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Interplay of genetic and epigenetic variation in evolutionary processes
Get PDFAdaptation and speciation are the fundamental processes shaping the biodiversity that surrounds us. The Modern Evolutionary Synthesis, merging Darwinian theory with Mendelian principles, requires an understanding of the genetic and epigenetic contributions to population divergence across micro- and macro-evolutionary scales to explain the maintenance of biodiversity. This dissertation examines the interplay of genetic and epigenetic variation with relevance to evolution across the dimensions of wild avian populations, sexes, and species. Utilizing natural variation in two avian systems, the scope of my investigation extends to: 1) the genetic architecture sustaining female-limited polymorphism in cuckoos, the 2) evolutionary maintenance of mimetic egg phenotypes and host specialization in cuckoos, the 3) the epigenetic factors regulating dosage compensation and dosage balance in crows, and 4) the relevance of DNA methylation to speciation in crows. In paper I, together with my colleagues I identify the genetic basis and evolutionary maintenance of a female-limited plumage polymorphism. While all male common cuckoos are grey, females are either monochromatic grey or rufous. We found that plumage polymorphism maps to the female-restricted W chromosome, and that these ancient maternal haplotypes have been maintained after descent from a common ancestor in two cuckoo sister taxa, likely through balancing selection. Our findings suggest that genetic variation residing on sex-limited chromosomes can be a key determinant in the maintenance of trait variation across species boundaries. In paper II, I examine the genetic basis of host specialization and egg mimicry resulting from a co-evolutionary arms race. Common cuckoos are generalist obligate brood parasites exhibiting an extreme diversity of mimetic eggs which they use to exploit numerous hosts across Eurasia. I identified that matrilineal haplotypes are associated with mimetic egg phenotypes, and found that these haplotypes are maintained across the species’ range from the combinatorial effects of balancing selection and gene flow. I identify mitochondrial OXPHOS genes as the nexus of egg diversification, working in concert with nuclear and W-linked genes to provide a fast-evolving substrate to facilitate phenotypic innovation for new mimetic eggs while ensuring stable transmission of phenotypes from mothers to daughters. In paper III, together with colleagues I shed light on the mechanisms underlying dosage balance and compensation in a female heterogametic system in Eurasian crow. While male heterogametic systems often exhibit inactivation of a female homogametic chromosome, dosage balance in avian systems is less clear. We identified a significant correlation between the upregulation of female Z-linked genes and increased chromatin accessibility, which appears to be the key driver of dosage balance between the sexes. In contrast to other systems, 5mC methylation did not covary with dosage, underlining the importance of chromatin accessibility over methylation in regulating gene dosage in crows. In my last chapter, manuscript IV, examines the extent to which 5mC methylation contributes to nascent species divergence in Eurasian crows. Using genome and methylome sequencing data from all-black carrion crows, grey-coated hooded crows, and their hybrids, we found that taxon-related methylation divergence is restricted to intergenic space within the region of genetic differentiation responsible for plumage polymorphism. While epigenetic factors may aid in translating genetic variation to phenotype and largely coincide with the ontogenetic program, its autonomous contribution to evolution is minimal in this system. Collectively, these studies show the complex interplay of genetic and epigenetic factors contributing to the maintenance of evolutionary patterns. These findings add to our understanding of how epigenetic and genetic mechanisms cooperate to generate and maintain evolutionarily relevant phenotypes across populations and species, and break new ground by exploring the hitherto poorly explored dynamics of sex-limited chromosomes and the contributions of epigenetic variation to evolution
Evaluating the therapeutic effects of a peptide-based inhibitor of amyloid aggregation in mouse models of Alzheimer's disease
Get PDFAlzheimer's disease (AD), a predominant neurodegenerative disorder, leads to cognitive impairment stemming from extensive neuronal loss with limited disease-modifying therapies (DMTs) available. Central to AD pathology is the accumulation of amyloid-beta (Aβ) peptide, a key player in disease progression. In addition to the Aβ-specific monoclonal antibodies, islet amyloid polypeptide (IAPP) has been found to attenuate amyloid self-assembly in vitro and in vivo. Building on IAPP's "cross-amyloid" inhibitor function, the macrocyclic 17-residue peptide 2E was designed as a mimic of the interaction surface of IAPP with Aβ. Encouragingly, 2E turned out to be a potent inhibitor with nanomolar affinity of Aβ amyloid self-assembly in vitro and, in addition, it exhibited significant proteolytic stability in human plasma and the ability to cross the blood-brain barrier (BBB) in a cell model. These favorable, drug-like, properties of 2E motivated us to test its effectiveness on amyloid pathology in AD mouse models.
