Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-12-11T16:01:41Z No. of bitstreams: 2 Tese -Cesar Augusto Sam Tiago Vilanova Costa - 2013.pdf: 2101811 bytes, checksum: 1cf67584701df4c2df1009b299703f7b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-12-11T19:00:48Z (GMT) No. of bitstreams: 2 Tese -Cesar Augusto Sam Tiago Vilanova Costa - 2013.pdf: 2101811 bytes, checksum: 1cf67584701df4c2df1009b299703f7b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-11T19:00:48Z (GMT). No. of bitstreams: 2 Tese -Cesar Augusto Sam Tiago Vilanova Costa - 2013.pdf: 2101811 bytes, checksum: 1cf67584701df4c2df1009b299703f7b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Não consta resumo em outro idioma. / Foi com a descoberta da atividade antimitótica da cisplatina por Rosenberg na década se 1960 e 70, em seu célebre estudo com bactérias Escherichia coli, que surgiu o interesse em sintetizar e entender as bases moleculares responsáveis pelo mecanismo de ação biológica dos compostos metálicos, visto que a própria cisplatina foi inicialmente sintetizada por Peyrone nos idos de 1840. Os primeiros estudos envolvendo o uso de complexos metálicos de rutênio como agentes antitumorais foram realizados por Tochter no final dos anos 1980 (Dale et al., 1992). Àquela época, foi inferido que todos os compostos de rutênio apresentavam como mecanismo de ação, a sua ligação com o DNA, formando adutos e desencadeando processos celulares de natureza deletéria que, por fim, levariam a morte celular. É interessante lembrar que esse é o mesmo mecanismo de ação dos compostos de platina mais aceitos nos dias atuais. Sadler e Dyson (2003) estudando compostos de rutênio que continham cloro em sua estrutura, como o cloreto de cis-(dicloro)tetraaminorutênio(III) [cis-[RuCl2(NH3)4]Cl], observaram que estes compostos apresentavam mecanismos de ação biológica muito parecidos com os apresentados pela cisplatina [Pt(NH3)2Cl2], onde a hidrólise da ligação Ru–Cl pode ser fortemente influenciada pela natureza dos coligantes presentes na estrutura do rutenato, como grupamentos amino ou até mesmo pela presença de átomos de carbono. A alta concentração de cloretos no sangue permite a esses compostos metálicos, levados por proteínas séricas, chegar até as células e atravessar sua membrana celular e nuclear. Uma vez no interior do núcleo, a ligação Ru–Cl é hidrolisada, devido a queda abrupta da concentração de cloretos (que é cerca de 25 vezes menor), levando o composto a se ligar ao DNA, mais especificamente à posição N7 da base nitrogenada guanina. Por outro lado, compostos que não possuem cloro em sua estrutura, parecem apresentar mecanismos de ação diferentes ao padrão "ligação ao DNA". Sabe-se que compostos que apresentam carboxilatos em sua molécula, como a carboplatina, oxaliplatina e o próprio ditionato de cis-tetraammino(oxalato)rutênio(III) [Cis-[Ru(C2O2)(NH3)4]2(S2O6)], uma vez no interior das células, são hidrolisados muito mais lentamente do que os compostos ricos em cloretos, o que leva a um acúmulo desses compostos no citoplasma, diminuindo sua migração até o núcleo e, assim reduzindo a sua capacidade de se ligar ao DNA. Mas se o DNA não é o alvo desses compostos, então, quem poderia ser? Essa pergunta está sendo respondida com recentes estudos, que revelaram a interação desses compostos, ricos em carboxilatos, com uma miríade de proteínas e enzimas, que vão desde catepsinas, chegando até mesmo à Pgp (Melchart & Sadler, 2008). Estudos realizados por Dyson e colaboradores (2007), utilizando alguns inibidores da proteína Pgp, como fenoxazinas e antracenos, coordenados com compostos de rutênio, observaram que estes novos complexos não somente inibiram a ação da enzima, como também induziram morte celular, demonstrando uma multifuncionalidade. Seguindo essa linha de pensamento, acreditamos que a capacidade do composto ditionato de cistetraammino(oxalato)rutênio(III) em induzir apoptose nas células tumorais, assim como os baixos níveis de expressão de Pgp apresentados pelas células tratadas, corroboram os resultados previamente observados por outros grupos, utilizando compostos de rutênio similares. A resistência a fármacos mediada por Pgp é o mecanismo de MDR mais estudado atualmente. Apesar do desenvolvimento de novos agentes antitumorais, a MDR mediada pela Pgp protege as células de possíveis agentes citotóxicos, limitando a eficácia dos tratamentos quimioterápicos em pacientes com câncer. Atualmente, a extensa maioria dos inibidores da Pgp disponíveis estão associados a vários inconvenientes, que limitam o seu uso no reestabelecimento da eficácia da quimioterapia antineoplásica, após o aparecimento do fenótipo MDR. A procura de inibidores de Pgp alternativos, com um processo sintético exequível e efeitos secundários reduzidos, continua a ser um desafio para os químicos, farmacêuticos e pesquisadores. É nesse contexto que estão sendo desenvolvidos e estudados novos agentes antitumorais que possam agir como inibidores de Pgp, apresentando um efeito dual, ou até mesmo multifuncional, no tratamento clínico das neoplasias malignas. Muito tem se discutido que a próxima geração de fármacos antitumorais poderá ser formada por substâncias que se ligam