Sjöfartshögskolan har under de senaste åren haft en trend av minskade antal ansökningar till sina sjöbefälsutbildningar. Detta har gjort att skolan inte kunnat fylla alla platser. Syftet med examensarbetet var att få en uppfattning om gymnasieelever i Kalmar känner till Sjöfartshögskolan i Kalmar och dess sjöbefälsutbildningar. Detta gjordes via en enkätundersökning och de svarande klasserna var avgränsade till avgångsklasser i de högskoleförberedande programmen som finns i Kalmar tätort. Resultaten visade att kunskapen om Sjöfartshögskolan utbildningar är låg. De har i många fall inte sett till skolan i samband med sitt val och de vet inte heller vilka utbildningar Sjöfartshögskolan har att erbjuda. Slutsatsen är att det kan vara marknadsföringen som brister och måste förändras om trenden ska vända. / Kalmar Maritime Academy have experienced a trend of falling number of applications to their Officer programmes during the last years. This fact have made the Academy unable to fill all seats with enrolled students. The purpose of the thesis was to get an understanding of how well the High School students in Kalmar knew the Maritime Academy and its Officer Programmes. This was made by a questionnaire and the answering classes were limited to those in their graduating year in one of the six university preparatory progammes and attending a High School in the City of Kalmar. The results showed that knowledge about the Academy’s programmes is low. The students have, in many cases, not seen the Academy in relation to their University choices and they also do not know what programmes and courses the Maritime Academy has to offer. The conclusion is it could be marketing that is failing and needs to be changed if the trend is to be turned around.

Os elementos de transposição são seqüências discretas que são capazes de mover- se de um lócus para outro, constituindo uma parte significante do genoma de eucariotos. São agrupados em duas classes principais, os elementos da Classe I, que se transpõem via um intermediário de RNA (retrotransposon), e os elementos da Classe II, que se transpõem via mecanismo de DNA do tipo corta e cola (transposon). A análise das seqüências de um banco de cDNA construído com RNA mensageiro da glândula salivar de Rhynchosciara americana mostrou a presença de representantes das duas classes de elementos. Nesse trabalho caracteriza-se quarto elementos de transposição tipo mariner, onde as seqüências consenso de nucleotídeos foram derivadas de múltiplas cópias defectivas contendo deleções, mudança no quadro de leitura e códons de terminação. Ramar1, um elemento full-length e Ramar2 um elemento defectivo que contém uma deleção na região interna da ORF da transposase, mas mantém e as extremidades intactas. Ramar3 e Ramar4 são elementos defectivos que apresentam muitas deleções no interior da ORF. As seqüências preditas das transposases demostraram que Ramar1 e Ramar2 estão filogeneticamente muito próximos dos elementos mariner da subfamília mauritiana. Enquanto, Ramar3 e Ramar4 pertencem às subfamílias mellifera e irritans, respectivamente. Hibridização in situ mostrou que Ramar1 localiza-se em muitas regiões do cromossomo, principalmente na heterocromatina pericentromérica, enquanto Ramar2 aparece em uma única banda no cromossomo A. Resultado ainda mais curioso foi a caracterização molecular de um elemento de retrotransposição, denominado RaTART, que provavelmente é o responsável pela reconstituição telomérica em R.americana, assim como os elementos TART, HeT-A e TAHRE de Drosophila. Experimentos de Southern Blots do retroelemento RaTART indicam que este está representado por seqüências repetidas no genoma de R.americana, enquanto que Northern Blots mostraram uma expressão em diferentes estágios do desenvolvimento e o transcrito de alto peso molecular detectado representa o retrotransposon non-LTR inteiro. Enquanto a localização cromossômica de RaTART por hibridização in situ mostrou uma marcação predominante nas extremidades dos cromossomos, indicando possivelmente o primeiro elemento de transposição descrito em R.americana com função definida na estrutura do cromossomo. O último retrotransposon, identificado nesse projeto, presente no genoma de R.americana, denominado R2Ra, foi isolado a partir de uma varredura em um banco genômico construído no bacteriófago lambda dash usando como sonda o recombinante pRa1.4 que contém a unidade de repetição do rDNA. A análise da seqüência mostrou a presença de regiões conservadas, como o domínio de transcriptase reversa e o motivo zinc finger na região amino-terminal. O sítio de inserção no gene 28S do rDNA é altamente conservado em R.americana e a análise filogenética mostrou que este elemento pertence ao grupo R2. A localização cromossômica confirma que o elemento móvel R2Ra se insere em um sítio específico no gene rDNA. / Transposable elements are discrete sequences that are able to move from one locus to another within the genome, constituting a significant part of eukaryotic genome. They are grouped into two main types, Class I elements transpose via an RNA intermediate (retrotransposon), and Class II elements transpose via a DNA \"cut-and-paste\" mechanism (transposons). The analysis of sequences of a cDNA bank constructed from mRNA of the salivary glands of Rhynchosciara americana showed the presence of putative types of two classes elements. In the present thesis we describe four mariner elements, where the nucleotides consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1, a full-length element and Ramar2 is a defective mariner element that contains a deletion overlapping most of the internal region of the transposase ORF and the extremities of the element maintain intact. Ramar3 e Ramar4 are defective mariner element that were impossible to predict a complete ORF. