Os mosquitos (Culicidae) compreendem um grupo monofilético, mas algumas relações dentro da família ainda não estão totalmente resolvidas. O objetivo deste trabalho foi elaborar uma hipótese filogenética para os gêneros de Culicidae baseada nos caracteres de variabilidade genética e perfil proteico. Além disso, foi analisado como a forma da asa evoluiu dentro desse grupo. Utilizou-se 76 espécies diferentes abrangendo 20 gêneros. O formato alar mostrou-se um bom marcador taxonômico, já que as análises em cada tribo ou gênero revelaram grupos naturais. Análises com genes nucleares mostraram Anophelinae como grupo irmão de todos os outros Culicinae. A topologia construída com genomas mitocondriais revelou grupos bem suportados, com alguns discordantes da filogenia atual. Foram identificados 24 biomarcadores nas espécies analisadas usando espectrometria MALDI-TOF, que podem ter potencial na identificação taxonômica. A morfologia, a genética e os perfis proteicos não foram concordantes, e isso pode estar ocorrendo devido a taxas evolutivas distintas entre eles. / Mosquitoes (Culicidae) comprise a monophyletic group, but some relationships within the family are not fully resolved. The aim of this study was to elaborate a phylogenetic hypothesis for the genera Culicidae based on the characters of genetic variability and protein profile. In addition, it was analyzed how wing shape evolved within this group. It was used 76 different species covering 20 genera. The wing shape showed to be a good taxonomic marker, because the analyzes in each tribe or genus revealed natural groups. Analyzes with nuclear genes showed Anophelinae as sister group of all other Culicinae. The topology constructed with mitochondrial genomes revealed well supported groups, with some discordant of the current phylogeny. Twenty-four biomarkers were identified in the species analyzed using MALDI-TOF spectrometry, which may have potential in taxonomic identification. Morphology, genetics, and protein profiles were inconsistent, and this may be occurring due to distinct evolutionary rates between them.

Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.

La tesi que teniu a les mans recull quatre treballs amb un objectiu comú; determinar si els patògens (virus, bacteris, paràsits.) han exercit pressions selectives sobre els genomes dels seus hostes (com per exemple els humans).Sabent que la detecció de l'empremta de la selecció permet identificar aquelles regions del genoma que han estat rellevants al llarg de l'evolució d'una espècie, ja que a nivell local és la variació funcional qui acaba essent objecte de la selecció, ens hem disposat a estudiar els possibles senyals de selecció en gens relacionats amb la interacció hoste-patògen. En concret, hem analitzat gens que codifiquen per: a) components del sistema immunitari innat i, b) enzims de glicosilació, la majoria dels quals s'inclouen en quatre de les principals rutes biosintètiques de glicans, en diferents poblacions humanes.Com a conclusió principal; ambdós conjunts de gens mostren clars senyals de selecció. A més hem vist que segons el context biològic on és troben certs gens és veuen més afectats per l'acció de la selecció natural. / The present thesis includes four studies with a common objective: determining whether pathogens (virus, bacteria, parasites.) have exerted selective pressures on the genome of their hosts (for example, humans).Detecting signatures of positive selection is a useful tool to identify functionally relevant genomic regions since selection locally shapes the functional variation. Based on this premise, we have studied the possible signatures of selection in genes related to host-pathogen interactions. Specifically, we have analyzed those genes encoding: a) components of the innate immunity response; and ii) glycosylation enzymes most of them involved in four major glycan biosynthesis pathways, in different human populations.The main conclusion obtained from these studies is that both studied gene categories show clear signatures of selection. Moreover, we have determined that according to their biological context certain genes are more prone to the action of selection.

