The process of evolution is of both scientific and medical interest. This thesis presents several studies using complete genomic reference sequences, comparative genomic data, and intraspecific diversity data to study the two key processes of evolution: mutation and selection. Large duplications, deletions, inversions, and translocations of DNA contribute to genomic variation both between and within species. Human chromosomes 15 and 17 contain a high percentage of dispersed, recently duplicated sequences. Examination of the relationships between these sequences showed that the majority of all duplications within each chromosome could be linked through core sequences that are prone to duplication. Comparison to orthologous sequences in other mammals allowed a reconstruction of the ancestral state of the human chromosomes, revealing that regions of rearrangement specific to the human lineage are highly enriched in chromosome-specific duplications. Comparison to copy number variation data from other studies also shows that these regions are enriched in current human structural variation. One specific region, the MAPT locus at 17q21.31, known to contain an inversion polymorphism in Europeans, was resequenced completely across both human orientation haplotypes and in chimpanzee and orangutan, revealing complex duplication structures at the inversion breakpoints, with the human region being more complex than chimpanzee or orangutan. Fluorescent in-situ hybridization analysis of human, chimpanzee, and orangutan chromosomes showed inversion polymorphisms of independent origin in all three species, demonstrating that this region has been a hotspot of genomic rearrangement for at least twelve million years. These results reveal a mechanistic relationship between sequence duplication and rearrangement in the great apes. We also generated a draft sequence of the chimpanzee genome and compared it to that of the human. Among other findings, this showed that CpG dinucleotides contribute 25% of all single base mutations, with a rate of mutation ~10-fold that of other bases, and that the male mutation rate in great apes is ~5-6 times the female rate, a higher ratio than had been observed in comparisons of primates and rodents. We detected six regions of probable recent positive selection in humans with a statistical method relying on chimpanzee sequence to control for regional variation in mutation rates. Finally, resequencing of several lines of domestic chicken and comparison to the reference chicken genome identified a number of gene deletions fixed in domestic lines and also several potential selective sweeps. Of particular interest are a missense mutation in TSHR nearly fixed in all domestic chickens and a partial deletion of SH3RF2 fixed in a high growth line. The TSHR mutation may play a role in relaxation of seasonal reproduction. A high-resolution QTL mapping experiment showed that the SH3RF2 deletion is significantly associated with increased growth. This work provides important new insights into the mechanics of evolutionary change at both the single nucleotide and structural level and identifies potential targets of natural and artificial selection in humans and chickens.

Sowohl in der Human- als auch in der Veterinärmedizin existieren mittlerweile zahlreiche Therapieverfahren, die immer öfter komplementär zur Schulmedizin eingesetzt werden. Zu den in der Tiermedizin besonders häufig angewandten Verfahren zählen die Neuraltherapie und die Akupunktur. Aber auch die Transkutane Elektrische Nerven Stimulation (TENS) und die Stimulation myofaszialer Triggerpunkte werden zu den segmentalen Therapiemethoden gerechnet. Die Linderung chronischer Schmerzzustände steht beim Einsatz dieser Verfahren im Vordergrund. Muskuloskelettale Erkrankungen der Lendenwirbelsäule und Lendenregion sowie der Gliedmaßen stellen beim Hund eine häufige Indikation für den Einsatz eines neuraltherapeutischen