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 are phylogenetically very close to mariner-like elements of mauritiana subfamily. However, Ramar3 and Ramar4 belong to mellifera and irritans subfamilies, respectively. In situ hybridisations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A. More interesting data were the molecular characterization of the non-LTR retrotransposon element, called as RaTART, that probably is the responsible by telomeric reconstruction in R.americana, as well as the telomeric retrotransposable elements TART, Het-A and TAHRE of Drosophila. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana, and Northern blot analysis showed a expression in different developmental stages and the transcript of high molecular mass detected represents the full-length non-LTR retrotransposon. However, the chromosomal localization of the retroelement by in situ hybridisation showed a labelling predominant on chromosome ends, indicating possibly the first transposable element described in R.americana with a defined role in chromosome structure. The last retrotransposon, identified in this project, present in the genome of Rhynchosciara americana, called R2Ra, was isolated from screening of a lambda dash genomic library using as probe the recombinant pRa1.4 of rDNA. The analysis of sequence showed the presence of conserved regions, like transcriptase reverse domain and zinc finger motif in the amino terminal region. The insertion site is high conserved in R.americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirm that the R2Ra mobile element insert into the site specific in rDNA gene.

Transposable elements are DNA sequences that can change their location within the genome and can be found in almost all organisms. The mariner transposable element belongs to Class II, Tc1-mariner superfamily. An inactive insertion of this element in the gene white (w+) located on the X chromosome, causes a phenotypic change in color's eyes becoming them a peach color in the place of the wild coloration, being for this reason called whitepeach (wpch). Crossing of mutant females with males obtained from nature, an offspring with eyes the color of peach or variegated with spots of wild color is formed enabling estimate transposicional activity in natural populations. From the use of this system, several studies have been performed to understand if the number of copies and the activity of the mariner element are involved with environmental factors, historical and/or populational. In this study, the existence of intrapopulation variability of D. simulans collected in two places at Santa Maria (RS) city was investigated by crossing with a line of D. simulans wpch. Moreover, data on another species, D. melanogaster, were also obtained and analyzed: interspecific crosses generated progeny with variegation. The isolines that showed these properties were studied using of molecular research tools, such as PCR, cloning and sequencing, it was also necessary. The results suggest the existence of intrapopulation variability in D. simulans and indicate the discovery of sequences very similar to the element mariner in the genome of natural populations of D. melanogaster. / Os elementos transponíveis são seqüências de DNA capazes de alterar sua localização dentro do genoma e podem ser encontrados em praticamente todos os organismos. O elemento transponível mariner pertence à classe II, superfamília Tc1-mariner. Uma inserção inativa deste elemento no gene white (w+) localizado no cromossomo X de Drosophila simulans, causa uma alteração fenotípica na cor dos olhos tornando-os cor de pêssego no lugar da coloração selvagem, sendo por este motivo denominada white-peach (wpch). Do cruzamento de fêmeas mutantes com machos coletados na natureza, uma prole com olhos cor de pêssego ou variegados com pontos de coloração selvagem é formada, possibilitando estimar a atividade transposicional em populações naturais. A partir do uso deste sistema, diversos estudos têm sido realizados para compreender se o número de cópias e a atividade do elemento mariner estão envolvidos com fatores ambientais, históricos e/ou populacionais. Neste trabalho, a existência de variabilidade intrapopulacional de D. simulans coletadas em dois pontos da cidade de Santa Maria RS foi investigada através de cruzamentos com uma linhagem de D. simulans wpch. Além disso, dados sobre uma outra espécie, D. melanogaster, também foram obtidos e analisados: cruzamentos interespecíficos que geraram descendência com variegação fez com que o uso de ferramentas de pesquisa molecular, como PCR, clonagem e seqüenciamento, também fossem necessárias. Os resultados obtidos sugerem a existência de variabilidade intrapopulacional em D. simulans e indicam a descoberta de seqüências semelhantes ao elemento mariner no genoma de populações naturais de D. melanogaster.