a mais do que um único alvo terapêutico, o que poderia acelerar tratamento contra a doença, reduzindo o número e a concentração de fármacos que deveriam ser administrados, como os coquetéis atualmente utilizados, e até mesmo aumentando a adesão ao tratamento por parte do paciente. No presente trabalho, estudamos dois complexos de rutênio, o cloreto e o ditionato de rutênio(III), que se apresentam como promissores no possível desenvolvimento de um novo fármaco antitumoral. Essa promessa transparece no fato de ambos serem de síntese química relativamente simples (processo sintético exequível) e, principalmente, por apresentarem efeito biológico de interesse em células tumorais, como citotoxicidade e indução de morte celular, especialmente por apoptose. Pelo que foi observado nos resultados de nossa pesquisa, os complexos aqui estudados, podem constituir um modelo para o estudo de novos agentes anticancerígenos com concomitante capacidade de não induzir MDR. Esta característica se mostrou muito evidente sobre a linhagem leucêmica K-562, onde os níveis de expressão de MDR1, após o tratamento com os rutenatos, foram muito inferiores aos apresentados pelas células tumorais tratadas com o fármaco controle Cisplatina. Ainda, é importante pontuar que o composto ditionato de cistetraammino(oxalato)rutênio(III) apresentou efeito citotóxico em ambas as linhagens tumorais K-562 e A549, sem contudo induzir altos níveis de expressão de Pgp (MDR1), apresentados pelos fármacos platinados. Assim, estudos mais aprofundados sobre a estrutura e funcionamento biológico desses complexos de rutênio, representam um ponto de partida interessante para o desenvolvimento de fármacos multifuncionais e de efeito desejável, auxiliando na delineação de estudos clínicos dirigidos a grupos selecionados de pacientes que reúnam características genotípicas e fenotípicas preditivas de máxima resposta terapêutica com mínima toxicidade. Posteriormente, estes estudos podem levar às realizações de testes diagnósticos e farmacológicos mais eficazes que poderão ser estabelecidos como rotina voltada para uma melhor definição de tratamentos. Isso traria um maior sucesso no teste de novos medicamentos e reduziria os custos e riscos, minimizando o tempo gasto para aprovação de um novo medicamento e a sua disponibilização para a sociedade.

Triquentrinas A são produtos naturais marinhos com atividade biológica e alta complexidade estrutural. Estes fatores tornam estes alcaloides e compostos análogos, como os herbindóis, alvos para a síntese total e plataforma para o desenvolvimento de novas metodologias sintéticas. Nesta Dissertação de Mestrado, procurou-se completar a síntese estereosseletiva da (+)-cis-triquentrina A empregando-se intermediários sintéticos protegidos com o grupo benzila que poderão ser usados para a futura prospecção de novos compostos com atividade biológica. A rota proposta baseia-se na obtenção do ácido (S)-3-(1-benzil-4-etil-1H-indol-7-il)butanóico, um intermediário-chave já descrito por Silva e colaboradores no percurso da síntese total da (+)-trans-triquentrina A, e da finalização da síntese de acordo com a abordagem proposta pelo grupo de RajanBabu para conversão do análogo protegido com grupo tosila à (+)-cis-triquentrina A. A resolução enzimática do intermediário-chave com lipase de Pseudomonas cepacia imobilizada em terra diatomácea foi otimizada, resultando em rendimentos de 32% e 99% ee na metade do tempo descrito anteriormente. Na etapa-chave da síntese, o (S)-ácido foi submetido a uma acilação de Friedel-Crafts intramolecular na presença de anidrido trifluoroacético que produziu o produto de ciclização desejado com 40% de rendimento. Na etapa final da síntese, o intermediário sintético protegido com grupo benzila não pode ser convertido à (+)-cis-triquentrina A, empregando-se a metodologia desenvolvida para a redução do composto análogo tosilado, dada a diferença de reatividade imposta pela troca do grupo protetor. As etapas realizadas até o penúltimo intermediário sintético (S)-8, consta com 10 etapas e rendimento global de 1,3%. viii As diferenças eletrônicas e estruturais relacionadas a diferentes grupos protetores poderão ser refletidas em variações na atividade antiproliferativa de indóis relacionados a triquentrinas. Ainda no interesse de preparar moléculas para envio à análises de atividade antiproliferativa preparou-se um composto relacionado à síntese da trans-triquentrina A tendo como etapa-chave uma contração de anel mediada por I(III) em 21% de rendimento, cujo trabalho foi incluído nos anexos. / Trikentrins A are marine natural products with biological activity and high structural complexity. These factors make these alkaloids and analogous compounds, such as herbidoles, targets for total synthesis and platform for the development of new synthetic methodologies. In this Master\'s Dissertation, we attempted to complete the stereoselective synthesis of (+)-cis-trikentrin A using synthetic intermediates protected with the benzyl group that could be used for the future prospection of new compounds with biological activity. The proposed route is based on the preparation of (S)-3-(1-benzyl-4-ethyl-1H-indol-7-yl) butanoic