In my thesis, I investigated the efficacy of two treatment paradigms of the macrocyclic peptide 2E in ameliorating amyloid pathology using both female and male 5XFAD mice, a commonly used animal model for AD. The findings revealed a significant reduction in cortical amyloid deposition, decreased plasma and CSF Aβ42 concentration, increased Aβ40 levels, shifted amyloid deposition from brain parenchyma to blood vessels, decreased neuronal damage, and enhanced astrocytic activation upon 2E administration. Similar effects of 2E on soluble Aβ42 and Aβ40 changes were observed in APPNL-G-F transgenic mice. Furthermore, treated 5XFAD mice displayed improvements in memory and motor functions without any alterations in anxiety or stress responses, as evidenced by various behavioral tests. Notably, I established a novel method to perform high-resolution RNA sequencing from PFA-fixed microscopy slices. RNA-seq analyses highlighted 2E's ability to bolster astrocytic activation while reversing AD-associated neuronal gene expression changes. Importantly, following intraperitoneal (i.p.) injection, 2E's presence in the brain attests to its biodistribution capability. Thus, the macrocyclic peptide 2E, either as a standalone or combined with other anti-amyloid strategies, emerges as a promising drug candidate to combat Aβ-driven AD pathogenesis
Visuo-vestibular sensorimotor plasticity in Xenopus and Axolotl larvae
Get PDFLocomotion causes disrupting consequences for sensory perception, which requires concurrent gaze stabilization to maintain visual acuity. Visuo-vestibular reflexes along with spinal efference copy signalling generate motor commands that enable the eyes to perceive a stable image during body/head movement. In vertebrates, these short-latency motor behaviors work synergistically and are evolutionarily well conserved. However, the underlying neural and circuit components must remain plastic and adapt to accommodate the eco-physiological requirements and locomotor characteristics of each species. This dissertation aimed to explore such adaptations of the oculomotor system ensuring gaze stabilization during self-motion. The following chapters focused on understanding how motor performances may be altered or improved in phylogenetically related species that share many similarities but also clear differences. This exploration got extended to pathological conditions, acutely after a severe loss of vestibular sensory input. For this, I profiled and compared the locomotion pattern of two amphibian species, the salamander Axolotl, and the frog Xenopus laevis. To ensure that potential differences were biologically meaningful I used similarly aged and sized animals of both species, at comparable developmental stages which were validated by comparing external morphological features. While Xenopus move more or less continuously, Axolotls exhibit interspersed, short bouts of locomotion followed by a passive glide. Moreover, Xenopus have longer bouts, while Axolotls display higher velocity bouts. In vitro whole-head recordings of the angular vestibulo-ocular reflex (VOR) in Axolotl were significantly lower in gain compared to Xenopus at identical stimulus conditions. Older staged Axolotls show an increase in gain but did only reach the level of stage 49 Xenopus hinting at a delayed developmental onset of angular gaze compensation. Further experiments on fictive locomotion revealed no compensation through efference copy derived eye motions. In addition, the capacity to stabilize gaze is critically dependent on the morphological parameters of inner ear structures. A comparative investigation of the horizontal canals revealed distinct differences in various parameters between Xenopus and Axolotl. These differences favor the dynamic of endolymph flow and, consequently, the capacity of semicircular canals to detect angular head accelerations in Xenopus.
In an additional set of experiments, I investigated the plasticity potential during pathological conditions, after the complete loss of unilateral vestibular input in Xenopus. Such a loss generates severe symptoms related to posture, eye movements, and higher- order perceptual deficits. While compensation of such injuries has been explored already extensively in various species, the novelty of my experiments involved in vitro whole head preparations. Such an approach enables a targeted nerve transection with a direct evaluation of its impact within minutes after the surgery, without the influence of anesthesia. Indeed, a severe impairment of the VOR could be observed after the lesion. However, a delayed further decline of both visual and vestibular reflexes persisted and did not show any signs of compensation. This led to the conclusion that the sensory loss was intensified by secondary neuronal effects that likely involve plasticity mechanisms evoked by the ongoing asymmetric activity in the shared visuo-vestibular circuits
Die Energiewende im Alltagsverstand
Get PDFFür die Begrenzung des Klimawandels ist ein schneller Umstieg auf erneuerbare Energien notwendig – doch die Akzeptanz der Energiewende in der Bevölkerung ist weitaus ambivalenter, als es die zahlreichen zustimmenden Umfragen vermuten lassen. Anhand verschiedener Gruppendiskussionen zeigt die vorliegende Dissertation: Obwohl explizit eine – mindestens „grundsätzliche“ – Zustimmung zur Energiewende kommuniziert wird, dominieren unbewusst Widerstände, Skepsis und Unvorstellbarkeit.