數年來台灣金控公司已如雨後春筍般冒出來，在金控公司底下含有產險公司、銀行、證券以及人壽公司等許多金融相關公司，因此，原本各自擺放於各子公司的資料庫可以通通整合在一起，當高階主管想提出決策時可利用資料庫進行資料採礦，以獲取有用的資訊。然而資料採礦的效果再怎麼神奇，也必須先有一個好的、完整的資料庫供使用，如果資料品質太差或者資料內容與研究目標無關，這是無法達成完美的資料採礦工作。 透過抽樣調查與函數映射的方法使得資料庫得以加值，因此當有目標資料庫與輔助資料庫時，可以利用函數映射方法使資料庫整合為一個大資料庫，再將資料庫中遺失值或稀少值作插補得到增值後的資料庫。在此給予這個整個流程一個名詞 ”Data SPA(Data Systematic Purifying Analysis)”，即「資料純化」。在本研究中，主要就是針對純化完成的資料進行結構的確認，確認經過這些過程之後的資料是效用且正確的。在本研究採用了橫向評估、縱向評估與全面性評估三種方法來檢驗資料。 資料純化後的資料經過三項評估後，可以發現資料以每個變數或者每筆觀察樣本的角度去查驗資料時，資料的表現並不理想，但是，資料的整體性卻是相當不錯。雖然以橫向評估和縱向評估來看，資料純化後的資料無法與原本完整的資料完全一致，但是透過資料純化的過程，資料得以插補且欄位得以擴增，這樣使得資料的資訊量增加，所以，資料純化確實有其效果，因為資訊量的增加對於要進行資料採礦的資料庫是一大助益。 / For the past few years, Taiwan has experienced a tremendous growth in its financial industry namely in banks, life and property insurances, brokerages and security firms. Needless to say the need to store the data produced in this industry has become an important and a primary task to accomplish. Originally, firms store the data in their own database. With the progressive development of data management, the data now can be combined and stored into one large database that allows the users an easy access for data retrieval. However, if the quality of the data is questionable, then the existence of database would not provide much insightful information to the users. To tackle the fore mentioned problem, this research uses functional mapping combining the goal and auxiliary database and then imputes the missing data or the rare data from the combined database. This whole process is called Data Systematic Purifying Analysis (Data SPA). The purpose of this research is to evaluate whether there is any improvement of the structure of the data when the data has gone through the process of systematic purifying analysis. Generally the resulting data should be within good quality and useful. After the assessments of the data structure, the behavior of the data with respect to their added variables and observations is unsatisfactory. However the manifestation of the data as a whole has seen an improvement. The modified database through Data SPA has augmented the database making it more efficient to the usage of data mining techniques.

