on projects completed during the fiscal year beginning July 1, 2008 and ending June 30, 2009. Detailed information on each of the research and development projects mentioned in this report is available from the Research and Technology Bureau, Highway Division, Iowa Department of Transportation. All approved reports are also online for viewing at: www.iowadot.gov/operationsresearch/reports.aspx.

Annual Report of the Iowa Highway Research Board – Research and Development Activities FY 2010

This report, entitled “Iowa Highway Research Board Research and Development Activities FY 2010” is submitted in compliance with Sections 301.35 and 312.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund, respectively. T is a report of the status of research and development projects in progress on June 20, 2010. It is also a report on projects completed during the fiscal year beginning July 1, 2009, and ending June 30, 2010. Detailed information on each of the research and development projects mentioned in this report is available from the Research and Technology Bureau, Highway Division, Iowa Department of Transportation. All approved reports are also online for viewing at www.iowadot.gov/operationsresearch/reports.aspx

Annual Report of the Iowa Highway Research Board Research and Development Activities FY 2010

The Highway Division of the Iowa Department of Transportation (Iowa DOT) engages in research and development for two reasons: first, to find workable solutions to the many problems that require more than ordinary, routine investigation; and second, to identify and implement improved engineering and management practices. This report, entitled ―Iowa Highway Research Board Research and Development Activities FY2009‖ is submitted in compliance with Sections 310.36 and 312.3A, Code of Iowa, which direct the submission of a report of the Secondary Road Research Fund and the Street Research Fund, respectively.