Transposable elements (TEs) are utilized for the creation of a wide range of transgenic organisms. However, in some systems, this technique is not very efficient due to low transposition frequencies and integration into unstable or transcriptionally inactive genomic regions. One approach to ameliorate this problem is to increase knowledge of how transposons move and where they integrate into target genomes. Most transposons do not insert randomly into their host genome, with class II TEs utilizing target sequences of between 2 – 8 bp in length, which are duplicated upon insertion. Furthermore, amongst insertion sites, certain sites are preferred for insertion and hence are classified as hot spots, while others not targeted by TEs are referred to as cold spots. The hypothesis tested in this analysis is that in addition to the primary consensus target sequence, secondary and tertiary DNA structures have a significant influence on TE target site preference. Bioinformatics was used to predict and analyze the structure of the flanking DNA around known insertion sites and cold spots for various TEs, to understand why insertion sites are used preferentially to cold spots for element integration. Hidden Markov Models were modeled and trained to analyze datasets of insertions of the P element in the Drosophila melanogaster genome, the Tc1 element in the Caenorhabditis elegans genome, and insertions of the Mos1, piggyBac and Hermes transposons into the target plasmid pGDV1. Analysis of the DNA structural profiles of the insertion sites for the P element and Hermes transposons revealed that both transposons targeted regions of DNA with a relatively high degree of bendability/flexibility at the insertion site. However, similar trends were not observed for the Tc1, Mos1 or piggyBac transposons. Hence, it is believed that the secondary structural features of DNA can contribute to target site preference for some, but not all transposable elements.

Transposable elements (TEs) are mobile DNA sequences that are a source of mutations and can target specific sites in host genome. Understanding the molecular mechanisms of TE target site preferences is a fundamental challenge in functional and evolutionary genomics. Here we used accurately mapped TE insertions in the Drosophila melanogaster genome, from large-scale gene disruption and resequencing projects, to better understand TE insertion site mechanisms. First we test predictions of the palindromic target site model for DNA transposon insertion using artificially generated P-element insertions. We provide evidence that the P-element targets a 14 bp palindromic motif that can be identified at the primary sequence level that differs significantly from random base composition in the D. melanogaster genome. This sequence also predicts local spacing, hotspots and strand orientation of P-element insertions. Next, we combine artificial P-element insertions with data from genome- wide studies on sequence properties of promoter regions, in an attempt to decode the genomic factors associated with P-element promoter targeting. Our results indicate that the P-element insertions are affected by nucleosome positioning and the presence of chromatin marks made by the Polycomb and trithorax protein groups. We provide the first genome-wide study which shows that core promoter architecture and chromatin structure impact P-element target preferences shedding light on the nuclear processes that influence its pattern of TE insertions across the D. melanogaster genome. In an effort to understand the natural insertion preferences of a wide range of TEs, we then used genome resequencing data to identify insertions sites not present in the reference strain. We found that both Illumina and 454 sequencing platforms showed consistent results in terms of target site duplication (TSD) and target site motif (TSM) discovery. We found that TSMs typically extend the TSD and are palindromic for both DNA and LTR elements with a variable center that depends on the length of the TSD. Additionally, we found that TEs from the same subclass present similar TSDs and TSMs. Finally, by correlating results on P-element insertion sites from natural strains with gene disruption experiments, we show that there is an overlap in target site preferences between artificial and natural insertion events and that P-element targeting of promoter regions of genes is a natural characteristic of this element that is influenced by the same features has the artificially generated insertions. Together, the results presented in this thesis provide important new findings about the target preferences of TEs in one of the best-studied and most important model organisms, and provide a platform for understanding target site preferences of TEs in other species using genomic data.