Os elementos de transposição são seqüências discretas que são capazes de mover- se de um lócus para outro, constituindo uma parte significante do genoma de eucariotos. São agrupados em duas classes principais, os elementos da Classe I, que se transpõem via um intermediário de RNA (retrotransposon), e os elementos da Classe II, que se transpõem via mecanismo de DNA do tipo corta e cola (transposon). A análise das seqüências de um banco de cDNA construído com RNA mensageiro da glândula salivar de Rhynchosciara americana mostrou a presença de representantes das duas classes de elementos. Nesse trabalho caracteriza-se quarto elementos de transposição tipo mariner, onde as seqüências consenso de nucleotídeos foram derivadas de múltiplas cópias defectivas contendo deleções, mudança no quadro de leitura e códons de terminação. Ramar1, um elemento full-length e Ramar2 um elemento defectivo que contém uma deleção na região interna da ORF da transposase, mas mantém e as extremidades intactas. Ramar3 e Ramar4 são elementos defectivos que apresentam muitas deleções no interior da ORF. As seqüências preditas das transposases demostraram que Ramar1 e Ramar2 estão filogeneticamente muito próximos dos elementos mariner da subfamília mauritiana. Enquanto, Ramar3 e Ramar4 pertencem às subfamílias mellifera e irritans, respectivamente. Hibridização in situ mostrou que Ramar1 localiza-se em muitas regiões do cromossomo, principalmente na heterocromatina pericentromérica, enquanto Ramar2 aparece em uma única banda no cromossomo A. Resultado ainda mais curioso foi a caracterização molecular de um elemento de retrotransposição, denominado RaTART, que provavelmente é o responsável pela reconstituição telomérica em R.americana, assim como os elementos TART, HeT-A e TAHRE de Drosophila. Experimentos de Southern Blots do retroelemento RaTART indicam que este está representado por seqüências repetidas no genoma de R.americana, enquanto que Northern Blots mostraram uma expressão em diferentes estágios do desenvolvimento e o transcrito de alto peso molecular detectado representa o retrotransposon non-LTR inteiro. Enquanto a localização cromossômica de RaTART por hibridização in situ mostrou uma marcação predominante nas extremidades dos cromossomos, indicando possivelmente o primeiro elemento de transposição descrito em R.americana com função definida na estrutura do cromossomo. O último retrotransposon, identificado nesse projeto, presente no genoma de R.americana, denominado R2Ra, foi isolado a partir de uma varredura em um banco genômico construído no bacteriófago lambda dash usando como sonda o recombinante pRa1.4 que contém a unidade de repetição do rDNA. A análise da seqüência mostrou a presença de regiões conservadas, como o domínio de transcriptase reversa e o motivo zinc finger na região amino-terminal. O sítio de inserção no gene 28S do rDNA é altamente conservado em R.americana e a análise filogenética mostrou que este elemento pertence ao grupo R2. A localização cromossômica confirma que o elemento móvel R2Ra se insere em um sítio específico no gene rDNA. / Transposable elements are discrete sequences that are able to move from one locus to another within the genome, constituting a significant part of eukaryotic genome. They are grouped into two main types, Class I elements transpose via an RNA intermediate (retrotransposon), and Class II elements transpose via a DNA \"cut-and-paste\" mechanism (transposons). The analysis of sequences of a cDNA bank constructed from mRNA of the salivary glands of Rhynchosciara americana showed the presence of putative types of two classes elements. In the present thesis we describe four mariner elements, where the nucleotides consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1, a full-length element and Ramar2 is a defective mariner element that contains a deletion overlapping most of the internal region of the transposase ORF and the extremities of the element maintain intact. Ramar3 e Ramar4 are defective mariner element that were impossible to predict a complete ORF. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 are phylogenetically very close to mariner-like elements of mauritiana subfamily. However, Ramar3 and Ramar4 belong to mellifera and irritans subfamilies, respectively. In situ hybridisations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A. More interesting data were the molecular characterization of the non-LTR retrotransposon element, called as RaTART, that probably is the responsible by telomeric reconstruction in R.americana, as well as the telomeric retrotransposable elements TART, Het-A and TAHRE of Drosophila. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana, and Northern blot analysis showed a expression in different developmental stages and the transcript of high molecular mass detected represents the full-length non-LTR retrotransposon. However, the chromosomal localization of the retroelement by in situ hybridisation showed a labelling predominant on chromosome ends, indicating possibly the first transposable element described in R.americana with a defined role in chromosome structure. The last retrotransposon, identified in this project, present in the genome of Rhynchosciara americana, called R2Ra, was isolated from screening of a lambda dash genomic library using as probe the recombinant pRa1.4 of rDNA. The analysis of sequence showed the presence of conserved regions, like transcriptase reverse domain and zinc finger motif in the amino terminal region. The insertion site is high conserved in R.americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirm that the R2Ra mobile element insert into the site specific in rDNA gene.

Les éléments transposables (ET) sont des séquences d‟ADN capables de se déplacer dans les génomes qui les hébergent. Les ET de type mariner (mariner-like element : MLE) ont été caractérisés chez les animaux et chez les plantes à fleurs terrestres mais pas chez les microalgues marines. Dans le présent travail, les MLE ont été recherchés chez les diatomées marines (Bacillariophycées) qui sont des microalgues possédant une enveloppe externe siliceuse. Elles ont colonisé tous les milieux aquatiques et constituent une part majeure du phytoplancton; de ce fait, elles jouent un rôle écologique clé dans le milieu marin.La caractérisation des MLE des diatomées a été réalisée au moyen d‟approches moléculaire et bio-informatique. La présence de MLE dans le génome de 10 espèces de diatomées a été mise en évidence grâce à l‟amplification de fragments d‟environ 380 pb. Ces fragments correspondent à une partie de la séquence conservée codant l‟enzyme responsable de la transposition des MLE : la transposase. L‟analyse des séquences obtenues, par des méthodes phylogénétiques, ainsi que la classification des MLE de diatomées mettent en évidence leur appartenance au groupe des MLE végétaux de la superfamille Tc1-mariner. Néanmoins, les séquences MLE de diatomées divergent considérablement par rapport aux MLE des plantes terrestres. Afin de déterminer si les diatomées ont la capacité de produire la transposase, l‟expression des MLE a été recherchée chez des diatomées soumises à des stress thermiques de courte durée (5 h). Nos travaux montrent que les MLE sont exprimés chez les trois espèces testées incluant la diatomée modèle Phaeodactylum tricornutum dont le génome a été séquencé récemment. L‟expression des MLE des diatomées est variable selon les conditions thermiques et selon les espèces.L‟ensemble de nos résultats suggère que les MLE sont ubiquistes dans les génomes de diatomées et qu‟ils sont présents de manière ancestrale dans la lignée végétale. Les MLE des diatomées forment trois nouvelles sousfamilles de la superfamille Tc1-mariner, la plus répandue des superfamilles d‟ET. De plus, leur expression suggère qu‟il existe des MLE capables de se déplacer dans le génome des diatomées. Si la transposition était vérifiée, ils pourraient alors être développés comme outils de mutagenèse. / Transposable elements (TE) are sequences able to move between two loci in the host genomes. Mariner-like element (MLE) are well characterized in animal and land plant genomes but not in marine microalgae. In this work, we have looked for MLE in marine diatoms (Bacillariophyceae) that are microalgae having a siliceous wall. They have colonized all aquatic environments and are a major component of the phytoplankton, so they play a major role in the ecology of marine environments.To characterize the diatom MLE, molecular and bio-informatics approaches were used. The presence of MLE was detected in ten diatom species which exhibited sequence fragments of about 380 pb. These fragments were identified as a section of the conserved sequence which encodes the