Verfahrens dar. In diesem Zusammenhang wird die Regio lumbalis des Hundes sehr häufig für eine Therapie herangezogen. Die Wirkung einer neuraltherapeutischen Behandlung wird in diesem Bereich segmental über die Spinalnerven vermittelt. Ziel der vorliegenden Dissertation war es, die Regio lumbalis des Hundes insbesondere im Hinblick auf das Innervationsmuster der Hautnerven detailliert darzustellen. Die erhobenen Befunde sollten einen Beitrag zum besseren Verständnis der Wirkmechanismen der verschiedenen Therapietechniken leisten. Außerdem sollte nach einem möglichen morphologischen Korrelat zu den in der TCM definierten Akupunkturpunkten gesucht werden. Zu diesem Zweck wurden der Verlauf und die Verzweigung der Nerven des Lendenbereiches von n=12 formalinfixierten Hunden beginnend von der dorsalen Medianlinie dargestellt. Eine fotografische Dokumentation fand in drei Schichten statt: im Bereich der Fascia thoracolumbalis (a), auf Niveau der langen Rückenmuskulatur (b) und über den Procc. transversi (c). Des Weiteren wurde bei n=2 Tieren eine Präparation des subkutanen Innervationsmusters vorgenommen. Um die präparatorischen Ergebnisse interindividuell vergleichen zu können, wurden sog. Kaudalverschiebungsindizes entwickelt und für die einzelnen Spinalnerven berechnet. Spinalnerven, welche die Haut der Regio lumbalis innervierten, stammten aus den Rückenmarkssegmenten Th12 bis L5. Nervi cutanei der Dorsaläste besaßen alle Hunde, bei n=7 Tieren beteiligten sich auch Hautäste von Rr. ventrales aus den Segmenten L3 bis L5 an der Innervation des dorsalen Lendenbereiches. Die Anzahl der Hautnervenäste variierte individuell zwischen drei und sieben, wobei die Nerven bei den Tieren mit einer geringeren Anzahl aus den weiter kranial gelegenen Segmenten stammten. Der Verlauf, die Verzweigung sowie der Perforation der Nervi cutanei durch die Fascia thoracolumbalis zeigten eine sehr hohe individuelle Variabilität. Im Bereich der langen Rückenmuskulatur wurde dagegen ein vergleichbares und segmental regelmäßiges Innervationsmuster angetroffen. Bei der Präparation der subkutanen Nervenverläufe fiel ein sehr irreguläres und segmental nicht nachvollziehbares Muster auf. Bei allen untersuchten Hunden zeigten die Dorsaläste der Spinalnerven eine Kaudalverschiebung um mindestens eine Wirbelkörperlänge. Die in der vorliegenden Arbeit eingeführten Kaudalverschiebungsindizes ermöglichen einen interindividuellen Vergleich des Verlaufes der Spinalnerven der Regio lumbalis. Durch die errechneten Indizes ist eine graphische Darstellung und aussagekräftige Interpretation der morphologischen Ergebnisse gegeben. Die zum Teil hohe interindividuelle Variation im Innervationsmuster der Hautnerven des Lendenbereiches in den oberflächlichen Körperschichten lässt den Schluss zu, dass die Hautareale des Lendenbereiches nicht bei jedem Tier von demselben Spinalnerven innerviert werden. Das bedeutet, dass die Dermatome bei verschiedenen Individuen nicht identisch sind. Des Weiteren schließt die unterschiedliche Lokalisation der Nerven und ihrer Durchtrittstellen durch die Fascia thoracolumbalis ein morphologisches Korrelat zu den in der TCM festgelegten Akupunkturpunkten aus. Die bei allen Hunden regelmäßige Anordnung der Spinalnerven auf Niveau der langen Rückenmuskulatur deutet vielmehr darauf hin, dass ein in der Körperperipherie gesetzter Reiz unabhängig von einer genauen Punktlokalisation einen segmental in das Rückenmark vermittelten Effekt bei einer neuraltherapeutischen Behandlung zur Folge hat. Für eine erfolgreiche Therapie sind daher die profunde Kenntnis der Segmentanatomie sowie der neuroanatomischen Verschaltungswege unbedingt erforderlich. In Zukunft sollte mehr Augenmerk auf individuelle Unterschiede im Innervationsmuster gelegt werden, da diese einen möglichen Grund für die individuell variable oder auch ausbleibende Wirkung bei einer neuraltherapeutischen Behandlung darstellen können.