Die Ruptur des vorderen Kreuzbandes (VKB) zählt zu den häufigsten Bandverletzungen des Menschen und wird vor allem bei jungen, aktiven Menschen primär operativ versorgt. Obwohl sich das Verständnis der Anatomie, Funktion und die Operationstechniken stetig verbessert haben ist heute die Revisionsrate nach VKB - Operation hoch. Die optimale Platzierung der Insertionspunkte ist unter arthroskopischen Bedingungen einer der kritischsten Schritte der Operation. Das Ziel der Studie war, ein Bildwandler (BV) basiertes Navigationsverfahren zu entwickeln und zu evaluieren, mit dem der femorale Bohrkanal anhand etablierter geometrischer Schablonen präzise navigiert angelegt werden kann. Die Anlage des tibialen Bohrkanals sollte in Relation zur Anatomie des Patienten auf BV-Bildern dargestellt werden können. Zur Ermittlung der Präzision des Systems wurden Insertionspunktlagen anhand abgewandelter geometrischer Schablonen mit dem Navigationssystem bestimmt, deren Lage am Modell radiologisch ausgewertet und mit den geplanten Sollwerten statistisch verglichen wurde. Außerdem wurden der Einfluss von Projektionsfehlern des BV-Bildes durch Rotation der Modelle um die Quer- und Längsachse und der Einfluss von Planungsungenauigkeiten untersucht. Das am Modell entwickelte System wurde anschließend im klinischen Einsatz auf Praktikabilität validiert. Es hat sich gezeigt, dass die femoralen Insertionspunkte am Modell unabhängig von der verwendeten Schablone mit einer hohen Präzision bestimmt werden können. Die medianen Abweichungen vom Sollwert in proximal-distaler (PD) - Richtung und in anterior-posteriorer (AP) - Richtung lagen bei allen Schablonen unter 1,00 mm. In PD-Richtung zeigte die Schablone nach Hertel et al. eine signifikant geringere Abweichung vom Sollwert als die Schablone nach Klos et al.. In AP-Richtung wurden mit der Schablone nach Hertel et al. signifikant geringere Abweichungen als mit den beiden anderen Schablonen erreicht. Bei Bestimmung des Projektionsfehlers durch Rotation lag die mediane Abweichung pro 1°-Rotation um die Längsachse in proximal-distaler (PD) - Richtung bei 0,31 % bzw. 0,07 mm und in anterior-posteriorer (AP) - Richtung bei 0,36 % bzw. 0,17 mm. Bei Quer-Rotation lag die mediane Abweichung pro 1°-Rotation in PD-Richtung bei 0,25 % bzw. 0,06 mm und in AP-Richtung bei 0,64 % bzw. 0,30 mm. Bei den Untersuchungen zur Abweichung durch Planungsungenauigkeit hat sich gezeigt, dass bei allen drei Schablonen Planungsfehler in anterior-posteriorer Richtung entlang der Blumensaat-Linie (BSL) die geringsten Auswirkungen auf die Lage des femoralen Insertionspunktes haben. Abweichungen bei der Planung in Richtung DISTAL / BSL(post.) und PROXIMAL / BSL(post.) haben die größten Fehler erbracht. / The Anterior Cruciate Ligament (ACL) is one of the most often torn ligaments of the musculoskeletal system. Especially in young and active people torn ACL are mainly treated surgically. Despite an increasing knowledge of the anatomical, functional and operative aspects about the ACL, there is still a high rate of long-time failures. Optimal positioning of the ACL-graft during arthroscopic reconstruction is of paramount importance in order to get good results. The aim of this project was to develop and evaluate a fluoroscopically based navigation system which could be used to precisely place the femoral drill hole with help of geometrical templates. The placement and orientation of the tibial tunnel was intended to be displayed on x-rays in relation to the patient’s anatomy. To determine the system’s precision, insertion sites where defined at the fluoroscopic image using geometrical templates and marked on plastic femur models with the help of navigation. The position of the insertion sites on the model was radiologically identified and statistically compared with the aimed ones. Additionally the influence of fluoroscopic projection errors due to axial and planar rotation of the models was tested. Following the model tests, the developed system was clinically tested in the OR to test its feasibility. The model tests revealed a high precision in femoral placement of the insertion sites independent from the three used templates (Hertel et al. / Klos et al. / Cazenave et al.). The median deviations from the aimed positions were in proximal-distal (PD) and in anterior-posterior (AP) direction both below 1.00 mm. In PD-direction the template by Hertel et al. showed a significantly lower deviation from the aimed position than the template developed by Klos et al.. In AP-direction the template by Hertel et al. showed a significantly lower deviation than the two others. The mean projection error due to longitudinal rotation per 1° was for PD-direction 0.31 % (0.07 mm) and in AP-direction 0.36% (0.17 mm). For planar rotation the median projection error per 1° was 0.25% (0.006 mm) for PD-direction and 0,64% (0.30 mm) for AP. The tests for deviation from the aimed position due to imprecise planning revealed for all templates least deviation along the Blumensaat-line (BSL) and most deviation in direction DISTAL / BSL(post.) and PROXIMAL / BSL (post.).