enzyme responsible for MLE transposition, the transposase. Phylogeny and classification analysis of these fragments revealed that diatom MLE belong to the Tc1-mariner superfamily, and more precisely to the vegetal MLE group. Nevertheless, diatom MLE sequences diverged from the flowering plant MLE. In order to determine if MLE transposase is produced in diatoms, MLE expression was looked for in diatoms under thermal stresses. Our results showed that diatom MLE were expressed in the three species tested, including the model diatom Phaeodactylum tricornutum which was completely sequenced recently. Diatom MLE expression was dependent on the thermal conditions and on the species.Our results suggest that MLE are widespread in diatom genomes and that they have an ancestral presence in the green lineage. Diatom MLE cluster in three subfamilies in the huge ET Tc1-mariner superfamilly. Finally, the diatom MLE expression detected could reflect the existence of active MLE in diatom genomes. If this hypothesis were verified, it could lead to the development of mutagenesis tools.

Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Detta arbete har haft som syfte att undersöka vilka orsaker som ligger bakom valet att avbryta sina studier i förtid vid sjökaptensprogrammet, samt att kartlägga problemområden som upplevs av de studenter som valt att avsluta sina studier i förtid. Arbetet har även syftat till att undersöka huruvida anledningarna skiljer sig från tidigare forskning av ämnet. Av särskilt intresse var praktikperioden och dess inverkan för studieavhopp. För att undersöka fenomenet närmare har en kvalitativ metod med semistrukturerade intervjuer använts. Respondenterna valdes ut från en och samma klass av sjökaptensprogrammet med ingångsår 2012 för att få undersökningen så tidsmässigt relevant som möjligt. Intervjuerna har fokuserats runt förbestämda teman, vilka tidigare forskning har identifierat som avgörande faktorer som leder till avhopp. Resultatet visar att beslut om studieavhopp uppstår som en kombination av flera faktorer, och är mycket sällan resultatet av en enskild faktor. Stickprovet som undersöktes var homogen med yrkesgruppen fartygsbefäl i resten av landet och även med sina före detta klasskamrater. Utbildningen beskrevs med positiva ordalag och det sociala nätverket ansågs verka skyddande. Praktiken har visat sig vara betydelsefull i beslutet, särskilt då verkligheten ombord kolliderar med studentens förskönade bild om livet till sjöss, och var ofta den utlösande faktorn i beslutet om avhopp samband med en negativ framtidstro och ett bristande intresse för yrket. / This study aims to examine underlying causes in cases of dropout from the Master mariner programme, as well as mapping out problem areas for those students who choose to drop out. The study also aims to examine whether these causes differ from previous research in the area. Of particular interest is the role work experience plays in the decision to drop out. In order to examine the subject further a qualitative method has been used and semi structured interviews have been held with respondents from a single year commencing their studies in 2012 in order for the study to be as relevant as possible. Interviews have focused around pre-determined themes, which previous research has concluded to be of importance. Results show dropout as a result of a combination of factors, and very rarely is a single factor significant enough to describe the phenomena. The sample group was homogenous to the larger population of Officers in the Swedish merchant navy, as well as to their previous class mates. The course was described in positive terms and the social network around a student was often a protective factor. Work experience is shown to be of vital importance when a student considers dropping out, especially when reality collide with the students´own glorified expectations of life at sea. This together with a negative view on future work prospects and a general lack of interest in shipping was commonly reason for dropping out.