I föreliggande single subject designstudie har tre personer med thai som modersmål och svenska som andraspråk tränats specifikt i svenskt uttal. Syftet med föreliggande studie var därför att utforma samt genomföra en uttalsintervention vilken delades in i en suprasegmentell och en segmentell modul. Syftet var vidare att undersöka hur suprasegmentella och segmentella förändringar i uttalet påverkade uttalets förståelighet. Uttalsträningen utfördes 14 gånger under en fem-veckors period. Interventionen delades upp i två moduler, en med suprasegmentellt fokus (B1) och en med segmentellt fokus (B2). I den suprasegmentella modulen ingick medvetandegörelse av stavelseuppbyggnad samt träning i svenska betoningsmönster. Den segmentella modulen innefattade träning av konsonantförbindelser, [ɧ] samt /r/. Efter varje tillfälle fick deltagarna repetera 50 ord som valts ut innan studiens början. Dessa ord analyserades utifrån deltagarnas förmåga att korrekt producera suprasegmentella och segmentella aspekter och utgjorde studiens mätpunkter. De suprasegmentella aspekterna bedömdes utifrån uppsatta bedömningskriterier och de segmentella aspekterna bedömdes med hjälp av Percentage Phonemes Correct (PPC). Efter genomförd intervention fick tre utomstående lyssnare skatta förståelighet för att undersöka om samband kunde ses mellan deras skattningar och de valda bedömningsverktygen. Enskilda ord ur ordlistorna valdes ut för att bedömningsverktygen även skulle jämföras mot akustiska mätningar. Resultaten i föreliggande studie visade på att samband kunde ses mellan specifik uttalsträning med segmentellt fokus och förbättrat uttal både vad gäller segmentella och suprasegmentella aspekter. Samma resultat visade den suprasegmentella uttalsträningen ge. Vidare kunde samband ses mellan bedömningen av de segmentella aspekterna utifrån mätpunkternas beräknade PPC och förståelighetsskattningarna. När PPC ökade förbättrades också förståeligheten. Då uttalsträning bör ha som fokus att uppnå god förståelighet hos andraspråksinläraren (Pennington, 1996; Levis, 2005) föreslås att segmentella aspekter bör ingå i uttalsträning. Mellan förbättringarna av suprasegmentella aspekter och förståelighet har inte liknande samband kunnat ses vilket dock kan bero på studiens design. Författarna till föreliggande studie anser därför att suprasegmentella aspekter i uttalsträning inte bör bortprioriteras. / Three native Thai speakers with Swedish as their second language have received special training in Swedish pronunciation in the current single-subject design study. The aim of the current study was therefore to devise and implement a set of pronunciation training exercises, divided into suprasegmental and segmental modules. The aim was also to examine how suprasegmental and segmental changes in pronunciation affected the intelligibility of the pronunciation. The pronunciation training consisted of 14 training sessions during a five-week period. The exercises were divided into two modules, one with focus on suprasegmental aspects (B1) and one with focus on segmental aspects (B2). The suprasegmental module included acquainting subjects with the syllabic structure of the Swedish language as well as training of the Swedish language stress pattern. The segmental module contained training of consonant clusters, the Swedish speech sound [ɧ] and /r/. At the end of each session the participants were asked to repeat 50 words chosen before the start of the study. These words constituted the measuring points of the study and were analyzed according to the participants' ability to produce suprasegmental and segmental aspects correctly. The suprasegmental aspects were assessed according to fixed criteria and the segmental aspects were rated by means of Percentage Phonemes Correct (PPC). After completion of the final training session, three independent listeners estimated intelligibility to determine if relationship existed between these and the chosen assessment tools. Single words were selected in order to compare their criteria-based and PPC-ratings with acoustic correlates. The results of the present study indicate that a relationship can be observed between specific pronunciation training with focus on segmental aspects and improved pronunciation in terms of segmental and suprasegmental aspects. The pronunciation training with suprasegmental focus indicated the same conclusion. Further, a relationship can be seen between the judgment of the segmental aspects based on the measuring points' PPC-rating and the ratings of intelligibility. The intelligibility improved when the PPC increased. The focus of pronunciation training should be to achieve good intelligibility (Pennington, 1996; Levis, 2005); therefore it is suggested that segmental aspects should be included in pronunciation training for native Thai speakers when learning Swedish as a second language. No similar relations have been discovered between the suprasegmental aspects and intelligibility, which, however, may be due to limitations in the design of the study. Therefore suprasegmental aspects should not be neglected in pronunciation training.