Durch eine Kombination von Gramscis Hegemonietheorie, Diskursanalyse und dokumentarischer Methode nach Bohnsack entwirft Julian Bothe ein Modell der Akzeptanz im Alltagsverstand der Bevölkerung, das auch Ambivalenzen, Widersprüche und Verdrängungsphänomene erfassen kann. Er analysiert verschiedene Energiewendetypen, zeigt, wie diese Ambivalenzen in breiteren gesellschaftlichen Umbrüchen begründet sind, und schlägt Wege vor, um die daraus resultierenden Blockaden zu überwinden.
Die skizzierten Erkenntnisse sind grundlegend, um die Akzeptanz von Transformationsprozessen zu verstehen. Dieses Buch richtet sich daher an alle, die die gesellschaftliche Akzeptanz von Veränderungen theoretisch analysieren, empirisch erforschen oder praktisch voranbringen wollen.
Julian Bothe studierte Geographie, Soziologie und Systemwissenschaft in Osnabrück und Hamburg. 2024 promovierte er mit der vorliegenden Dissertation an der Fakultät für Geowissenschaften der LMU. Sein Interesse gilt den gesellschaftlichen Dynamiken aktueller Krisen und sozio-technischer Veränderungen, mit Schwerpunkt auf Klima, Energiewende und sozial-ökologischer Transformation.The mitigation of climate change requires a rapid transition to renewable energy - but social acceptance of the energy transition is far more ambivalent than surveys suggest. Based on a series of group discussions, this thesis shows: Scepticism, resistance and an inability to imagine a successful transition unconsciously dominate all discussions, even though most groups explicitly communicate their approval - at least "in principle" - of the energy transition.
Using a combination of Gramsci’s theory of hegemony, discourse analysis, and Bohnsack’s documentary method, Julian Bothe develops a model of social acceptance that accounts for ambivalence, contradictions, and denial. He identifies acceptance as rooted in common sense, analyses different ideal types of how people construct the energy transition, shows how ambivalence and resistance are linked to broader social change, and suggests ways to overcome the resulting barriers to transformation.
The book outlines insights fundamental for understanding the acceptance of transformation processes. It is therefore useful for anyone who wishes to analyse theoretically, research empirically, or promote practically the social acceptance of change.
Julian Bothe has studied geography, sociology, and systems science in Osnabrück and Hamburg. In 2024, he received a PhD from the Faculty of Geosciences at LMU Munich. He is interested in the social dynamics of current crises and socio-technical changes, with a focus on climate change, the energy transition, and socio-ecological transformation
Proteomic insights into cardiometabolic health: association of protein biomarkers with type 2 diabetes, coronary heart disease, and mortality
Get PDFNew reactivities and functionalities introduced by mimics of protein post-translational modifications
Get PDFProtein post-translational modifications (PTMs) play crucial roles in regulating protein function, stability, localization, and interactions. These modifications, including phosphorylation, glycosylation, ubiquitinoylation, acetylation, and methylation, among others, are able to activate or deactivate enzymatic activities, dictate subcellular localization, mediate protein-protein interactions, and label proteins for degradation. By dynamically altering protein properties, PTMs enable cells to respond rapidly to environmental changes and maintain homeostasis, thereby contributing to processes such as signal transduction, immune response, cell cycle control, and apoptosis.
To study protein PTMs, scientists employ a variety of methods such as high-resolution tandem mass spectrometry that allows for the precise identification and quantification of PTMs by analyzing peptide fragments. Western blotting, using specific antibodies recognizing modified residues, is another common method to detect and analyze PTMs. These methods, combined with bioinformatics tools, offer comprehensive approaches to understanding the complex roles of PTMs in protein regulation.