acid, a key intermediate already described by Silva et al., in the course of total synthesis (+)-trans-triquentrin A, and the final part of the synthesis according to the approach proposed by the RajanBabus group for conversion of the protected analogue with tosyl group to (+)-cis-trikentrin A. The enzymatic resolution of the key intermediate with Pseudomonas cepacia lipase immobilized on diatomaceous earth was optimized, resulting in 32% yield and 99% ee in half the time described above. In the key step of the synthesis, the (S)-acid was subjected to an intramolecular Friedel-Crafts acylation in the presence of trifluoroacetic anhydride which yielded the desired cyclization product in 40% yield. In the final step of the synthesis, the synthetic intermediate protected with benzyl group couldnt be converted to (+)-cis-trikentrin A, employing the methodology developed for the reduction of the tosylated analogous compound, given the difference of reactivity imposed by exchange of the protective group. The steps carried out up to the synthetic intermediate (S)-8, consists of 10 steps and overall yield of 1.3%. Electronic and structural differences related to different protective groups may be reflected in variations in the antiproliferative activity of indoles related to trikentrins. A compound related to the synthesis of trans-triquentrin A having as its key step an I(III) mediated ring contraction in 21% yield was also prepared in the interest of preparing molecules for antiproliferative activity analysis, whose work was included in the appendix.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 dissertacao_lara_dias.pdf: 2572863 bytes, checksum: 41207696775098ff734edf871864bf12 (MD5) Previous issue date: 2011-02-14 / The current challenges in plant breeding are to maximize the productivity of major crop species and to create means for exploring novel crop environments. One of these environments is the lowland hydromorphic soils that are proper for the irrigated rice crop. Adapting other crops to this environment could reduce the incidence of diseases, pests and weeds, therefore benefiting from a crop rotation system. When a plant is exposed to abiotic stresses, it has to cope with environmental changes through physiological and anatomic changes that need quick gene expression responses, i.e., changes in active/silenced status as well as in the rates of transcription. Cis-acting regulatory elements have straight relationship with transcription factors (TF) in complex signaling networks. This TF binding sites (cis-elements) are the functional DNA elements that influence temporal and spatial transcriptional activity. We investigated possible patterns of sequences that can be inferred about the mechanisms that plants use to develop under flooding stress. This search for possible homologies between the various cis-elements would lead us to performed interactive analyses about how plants use their molecular mechanisms responding to abiotic stresses. Online databases were searched, looking for genes previously described in literature which are expressed in response to flooding in Oryza sativa, Arabidopsis thaliana and their homologous in Glycine max and Zea mays. The 1.0 Kb upstream portion of each gene was extracted and analyzed in silico. Besides, all the promoters of these four species were subjected to a tool for searching for novel signals, intending to find new motif patterns. Our in silico analysis shows that from 259 cis elements found in PLACE for all promoters of Arabidopsis and rice, 12 of them are common to both species, and are distinguished by having high frequency. Using the MEME program two consensus motifs could be found among the species Oryza sativa and Zea mays. These could represent new cis elements patterns, because they had relatively high occurrences in the gene promoters and they are related to conserved sequences in monocots. The analysis here presented shows important points for future studies related to the waterlogging stress and unmasking molecular tolerance mechanisms to this typical stress. From the data generated, it will be possible to direct experiments on genetic transformation with target genes and/or cis elements in order to attribute some characteristic in plants, such as those found in rice, so they can develop in an environment with O2 deprivation. / Os atuais desafios no melhoramento de plantas são maximizar a produtividade das principais espécies cultivadas e criar meios para explorar uma variedade de ambientes de cultivo. Um desses ambientes são os solos hidromórficos de planícies alagadas. A adaptação de outras culturas a este ambiente poderia reduzir a incidência de doenças, pragas e plantas daninhas, se beneficiando de um sistema de rotação de culturas. Quando uma planta é exposta a estresses abióticos ela tem que lidar com as alterações ambientais através de mudanças fisiológicas e anatômicas, as quais necessitam de rápidas mudanças na expressão gênica, ou seja, no seu estado inicial, bem como nas taxas de transcrição. Elementos regulatórios de ação cis têm relação direta com fatores de transcrição (FT) em complexas redes de sinalização. Estes sítios de ligação de FTs são