Segmental long spacing (SLS) collagen self-assembly was studied by analyzing aggregates formed from different nucleoside triphosphates at various protonation stages. Triple-negatively charged triphosphate groups were determined to be critical for SLS assembly, electrostatically bridging basic residues between collagen monomers. In the second part of this thesis, the nominal elastic modulus for each of the three forms of Type I collagen aggregate was measured and compared. Fibrous long spacing collagen, often associated with diseased tissues, exhibited lower stiffness in comparison to the other forms, native and SLS, suggesting decreased structural stability in diseased tissues. In the last section, a unidirectional pattern of native fibrils was assembled using mica as a template; the ability to customize and change the surface morphology was also demonstrated. For the first time, collagen monomers deposited on the mica were demonstrated to gain lateral mobility.

Segmental long spacing (SLS) collagen self-assembly was studied by analyzing aggregates formed from different nucleoside triphosphates at various protonation stages. Triple-negatively charged triphosphate groups were determined to be critical for SLS assembly, electrostatically bridging basic residues between collagen monomers. In the second part of this thesis, the nominal elastic modulus for each of the three forms of Type I collagen aggregate was measured and compared. Fibrous long spacing collagen, often associated with diseased tissues, exhibited lower stiffness in comparison to the other forms, native and SLS, suggesting decreased structural stability in diseased tissues. In the last section, a unidirectional pattern of native fibrils was assembled using mica as a template; the ability to customize and change the surface morphology was also demonstrated. For the first time, collagen monomers deposited on the mica were demonstrated to gain lateral mobility.

En la presente tesis doctoral se ha realizado un estudio de investigación encaminado a definir los genes responsables que causan hipercrecimiento en humanos, así como la identificación y caracterización de las mutaciones de pacientes españoles con síndromes de hipercrecimiento, con especial atención a los afectos de síndrome de Sotos. Los resultados obtenidos han permitido definir el espectro mutacional en un considerable número de pacientes con síndrome de Sotos, caracterizar en detalle estas mutaciones así como muchos polimorfismos intragénicos, en ambos casos, algunos cambios no han sido previamente descritos y otros son recurrentes. Se ha verificado la escasez de microdeleciones en población española, como en otros estudios europeos, y en los casos en los que se han identificado, se han caracterizado en detalle el tamaño y origen parental de las mismas. Se ha corroborado la validez de los criterios clínicos como indicación para estudio molecular. Se han diseñado estudios para buscar grandes reordenamientos genómicos en pacientes sin mutación previamente identificada, llegándose a caracterizar de estar manera varios reordenamientos genómicos en pacientes con sobrecremiento. / This doctoral thesis presents a research study designed to identify genes causing overgrowth in humans. The indentification and the characterization of mutations in spanish patients with overgrowth syndromes, mainly focusing on the patients with Sotos syndrome, were permormed. The obtained results helped to define the mutational spectrum in considerable amount of patients with Sotos syndrome, the detailed characterization of these mutations and, also, many intragenic polymorphisms. In both cases some of the identified changes were not previously reported and others were recurrent. It was verified that in spanish population the ratio of microdeletions is low, being similar to those previously reported in other european studies. In the cases that were identified, the detailed analysis of the size and the parental origin of mutations was carried out. The validity of clinical criteria as indicators for molecular study was confirmed. For the patients without known mutations, the experiments exploring large genomic rearrangements were designed and, consiquently, several genomic rearrangements in the patients with overgrowth were discovered.