We started to investigate a newly discovered PTM called adenylylation (AMPylation) by developing halogen-modified AMPylation mimics for subsequent cross-coupling via Suzuki-Miyaura reactions in the living cells. However, the downstream chemical biology analysis using functionalized aryl pinacol boronates revealed an unknown reactivity. For example, incubation between cell lysates and a fluorescent aryl pinacol boronate has brought significant labelling regardless of the addition of artificial AMPylation probes or palladium catalysts. To our delight, we found that the unknown reactivity was due to selective protein labelling (S-arylation on disulfides) triggered by ammounium persulfate (APS). Then oxidized glutathione and two more short peptides were applied to verify the S-arylation product formation, which was further fragmented in high-resolution MS2 to identify diagnostic ions. The radical-based mechanistic pathway of this reaction was confirmed by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap experiments, and the reaction was found to proceed only when aryl moieties were substituted with electron-donating groups, which was consistent with competition studies in cell lysates. This method was the first study to perform selective disulfide S-arylation with aryl radicals in mild aqueous conditions, and there was no need to use metal catalyst or photocatalysis.
On the other hand, we took advantage of protein glycosylation and ubiquitinoylation to develop a two-component proteolysis targeting chimeras (PROTACs) strategy, which was proved to be able to selectively target O-GalNAcylated and O-GlcNAcylated proteins for proteasomal degradation. As a result, the critical metabolic and signaling pathways governed by glycoproteins were heavily perturbated, triggering severe cytotoxicity in human cancer cell lines. The approach termed GlyTACs leveraged from metabolic incorporation of easily accessible and cell-permeable peracetylated N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) mimics bearing an azide group into glycoproteins. In the living cells, the azido-modified glycoproteins served as covalent anchors for the introduction of thalidomide moiety by strain-promoted azide-alkyne cycloaddition (SPAAC) to recruit E3 ligase cereblon, resulting in stepwise ubiquitinoylation of ‘sensitized’ proteins and their degradation by proteasome. The efficiency of the GlyTAC system was shown in a series of human cancer cell lines and the mechanistic pathway was verified by performing control experiments at each stage of the process. Given the characteristic features of cancer cells including fast nutrient turnover, and overall increase of protein glycosylation, as well as the low cytotoxicity of the individual components, this GlyTAC approach may open a feasible strategy in cancer therapy.
In summary, these studies have discovered unexplored reactivities and functionalities of protein PTMs, helping chemical biologists utilize bioorthogonal reactions by avoiding unwanted side reactions and introducing therapeutic potentials
The neural and behavioral basis of serial dependence in time perception
Get PDFSequential bias influences decision-making by leveraging past experiences to shape current perception. This phenomenon has been extensively studied in the visual domain, however, only a handful of recent behavioral studies have explored trial-to-trial sequential effects on timing, and even fewer have linked these effects to specific neural signatures. This leaves a significant gap in our understanding of neural mechanisms underlying temporal sequential effects at play. Moreover, the underlying mechanisms of whether serial dependence arises from perceptual or post-perceptual processes remain debatable. One perspective posits it as a perceptual mechanism aiding perceptual stability and temporal continuity by integrating past and current information to filter out abrupt noises. Alternatively, recent insights link it to decision-related post-perceptual factors, with working memory playing a crucial role in integrating preceding stimuli with current sensory inputs for decision-making and motor plans.
Therefore, this thesis aims to investigate the serial dependence effect in time perception. To begin with, Chapter 2 explores the impact of task measurements and task relevance on temporal sequential biases using a dual-feature random dot kinematogram. Participants encoded both features (duration and direction) and reported one based on a post-cue. The preceding duration-report trials were task-relevant, while the previous direction-report trials were considered task-irrelevant. Two experiments, employing time discrimination task and duration reproduction tasks, were conducted to further explore the influence of task measurements. Chapter 3 addresses sequential dependence in both motion direction and temporal perception concurrently. It utilized a unified experimental paradigm employing coherent motion stimulus for direction and time reproduction tasks. Additionally, two experiments varied the cue setting where participants were informed about the task before (pre-cue setting) or after (post-cue setting) the stimulus to further explore the influence of working memory underlying spatial and temporal serial dependence effect. Chapter 4 investigates the neural mechanisms underlying serial dependence in a duration reproduction task in conjunction with functional Magnetic Resonance Imaging (fMRI) scanning. Participants were required to remember the stimulus duration, and then either reproduce it or passively observe it, as instructed by the cue. This design allowed us to compare how the post-perceptual factors contribute to sequential dependence and further explore the neural representations underlying such bias.