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Transposition elements (TEs) are DNA sequences with the ability to move from one chromosome region to another. These elements have different mechanisms of transposition and diversity in their sequences, giving them wide distribution among most organisms. The DNA transposon mariner is found in a range of bodies, being ubiquitous in eukaryotes and is excellent for studying the behavior of TE within natural populations of Drosophila. Among the natural populations of D. simulans there are variations in the number of copies of mariner and, hence, in its activity. But within a same population such variations are scarcely assessed. To estimate the activity in natural populations of D. simulans, crosses were made between these and the mutant strain, D. simulans white-peach. The F1 males have spots in the eyes that indicate reversion to the wild condition in a white-peach context, which are used to estimate the activity. In this work, isofemale strains were prepared from recently collected natural populations and from strains maintained in the laboratory. The variables assessed the activity within and between isofemale strains. The number of spots in F1 revealed how many transposition events occurred, and there was no difference in variances between the isofemale strains of the same collection site. The area of the spots showed in what phase of development transposition occurred more frequently. In the test experiment, the embryo stage showed the large patches. Blotches indicated that mobilization occurred in the early stages of cellular determination. In crosses between the evaluated isofemale strains and the white-peach strains, mobilization occurred at all stages of development. Molecular analysis of the copies by qPCR showed variation. But there was no correspondence between the high number of copies and patches most isofemale strains. These differences may be due to a single highly active element or of many elements with low activity. Some already described mariner regulation mechanisms may be acting in isofemale strains evaluated by inhibiting or decreasing the activity of some copies. Additional analyses are also needed to investigate how many copies are actually active in the genome of natural and recently collected strains. / Os elementos de transposição (TEs) são seqüências de DNA com a capacidade de moveremse de uma região cromossômica para outra. Estes elementos apresentam mecanismos de transposição diferenciados e diversidade em suas sequências conferindo-lhes ampla distribuição entre a maioria dos organismos. O transposon de DNA mariner é encontrado numa gama de organismos, sendo ubíquo nos eucariotos e é excelente para estudar o comportamento dos TE dentro de populações naturais de Drosophila. Entre as populações naturais de D. simulans há variações no número de cópias de mariner e, consequentemente, na sua atividade. Porém, dentro de uma mesma população tais variações são pouco avaliadas. Para estimar a atividade em populações naturais de D. simulans, cruzamentos foram realizados entre estas e a linhagem mutante, D. simulans white-peach. Os machos da F1 apresentam nos olhos manchas de reversão à condição selvagem num contexto white-peach, que são utilizadas para estimar a atividade. Nesse trabalho, foram estabelecidas isolinhagens de populações naturais, recentemente coletadas e utilizadas linhagens mantidas no laboratório. As variáveis avaliaram a atividade dentro e entre as isolinhagens. O número de manchas da F1 revelou quantos eventos de transposição ocorreram e através da análise não apresentou diferenças nas variâncias entre as isolinhagens de um mesmo local de coleta. A área das manchas representa em qual fase do desenvolvimento mais ocorreu transposição. No experimento teste, a fase de embrião foi a que apresentou manchas grandes. Manchas grandes indicam que a mobilização ocorreu nos estágios iniciais de determinação celular. Nos cruzamentos entre as isolinhagens avaliadas e a linhagem white-peach, a mobilização ocorreu em todas as fases do desenvolvimento. A análise molecular do número de cópias de mariner por qPCR mostrou variação no número de cópias, porém não houve correspondência entre a alta quantidade de cópias e de manchas na maioria das isolinhagens. Essas diferenças podem ser devidas a um único elemento altamente ativo ou de vários com baixa atividade. Mecanismos de regulação de mariner já descritos podem estar atuando nas isolinhagens avaliadas, inibindo ou diminuindo a atividade de algumas cópias. Também são necessárias análises adicionais que investiguem quantas cópias realmente estão ativas no genoma das linhagens naturais e recentemente coletadas.