elementos funcionais de DNA que influenciam a atividade transcricional de forma temporal e espacial. Neste trabalho, foram investigados possíveis padrões de seqüências que se possam inferir sobre os mecanismos que as plantas utilizam para se desenvolver na condição do alagamento. Essa busca por possíveis homologias entre os vários elementos cis permite realizar análises interativas sobre como as plantas utilizam seus mecanismos moleculares para responder a estresses abióticos. Bancos de dados online foram utilizados, na busca de genes previamente descritos em literatura e que são expressos em resposta ao alagamento em Oryza sativa, Arabidopsis thaliana e seus homólogos em Glycine max e Zea mays. A porção de 1,0 Kb a montante de cada gene foi extraída e analisada in silico. Além disso, todos os promotores das quatro espécies foram submetidos a busca por uma variedade de sinais, com a intenção de encontrar novos padrões de motivos de DNA. Esta análise mostrou que, dos 259 elementos cis encontrados em promotores de Arabidopsis e arroz, 12 deles são comuns a ambas as espécies e se distinguem do restante por terem alta freqüência. Utilizando o programa MEME dois motivos consenso foram encontrados entre as espécies Oryza sativa e Zea mays. Estes podem representar novos padrões de elementos cis, pois apresentaram ocorrências relativamente elevada nos promotores de genes e são relacionados com seqüências conservadas em monocotiledôneas. A análise aqui apresentada mostra pontos importantes para futuros estudos relacionados ao estresse por alagamento e auxilia na descoberta de mecanismos moleculares de tolerância ao alagamento nas plantas. A partir dos dados gerados, será possível direcionar experimentos de transformação genética a fim de atribuir alguma característica às plantas, tais como aqueles encontrados no arroz, para que possam se desenvolver em um ambiente com privação de O2.

L’innovation est devenue d’une nécessité vitale pour la firme afin de s’adapter à un environnement en changement perpétuel. La théorie évolutionniste a mis en exergue le rôle des compétences en matière d’analyse de la firme. Des compétences spécifiques sont ainsi indispensables à la réalisation des projets d’innovation.Selon cette théorie, les compétences internes occupent une place primordiale au sein de la firme mais elles sont parfois insuffisantes à l’entreprise pour innover ; ce qui nécessite de faire appel à l’environnement externe. Outre que les compétences employées en interne, les compétences peuvent être acquises de l’externe à travers des travailleurs indépendants ou par l’intermédiaire des relations de coopération. En mobilisant un cadre d’analyse évolutionniste soutenu par des éléments de la théorie des compétences, nous cherchons à déterminer les facteurs impactant la propension à innover des firmes française tout en soulignant l’influence des compétences employées en interne ou issues de l’extérieur sur la probabilité de s’engager dans des activités d’innovation.Dans cette lignée, nous évoquons d’abord les approches théoriques de l’innovation pour ensuite souligner l’importance des compétences de l’entreprise et de son personnel et la nécessité d’entretenir des accords de coopération pour des fins d’innovation.Pour compléter notre raisonnement nous s’aidons des analyses descriptives suivies par une analyse économétrique effectuée sur des données de l’enquête CIS 2010.L’achèvement de ce travail démontre que la probabilité de s’engager dans des activités d’innovation est affectée par des éléments issus de l’environnement interne et externe de la firme. / Innovation has become a vital need for the firm to deal with a continuously changing environment. Evolutionary theory has emphasized the role of competences in firm analysis. Therefore, specific competences are essential for the fulfillment of firms’ innovation activities.According to evolutionary theory, internal competences are central to the firm. Though, they can be sometimes insufficient for it to innovate. Thus, innovation requires from the firm to look for complementary external competences to reinforce those existing internally.Besides the competences used in-house, competences can be acquired externally through freelancers or cooperative relationships. Basing on an evolutionary analytical framework enhanced with elements of competence-based view, the main body of this thesis analyses the behavior of French companies in terms of innovation. In other words, we seek to determine the factors increasing the innovation capacity of firms while stressing the influence of competences used in-house or acquired externally on the probability for firms to engage in innovation projects.Within this scope, we first invoke different approaches of innovation and then emphasize the importance of firm competences and its staff in addition to their participation into cooperative agreements for innovation. To finalize our interpretation, we conduct a descriptive analysis followed by a study based on the survey data of Innovation CIS 2010. The results show that the probability for firms to engage in innovation activities is affected by elements from the internal and external environment of the firm.