In post-tensioned (PT) bridges, prestressing steel tendons are the major load carrying components. These tendons consist of strands, ducts, and cementitious grout that fill the interstitial space between the strands and ducts. However, inspections on PT bridges have reported the presence of voids, moisture, and chlorides inside grouted ducts as the major cause of accelerated corrosion of strands. Corrosion of the strands has resulted in PT bridge failures in Europe and tendon failures in the United States. As most of the PT bridges have high importance measures and the consequences of failure are significant, it is important to maintain high levels of safety and serviceability for these bridges. To meet this goal, bridge management authorities are in dire need of tools to quantify the long-term performance of these bridges. Time-variant structural reliability models can be useful tools to quantify the long-term performance of PT bridges. This doctoral dissertation presents the following results obtained from a comprehensive experimental and analytical program on the performance of PT bridges. 1) Electrochemical characteristics of PT systems 2) Probabilistic models for tension capacity of PT strands and wires exposed to various void and environmental conditions 3) Time-variant structural reliability models (based on bending moment and stress limit states) for PT bridges 4) Time-variant strength and service reliabilities of a typical PT bridge experiencing HS20 and HL93 loading conditions and different exposure conditions for a period of 75 years The experimental program included exposure of strand specimens to wet-dry and continuous-atmospheric conditions. These strand specimens were fabricated to mimic void and/or grout-air-strand (GAS) conditions inside the tendons. It was found that the GAS interface plays a major role in strand corrosion. The GAS interfaces that are typically located in the anchorage zones of harped PT girders or vertical PT columns can cause aggressive strand corrosion. At these locations, if voids are present and the environment is relatively dry, then limited corrosion of the strands occurs. However, if the presence of high relative humidity or uncontaminated and chloride-contaminated water exists at these interfaces, then corrosion activity can be high. The strands were exposed for a period of 12, 16, and 21 months, after which the remaining tension capacity was determined. The analytical program included the development of probabilistic strand capacity models (based on the experimental data) and the structural reliability models. The timevariant tension capacity predicted using the developed probabilistic models were reasonably consistent with the tendon failures observed in PT bridges in Florida and Virginia. The strength reliability model was developed based on the moment capacity and demand at midspan. Service reliability model was developed based on the allowable and applied stresses at midspan. Using these models, the time-variant strength and service reliabilities of a typical PT bridge were determined based on a set of pre-defined constant and random parameters representing void, material, exposure, prestress, structural loading, and other conditions. The strength and service reliabilities of PT bridges exposed to aggressive environmental conditions can drop below the recommended values at relatively young ages. In addition, under similar conditions the service reliability drops at a faster rate than the strength reliability.

Contrary to the linings of conventionally driven tunnels, the linings of tunnels bored by tunnel boring machines (TBMs) consist of precast concrete segments which are articulated or coupled at the longitudinal and circumferential joints. There are several analytical and numerical structural analysis methods proposed for the design of TBM segmental linings. In this thesis study, different calculation methods including elastic equation method and two dimensional (2D) and three dimensional (3D) beam &ndash / spring methods are compared and discussed. This study shows that in addition to the characteristics of concrete segments, the mechanical and geometrical properties of longitudinal and circumferential joints have significant effects on the structural behavior of segmental lining.

Non-healing bone defects have a significant socioeconomic impact in the U.S. with approximately 600,000 bone grafting procedures performed annually. Autografts and allografts are clinically the most common treatments; however, autologous donor bone is in limited supply, and allografts often have poor mechanical properties. Therefore, tissue engineering and regenerative medicine strategies are being developed to address issues with clinical bone grafting. The overall objective of this work was to develop bone tissue engineering strategies that enhance healing of orthotopic defects by targeting specific osteogenic cell signaling pathways. The general approach included the investigation of two different tissue engineering strategies, which both focused on directed osteoblastic differentiation to promote bone formation. In the first cell-based strategy, we hypothesized that constitutive overexpression of the osteoblast-specific transcription factor, Runx2, in bone marrow stromal cells (BMSCs) would promote orthotopic bone formation in vivo. We tested this hypothesis by delivering Runx2-modified BMSCs on synthetic scaffolds to critically-sized defects in rats. We found that Runx2-modified BMSCs significantly increased orthotopic bone formation compared to empty defects, cell-free scaffolds and unmodified BMSCs. This gene therapy approach to bone regeneration provides a mineralizing cell source which has clinical relevance. In the second biomaterial-based strategy, we hypothesized that incorporation of the collagen-mimetic peptide, GFOGER, into synthetic bone scaffolds would promote orthotopic bone formation in vivo without the use of cells or growth factors. We tested this hypothesis by passively adsorbing GFOGER onto poly-caprolactone (PCL) scaffolds and implanting them into critically-sized orthotopic defects in rats. We found that GFOGER-coated scaffolds significantly increased bone formation compared to uncoated scaffolds in a dose dependent manner. Development of this cell-free strategy for bone tissue engineering provides an inexpensive therapeutic alternative to clinical bone defect healing, which could be implemented as a point of care application. Both strategies developed in this work take advantage of specific osteoblastic signaling pathways involved in bone healing. Further development of these tissue engineering strategies for bone regeneration will provide clinically-relevant treatment options for healing large bone defects in humans by employing well-controlled signals to promote bone formation and eliminating the need for donor bone.