Les plantes produisent une remarquable diversité de métabolites pour faire face aux contraintes d’un environnement en constante fluctuation. Cependant la manière dont les plantes ont atteint un tel degré de complexité métabolique et les forces responsables de cette diversité chimique reste largement incomprise. On considère généralement que le mécanisme de duplication des gènes contribue pour une grande part à l’évolution naturelle. En absence de transfert horizontal, les gènes d’évolution récente se cantonnent généralement chez quelques espèces et sont soumis à une évolution rapide, alors que les gènes conservés et plus anciens ont une distribution beaucoup plus large et sont porteurs de fonctions essentielles. Il est donc intéressant d’étudier l’adaptation des plantes en analysant parallèlement les gènes qui présentent soit une large distribution taxonomique, soit une distribution plus restreinte, de type lignée-spécifique. Les cytochromes P450 (CYP) constituent l’une des plus vastes familles de protéines chez les plantes, présentant des phylogénies très conservées ou très branchées qui illustrent la plasticité métabolique et la diversité chimique. Pour illustrer l’évolution des fonctions des cytochromes P450 dans le métabolisme végétal, nous avons sélectionné trois gènes, l’un très conservé au cours de l’évolution, CYP715A1 et les deux autres, CYP98A8 et CYP98A9, très récemment spécialisés de manière lignée spécifique chez les Brassicaceae. Les gènes appartenant à la famille CYP715 ont évolué avant la divergence entre gymnospermes et angiospermes, et sont le plus souvent présent en copie unique dans les génomes végétaux. Ceci suggère que leur fonction est essentielle et très conservée chez les plantes à graines (spermaphytes). Sur la base d’une analyse transcriptionnelle et de l’expression du gène GUS sous le contrôle du promoteur de CYP715A1, il est apparu que ce gène est spécifiquement exprimé au cours du développement floral, dans les cellules tapétales des jeunes boutons floraux ainsi que dans les filaments lors de l’anthèse. CYP715A1 est également fortement induit dans les cellules du péricycle de la zone d’élongation racinaire en réponse au stress salin. L’induction par le sel nécessite une région promotrice située entre 2 et 3 kb en amont de la région codante (i.e ; codon START), ce qui suggère la présence d’un facteur cis à cet endroit. Afin de déterminer la fonction de CYP715A1 chez Arabidopsis thaliana, j’ai identifié deux mutants d’insertion de T-DNA par génotypage et complémenté ces mutants avec le gène natif. La perte de fonction de CYP715A1 n’a pas d’impact sur la croissance et la fertilité de la plante en conditions de laboratoire. Cependant, une analyse par microscopie électronique en transmission montre un phénotype d’intine ondulée. La perte de fonction du gène CYP715A1 a également entraîné une réduction de la taille des pétales et un défaut d’anthèse. [...] / Plants produce a remarkable diversity of secondary metabolites to face continually challenging and fluctuating environmental constraints. However, how plants have reached such a high degree of metabolic complexity and what are the evolutionary forces responsible for this chemodiversity still remain largely unclarified. Gene evolution based on gene birth and extinction has been reported to nicely reflect the natural evolution. Without horizontal gene transfer, young genes are often restricted to a few species and have undergone rapid evolution, whereas old genes can be broadly distributed and are always indicative of essential housekeeping functions. It is thus of interest to study plant adaptation with parallel focus on both taxonomically widespread and lineage-specific genes. P450s are one of the largest protein families in plants, featuring both conserved and branched phylogenies. Examples of P450 properties reflecting metabolic versatility, chemodiversity and thus plant adaptation have been reported. To illustrate evolution of P450 functions in plant metabolism, we selected two P450 genes, one evolutionary conserved CYP715A1 and the second a recently specialized lineage-specific gene CYP98A9 in Arabidopsis thaliana.CYP715s evolved before the divergence between gymnosperms and angiosperms and are present in single copy in most sequenced plant genomes, suggesting an essential housekeeping function highly conserved across seed plants. Based on transcriptome analysis and promoter-driven GUS expression, CYP715A1 is selectively expressed in tapetal cells of young buds and filaments of open flowers during flower development. In addition, CYP715A1 is highly induced in the pericycle cells of the root elongation zone upon salt stress. The salt induction relies on the 2-3kb region of CYP715A1 promoter, suggesting some salt-response elements may exist in this area. To characterize the function of CYP715A1 in Arabidopsis, I identified two T-DNA insertion mutants by genotyping and confirmed by complementation with native CYP715A1 gene. Loss of function of CYP715A1 has no impact on plant growth and fertility in laboratory conditions. However, transmission electron microscopy (TEM) analysis has shown constant undulated intine phenotype in two knockout mutants and also the petal growth is significantly inhibited. These two phenotypes nicely match the native expression pattern of CYP715A1. Gene co-expression analysis suggests involvement of CYP715A1 in gibberellin (GA) metabolism under salt treatment. GAs profiling on mutant flowers also indicates reduced accumulation specific GAs. Unfortunately, no significant phenotype either related to root growth or root architecture under salt treatment can be observed. Recombinant expression of the CYP715A1 enzyme in yeast so far does not allow confirming GAmetabolism. However, metabolic profiling of inflorescences in mutants and over-expression lines, together with transcriptome analysis of the loss of function cyp715a1 mutants strongly support a CYP715A1 role in signaling, hormone homeostasis and volatile emission in agreement with the purifying selection leading to gene conservation observed in spermatophytes.[...]

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We now report an in depth analysis of the successful in vitro enzymatic synthesis of PHB utilizing the three-enzyme system from the bacteria Cupriavidus necator. Using HPLC methodology developed in this laboratory, and by adding each enzyme in a step-wise manner, we follow each individual stage in the three-enzyme route for PHB synthesis and delineate all stoichiometric relationships. We report the construction of the first metabolic model developed specifically for analyzing in vitro enzymatic PHB synthesis. We developed a hands-on student laboratory for culturing, producing, isolating, and purifying the bacterial biopolyesters PHB. We now report the first structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using MS, MS/MS, and homo- and hetero-nuclear NMR analyses.We describe HPLC methodology to separate the isomers of several iso-CoA-containing compounds and report the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl-transfer reactions. We describe a simple regioselective synthesis of iso-CoA from CoA. We also demonstrate a plausible mechanism, which accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. Herein we report that phenylaminoethyl selenide compounds protect DNA from peroxynitrite-mediated single-strand breaks. The mechanism of protection against peroxynitrite mediated DNA damage was investigated by HPLC. The chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. The unique chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. We report the development of novel CDB derivatives, which are selective COX-II inhibitors. A series of compounds were assayed with an in vitro colorimetric inhibitor screening and with a whole blood ELISA screening and the results indicate that MST is a selective inhibitor of COX-II.