Les switches multimodulables sont des systèmes qui, sous l'action d'un ou de plusieurs stimuli extérieurs, donnent naissance à un ensemble d'états aux propriétés physico-chimiques différentes. Au cours de cette thèse, plusieurs voies de synthèse permettant l'obtention d'une nouvelle famille de biphotochromes organiques constitués d'un dithiényléthène relié à une indolino[2,1-b]oxazolidine ont été mises au point. Les dithiényléthènes préparés présentent d'excellentes performances photochromiques et s'interconvertissent de façon réversible entre deux états stables thermiquement. Les 10-styryl-indolino[2,1-b]oxazolidines sont des molécules acidochromiques et doublement photochromiques par ouverture du cycle oxazolidine ou isomérisation de la double liaison. Ce système moléculaire compte jusqu'à huit états multicolores aux propriétés physico-chimiques différentes, accessibles par irradiations lumineuses et variation de l'acidité. Les propriétés ont été étudiées en solution et en matrice polymère, tous les états des biphotochromes étant caractérisés par spectroscopie d'absorption électronique et RMN. Cette thèse a été l'occasion de développer le premier système multimodulable incorporant huit états différents au sein d'une même molécule. / Multi-addressable switches are systems that give rise to a set of states with different physical-chemical properties upon the effect at least one external stimulus. During this thesis, several synthetic routes to a new family of organic biphotochromes composed of a dithienylethene linked to an indolino[2,1-b]oxazolidine have been developed. Synthesized dithienylethenes exhibit excellent photochromic properties and can be reversibly photoswitched between two thermally stable states. 10-styryl-indolino[2,1-b]oxazolidines are acidochromic molecules and photochromic by opening of the oxazolidine ring or cis/trans isomerization of the double bond. This molecular system exhibits up to eight multicoloured states with different physical-chemical properties, interchangeable by different sequences of light irradiation and pH variation steps. Properties were studied in solution and in a polymer matrix and all biphotochromes states have been characterised by electronic absorption spectroscopy and NMR. Additionally, the first multi-addressable system incorporating eight different states within the same molecule is described.

La régulation des gènes est à l’origine de la diversité cellulaire en permettant aux cellules de se différencier et de se spécialiser. La régulation génique repose largement sur l’existence de séquences d’ADN non codantes dans le génome, appelées "éléments cis-régulateurs", qui vont permettre de recruter de nombreux facteurs de transcription afin de former d’importants complexes (nucléo)protéiques qui vont agir sur le niveau de transcription des gènes. Ce recrutement est notamment contrôlé par des modifications épigénétiques. Le développement des techniques de séquençage et des méthodes d’analyse bioinformatiques permettent d’intégrer de grandes quantités de données pour étudier le fonctionnement des éléments régulateurs. Dans un premier temps, l’intégration de l’ensemble des données ChIP-seq disponibles dans les bases de données nous a permis de créer un catalogue d’éléments régulateurs putatifs chez l’Homme. L’analyse de ce catalogue nous a alors mené à caractériser ces éléments et à mettre en évidence la complexité combinatoire des facteurs de transcription. Dans un deuxième temps, nous avons réalisé une étude basée sur l’analyse des éléments régulateurs impliqués dans la différenciation précoce des lymphocytes T chez la souris. Cette étude a permis de mettre en évidence deux niveaux de complexité impliqués dans la régulation des gènes : le premier est basé sur la combinatoire des facteurs de transcription au sein des éléments régulateurs et le second repose sur la combinatoire des éléments eux-mêmes. Finalement, nous avons développé une nouvelle technique d’analyse quantitative et à haut débit de l’activité régulatrice de régions génomiques chez les mammifères. / Gene regulation is responsible for cell diversity by allowing cell differentiation and specialisation. Gene expression regulation relies mainly on the existence of non-coding DNA sequences in the genome, called "cis-regulatory elements", which recruit numerous transcription factors to form (nucleo)protein complexes which act on the gene transcription level. This recruitment is controlled in particular by epigenetic modifications. The rapid development of sequencing technologies and bioinformatics methods makes possible the integration of large amounts of data to study regulatory elements. First, the integration of ChIP-seq data for all transcription factors available in public databases has allowed us to create an extensive catalogue of putative regulatory elements in the human genome. The overall analysis of this catalogue led us to further characterize these elements and to highlight the high level of combinatorial complexity of transcription factors in the genome. Secondly, we conducted a more specific study based on the analysis of the regulatory elements involved in the early differentiation of T-cells in mice. This study provided an opportunity to highlight two levels of complexity based on regulatory elements and involved in gene regulation: the first rests on the transcription factor combinatorial in regulatory elements and the second is based on the combinatorial of elements themselves within loci. Finally, to validate experimentally the regulatory elements, we have developed a new quantitative and high-throughput technique to assess the regulatory activity of genomic regions in mammals.