Background: In a retrospective study, risk factors for complications after the bridging of mandibular defects using reconstruction plates were reviewed. Especially the loosening of the plate-screw-mandible complex should be analyzed with a finite element model in order to reduce plate complications in future. Patients and Methods: We examined 60 patients who underwent a treatment with reconstruction plates after tumor resection during a period of 10 years. The problem of screw loosening was additionally reviewed by means of a finite element study, and a model for the loosening process was developed. Results: Our postoperative examination showed that 26 patients suffered from complications that required an early removal of the plate. These complications were oral or extraoral plate exposures, the looseness of screws with or without plate displacement, and plate fractures. Thereby, we noticed that maxillary and mandibular areas of opposing teeth, the size of the mandible defect, and the crossing of the orofacial midline are all risk factors for plate complications. On the basis of the finite element model, a modified arrangement of the screws was derived. Hence, a new type of resection plate was established. Conclusions: By repositioning the screw holes along the long axis of the plate, the transition from tensile force to torque force of the screws in the screw-plate-bone complex can be minimized. Thereby, the complication of screw loosening will be considerably reduced. / Hintergrund: In einer retrospektiven Studie wurden Risikofaktoren für Komplikationen nach Überbrückung von Unterkieferdefekten mit Rekonstruktionsplatten geprüft. Insbesondere die Lockerungsvorgänge des Schrauben- Platten-Unterkiefer-Verbundes sollten mit einer Finite- Elemente-Modellierung analysiert werden, um in Zukunft eine Reduzierung der Plattenkomplikationen erreichen zu können. Patienten und Methoden: Es wurden 60 Patienten untersucht, welche im Zeitraum von 10 Jahren im Rahmen von Tumoroperationen mit Rekonstruktionsplatten versorgt wurden. Das Problem der Lockerung der Plattenschrauben wurde zusätzlich mittels einer Finite-Elemente-Studie überprüft und ein Modell für den Lockerungsvorgang erarbeitet. Ergebnisse: Die Nachuntersuchungen ergaben, dass bei 26 Patienten die Platte wegen Komplikationen vorzeitig entfernt werden musste. Die Komplikationen traten als orale und extraorale Plattenfreilage, als Schraubenlockerung ohne oder mit Plattendislokationen und als Plattenbrüche auf. Dabei konnte festgestellt werden, dass bestehende Stützzonen des körpereigenen Restgebisses, die Größe des Unterkieferdefektes und dessen Mittellinienüberschreitung Risikofaktoren für Plattenkomplikationen darstellen. Anhand der Finite-Elemente-Modellierung wurde eine veränderte Schraubenanordnung abgeleitet. Daraus resultiert eine neue Form der Resektionsplatte. Schlussfolgerungen: Durch die Verschiebung der Schraubenlöcher aus der Längsachse der Platte kann der Übergang von der Zugbelastung zur Drehmomentbelastung der Schrauben im Schrauben-Platten-Knochen-Verbund der Platte minimiert werden. Dadurch werden Schraubenlockerungen als Komplikationen wesentlich seltener auftreten. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.