Transposons sind genetische Elemente, die fähig sind, sich innerhalb des Genoms zu bewegen. Sleeping Beauty (SB) gehört zur Tc1/mariner-Superfamilie von Transposons. SB wurde aus molekularen Fossilien rekonstruiert um u.a. einen sicheren und effizienten Vektor für die Gentherapie zu schaffen. Zu diesem Zweck ist es notwendig, den Mechanismus der SB-Transposition und deren Regulation, die Aktivitäten des Proteins und den Einfluss von Wirtsfaktoren genau zu verstehen. In meiner Arbeit habe ich die einzelnen Schritte des Transpositionsprozesses und die Bildung des sogenannten paired-end complex (PEC) – eine Voraussetzung für die folgenden katalytischen Reaktionen – untersucht. Zusätzlich habe ich versucht, einen in vitro Transpositionstest für SB zu etablieren. SB gehört zur IR/DR-Gruppe der Tc1/mariner-Superfamilie. Im Gegensatz zu mariner-like-Elementen ist die IR/DR-Struktur von SB durch lange IRs mit insgesamt vier Bindestellen für die Transposase gekennzeichnet. Ich habe die Fähigkeit dieser beiden Transposon-Systeme zum Ausschneiden eines Transposonendes ohne die Beteiligung des anderen Endes im PEC getestet. Solche unpräzise Transposition kann zu genomic rearrangements führen. Meine Ergebnisse zeigen, dass SB zwar imstande ist, ein einzelnes Transposonende auszuschneiden, dies geschieht jedoch weit weniger effizient als bei mariner-like-Elementen. Die Unterdrückung unpräziser Transpositionsereignisse ist ein Ergebnis der besseren durch die IR/DR-Struktur bedingten Regulation von SBs Transposition. Die Komplexität der IRs in Kombination mit der zweiteiligen DNA-Bindedomäne von SB kann als Mittel einer raffinierten Regulation des Transpositionsprozesses angesehen werden, welche das Genom vor anormalen Transpositionsereignissen schützt. Die Ergebnisse meiner Arbeit legen ein Modell nahe, in dem die Bildung des PEC während der Transposition von SB ein höchst genau regulierter Prozess ist, der durch die DNA-Protein- und Protein-Protein-Bindeaffinitäten geleitet wird. / Transposons are pieces of DNA able to move within the genomes. Sleeping Beauty is a verterbrate Tc1/mariner transposon reconstructed from molecular fossils to create a safe and efficient vector for gene therapy. For that purpose it is important to deeply understand the mechanism and regulation of the SB transposition, the activities of the transposase and influence of host factors on the process. Therefore, in this project I studied the single steps of the transposition reaction and formation of the paired-end complex (PEC) which is a prerequisite for the subsequent catalytic steps. Additionally, I tried to establish an in vitro transposition assay for Sleeping Beauty that would serve an easy assay for testing the system and probe mechanisms affecting the regulation of transposition activity. Sleeping Beauty belongs to the IR/DR subfamily of the Tc1/mariner-like transposons. In contrast to mariner-like elements the IR/DR structure of SB is characterized by long IRs with four binding sites for the transposase. I compared the ability of the two systems to perform cleavage of the single transposon end without including the second end in the PEC. Such imprecise transposition can lead to genome rearrangements. My results show that SB is capable of single-end cleavage; however, to much lower extent than the mariner-like element. Lower number of imprecise transposition events is a result of better regulation of the SB transposition imposed by the IR/DR stucture. The complexity of the inverted repeats together with the bipartite DNA-binding domain of SB might offer means for more sophisticated regulation of the transposition process, thereby protecting the genome from aberrant transposition events. I propose that complex formation in SB transposition is a strictly regulated ordered assembly process, guided by DNA-protein and protein-protein interaction interfaces of the DNA-binding subdomains. Obtained results allowed me to draw a model how the paired-end complex is formed.