Changes in cis- and trans-regulatory elements are among the prime sources of genetic and phenotypical variation at species level. The introduction of cis- and trans- regulatory variation has played important roles in driving diversity, phenotypical differentiation, and evolution of humans. Therefore, variation that occurs on cis- and trans- regulatory elements becomes imperative to better understanding of human genetic diversity and its evolution. In this research, around 3360 gene regulatory factors (GRF) from the human genome were catalogued. This catalog includes genes that code for proteins that perform gene regulatory activities such DNA-depending transcription, RNA polymerase II transcription cofactor and co-repressor activity, chromatin binding and remodeling, among other 218 regulatory functions. This GRF catalog allowed us to initially explore how some GRF genes have evolved in humans, archaic humans (Neandertal and Denisovan) and non-human primate species. We discussed the likely phenotypical and medical effects that evolutionary changes in GRF genes may have introduced into the human genome; for instance, traits associated to speech and language capabilities, genomic recombination hotspots, diseases, among others. By using genome-wide datasets, we additionally looked for GRFs likely to be candidates for positive selection in three human populations: Utah Residents with Northern and Western Ancestry (CEU), Han Chinese in Beijing (CHB), and Yoruba in Ibadan (YRI). As result, we produced a set of candidates that gathers genes that may have contributed in shaping the phenotypical diversity currently observed in these populations; for instance, by introducing regulatory diversity at population-specific level. We additionally identified six GRF classes enriched for genes located in regions that are likely candidates for positive selection at population specific level. We found that out of the 41 DNA-binding GRF classes classified so far, six groups exhibited enrichment for genes located on regions that may have been under positive selection: C2H2 zinc finger, KRAB-ZNF zinc finger, Homeo domain, Tryptophan cluster, Fork head/winged helix and, and High-mobility HMG domain. We additionally identified three KRAB-ZNF gene clusters, in the chromosomes one, three, and 16, for the Asian population that exhibit regions with extended haplotype homozygosity EHH (larger than 100 kb). This EHH suggests that these regions have undergone positive selection in CHB population. Finally, considering that a representative fraction of the phenotypic diversity observed between humans and its closely related species are likely explained by changes in cis-regulatory elements (CREs), we investigated putative binding sites for the transcription factor GABPa. Using ChIP-Seq data generated from a human cell line (HEK293T), 11,619 putative GABPa CREs were found, Out of which 224 are putative human-specific. To experimentally validate the transcriptional activity of these human-specific CREs, reporter gene essays and knock-down experiments were performed. Our results supported the functionality of these human-specific GABPa CREs and suggest that at least 1,215 genes are primary targets of GABPa. Finally, further analyses depict scenarios that put together transcriptional regulation by GABPa and the evolution of particular human traits; for instance, cognitive abilities, breast morphology, lipids and glucose metabolic pathways, among others.

Les ascidies présentent des propriétés remarquables au sein des métazoaires qui en font un modèle particulièrement intéressant pour étudier le fonctionnement et l’évolution des éléments cis-régulateurs dans un contexte développemental. Ciona intestinalis et Phallusia mammillata, deux espèces d’ascidies qui ont divergé il y a environ 300 millions d’années, combinent une grande conservation de leurs processus développementaux avec une grande divergence de leur séquence génomique. Pour comprendre comment « fabriquer » des embryons similaires avec des génomes divergents, nous avons identifié les éléments cis-régulateurs actifs au cours du développement de Ciona intestinalis et Phallusia mammillata en développant et en appliquant la méthode de ChIP-Seq sur des modifications d’histones sur des jeunes gastrulae. La définition puis la validation fonctionnelle de différentes catégories d'éléments cis-régulateurs nous a permis de révéler quelques propriétés de la cis-régulation au sein de génomes compacts et intensément remaniés. En sus, les données que nous avons produites constituent une resource fonctionnelle unique pour la caractérisation des éléments cis-régulateurs chez les ascidies et l'étude de leur évolution au sein des Chordés. / Ascidians display remarkable features within metazoans making them particularly suited for the study of function and evolution of cis-regulatory elements in the context of embryonic development. Ciona intestinalis and Phallusia mammillata, two ascidian species that diverged about 300M years ago, combine high conservation of their developmental processes with high divergence of their genome sequence. To understand how to “make” similar embryos with divergent genomes, we identified active cis-regulatory elements during Ciona intestinalis and Phallusia mammillata development by developing and applying the ChIP-Seq method on histone modifications in early-gastrula embryos. Definition then functional validation of different categories of cis-regulatory elements led us to reveal some features of cis-regulation within compact and highly dynamic genomes. Together, our data constitute a unique functional resource for characterizing cis-regulatory elements in ascidians and questioning their evolution within the Chordates.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Modern surgery of the 21st century relies in many aspects on computers or, in a wider sense, digital data processing. Department administration, OR scheduling, billing, and - with increasing pervasion - patient data management are performed with the aid of so called Surgical Information Systems (SIS) or, more general, Hospital Information Systems (HIS). Computer Assisted Surgery (CAS) summarizes techniques which assist a surgeon in the preparation and conduction of surgical interventions. Today still predominantly based on radiology images, these techniques include the preoperative determination of an optimal surgical strategy and intraoperative systems which aim at increasing the accuracy of surgical manipulations. CAS is a relatively young field of computer science. One of the unsolved "teething troubles" of CAS is the absence of technical standards for the interconnectivity of CAS system. Current CAS systems are usually "islands of information" with no connection to other devices within the operating room or hospital-wide information systems. Several workshop reports and individual publications point out that this situation leads to ergonomic, logistic, and economic limitations in hospital work. Perioperative processes are prolonged by the manual installation and configuration of an increasing amount of technical devices. Intraoperatively, a large amount of the surgeons'' attention is absorbed by the requirement to monitor and operate systems. The need for open infrastructures which enable the integration of CAS devices from different vendors in order to exchange information as well as commands among these devices through a network has been identified by numerous experts with backgrounds in medicine as well as engineering. This thesis contains two approaches to the integration of CAS systems: - For perioperative data exchange, the specification of new data structures as an amendment to the existing DICOM standard for radiology image management is presented. The extension of DICOM towards surgical application allows for the seamless integration of surgical planning and reporting systems into DICOM-based Picture Archiving and Communication Systems (PACS) as they are installed in most hospitals for the exchange and long-term archival of patient images and image-related patient data. - For the integration of intraoperatively used CAS devices, such as, e.g., navigation systems, video image sources, or biosensors, the concept of a surgical middleware is presented. A c++ class library, the TiCoLi, is presented which facilitates the configuration of ad-hoc networks among the modules of a distributed CAS system as well as the exchange of data streams, singular data objects, and commands between these modules. The TiCoLi is the first software library for a surgical field of application to implement all of these services. To demonstrate the suitability of the presented specifications and their implementation, two modular CAS applications are presented which utilize the proposed DICOM extensions for perioperative exchange of surgical planning data as well as the TiCoLi for establishing an intraoperative network of autonomous, yet not independent, CAS modules. / Die moderne Hochleistungschirurgie des 21. Jahrhunderts ist auf vielerlei Weise abhängig von Computern oder, im weiteren Sinne, der digitalen Datenverarbeitung. Administrative Abläufe, wie die Erstellung von Nutzungsplänen für die verfügbaren technischen, räumlichen und personellen Ressourcen, die Rechnungsstellung und - in zunehmendem Maße - die Verwaltung und Archivierung von Patientendaten werden mit Hilfe von digitalen Informationssystemen rationell und effizient durchgeführt. Innerhalb der Krankenhausinformationssysteme (KIS, oder englisch HIS) stehen für die speziellen Bedürfnisse der einzelnen Fachabteilungen oft spezifische Informationssysteme zur Verfügung. Chirurgieinformationssysteme (CIS, oder englisch SIS) decken hierbei vor allen Dingen die Bereiche Operationsplanung sowie Materialwirtschaft für spezifisch chirurgische Verbrauchsmaterialien ab. Während die genannten HIS und SIS vornehmlich der Optimierung administrativer Aufgaben dienen, stehen die Systeme der Computerassistierten Chirugie (CAS) wesentlich direkter im Dienste der eigentlichen chirugischen Behandlungsplanung und Therapie. Die CAS verwendet Methoden der Robotik, digitalen Bild- und Signalverarbeitung, künstlichen Intelligenz, numerischen Simulation, um nur einige zu nennen, zur patientenspezifischen Behandlungsplanung und zur intraoperativen Unterstützung des OP-Teams, allen voran des Chirurgen. Vor allen Dingen Fortschritte in der räumlichen Verfolgung von Werkzeugen und Patienten ("Tracking"), die Verfügbarkeit dreidimensionaler radiologischer Aufnahmen (CT, MRT, ...) und der Einsatz verschiedener Robotersysteme haben in den vergangenen Jahrzehnten den Einzug des Computers in den Operationssaal - medienwirksam - ermöglicht. Weniger prominent, jedoch keinesfalls von untergeordnetem praktischen Nutzen, sind Beispiele zur automatisierten Überwachung klinischer Messwerte, wie etwa Blutdruck oder Sauerstoffsättigung. Im Gegensatz zu den meist hochgradig verteilten und gut miteinander verwobenen Informationssystemen für die Krankenhausadministration und Patientendatenverwaltung, sind die Systeme der CAS heutzutage meist wenig oder überhaupt nicht miteinander und mit Hintergrundsdatenspeichern vernetzt. Eine Reihe wissenschaftlicher Publikationen und interdisziplinärer Workshops hat sich in den vergangen ein bis zwei Jahrzehnten mit den Problemen des Alltagseinsatzes von CAS Systemen befasst. Mit steigender Intensität wurde hierbei auf den Mangel an infrastrukturiellen Grundlagen für die Vernetzung intraoperativ eingesetzter CAS Systeme miteinander und mit den perioperativ eingesetzten Planungs-, Dokumentations- und Archivierungssystemen hingewiesen. Die sich daraus ergebenden negativen Einflüsse auf die Effizienz perioperativer Abläufe - jedes Gerät muss manuell in Betrieb genommen und mit den spezifischen Daten des nächsten Patienten gefüttert werden - sowie die zunehmende Aufmerksamkeit, welche der Operateur und sein Team auf die Überwachung und dem Betrieb der einzelnen Geräte verwenden muss, werden als eine der "Kinderkrankheiten" dieser relativ jungen Technologie betrachtet und stehen einer Verbreitung über die Grenzen einer engagierten technophilen Nutzergruppe hinaus im Wege. Die vorliegende Arbeit zeigt zwei parallel von einander (jedoch, im Sinne der Schnittstellenkompatibilität, nicht gänzlich unabhängig voneinander) zu betreibende Ansätze zur Integration von CAS Systemen. - Für den perioperativen Datenaustausch wird die Spezifikation zusätzlicher Datenstrukturen zum Transfer chirurgischer Planungsdaten im Rahmen des in radiologischen Bildverarbeitungssystemen weit verbreiteten DICOM Standards vorgeschlagen und an zwei Beispielen vorgeführt. Die Erweiterung des DICOM Standards für den perioperativen Einsatz ermöglicht hierbei die nahtlose Integration chirurgischer Planungssysteme in existierende "Picture Archiving and Communication Systems" (PACS), welche in den meisten Fällen auf dem DICOM Standard basieren oder zumindest damit kompatibel sind. Dadurch ist einerseits der Tatsache Rechnung getragen, dass die patientenspezifische OP-Planung in hohem Masse auf radiologischen Bildern basiert und andererseits sicher gestellt, dass die Planungsergebnisse entsprechend der geltenden Bestimmungen langfristig archiviert und gegen unbefugten Zugriff geschützt sind - PACS Server liefern hier bereits wohlerprobte Lösungen. - Für die integration intraoperativer CAS Systeme, wie etwa Navigationssysteme, Videobildquellen oder Sensoren zur Überwachung der Vitalparameter, wird das Konzept einer "chirurgischen Middleware" vorgestellt. Unter dem Namen TiCoLi wurde eine c++ Klassenbibliothek entwickelt, auf deren Grundlage die Konfiguration von ad-hoc Netzwerken während der OP-Vorbereitung mittels plug-and-play Mechanismen erleichtert wird. Nach erfolgter Konfiguration ermöglicht die TiCoLi den Austausch kontinuierlicher Datenströme sowie einzelner Datenpakete und Kommandos zwischen den Modulen einer verteilten CAS Anwendung durch ein Ethernet-basiertes Netzwerk. Die TiCoLi ist die erste frei verfügbare Klassenbibliothek welche diese Funktionalitäten dediziert für einen Einsatz im chirurgischen Umfeld vereinigt. Zum Nachweis der Tauglichkeit der gezeigten Spezifikationen und deren Implementierungen, werden zwei modulare CAS Anwendungen präsentiert, welche die vorgeschlagenen DICOM Erweiterungen zum perioperativen Austausch von Planungsergebnissen sowie die TiCoLi zum intraoperativen Datenaustausch von Messdaten unter echzeitnahen Anforderungen verwenden.