Our studies consistently identify an attractive sequential bias in time perception, where durations are perceived as longer following longer previous durations, and vice versa. In Chapter 2, task measurements significantly influence sequential dependence in time perception. The discrimination task shows consistent sequential effects regardless of whether it follows a timing or direction task. Conversely, the reproduction task exhibits a more pronounced sequential effect following the same timing task compared to the direction task. Findings in Chapter 3 reveal an attraction bias in time reproduction and a repulsion in direction estimation. The temporal attraction was more pronounced when the preceding task was also time-related, while direction repulsion remains unaffected by the preceding task. Additionally, both attraction and repulsion biases were intensified by the post-cue compared to the pre-cue. These results suggest that opposing sequential effects in spatial and temporal domains may originate from different processing stages linked to sensory adaptation and post-perceptual processes involving working memory. In Chapter 4, at the neural level, our results link striato-thalamo-cortical and performance monitoring networks to time perception and prior tasks, respectively. Notably, we observed that hippocampus activity was directly linked with the sequential bias on both prior tasks and prior duration. This hippocampal activation was particularly evident during the encoding phase following passive viewing trials and led to a decrease in sequential bias. These findings highlight the involvement of post-perceptual stages that link sensory representations to responses and underscore the critical role of active timing-related and memory networks in the temporal sequential dependence.Der sequenzielle Bias beeinflusst die Entscheidungsfindung, indem vergangene Erfahrungen genutzt werden, um die aktuelle Wahrnehmung zu formen. Dieses Phänomen wurde im visuellen Bereich intensiv erforscht. Allerdings haben nur wenige neuere Verhaltensstudien die Versuch-zu-Versuch-sequenziellen Effekte auf das Zeitverhalten untersucht, und noch weniger Arbeiten haben diese Effekte mit spezifischen neuronalen Signaturen in Verbindung gebracht. Dies führt zu einer erheblichen Wissenslücke hinsichtlich der neuronalen Mechanismen, die den zeitlichen sequenziellen Effekten zugrunde liegen. Zudem bleibt die Frage offen, ob die serielle Abhängigkeit aus perzeptuellen oder postperzeptuellen Prozessen resultiert. Ein Ansatz sieht die serielle Abhängigkeit als perzeptuellen Mechanismus, der die Wahrnehmungsstabilität und zeitliche Kontinuität unterstützt, indem frühere und aktuelle Informationen integriert werden, um plötzliche Störungen herauszufiltern. Alternativ wird die serielle Abhängigkeit mit entscheidungsbezogenen postperzeptuellen Prozessen in Verbindung gebracht, wobei das Arbeitsgedächtnis eine entscheidende Rolle bei der Integration früherer Reize mit aktuellen sensorischen Eingaben für die Entscheidungsfindung und Handlungsplanung spielt.
Ziel dieser Arbeit ist es, den Effekt der seriellen Abhängigkeit in der Zeitwahrnehmung zu untersuchen. In Kapitel 2 wird untersucht, wie Aufgabenmessungen und Aufgabenrelevanz zeitliche Verzerrungen beeinflussen. Die Teilnehmenden kodierten sowohl Dauer als auch Richtung und gaben eines dieser Merkmale nach einem Hinweis (Post-Cue) an. Zwei Experimente – eine Zeitdiskriminationsaufgabe und eine Dauerreproduktionsaufgabe – wurden durchgeführt, um den Einfluss dieser Messungen zu erforschen. Kapitel 3 untersucht die serielle Abhängigkeit in der Bewegungsrichtung und Zeitwahrnehmung mit einem kohärenten Bewegungsstimulus. Zwei Experimente variierten, ob die Teilnehmenden vor (Pre-Cue) oder nach (Post-Cue) dem Stimulus über die Aufgabe informiert wurden, um den Einfluss des Arbeitsgedächtnisses zu testen. Kapitel 4 erforscht die neuronalen Mechanismen der seriellen Abhängigkeit bei einer Dauerreproduktionsaufgabe mittels fMRI. Die Teilnehmenden mussten die Stimulusdauer entweder reproduzieren oder passiv beobachten, was half, den Einfluss postperzeptueller Faktoren und die zugrunde liegenden neuronalen Repräsentationen zu untersuchen.
Unsere Studien zeigen durchgängig einen attraktiven sequenziellen Bias in der Zeitwahrnehmung, bei dem Zeitspannen nach längeren vorherigen Zeitspannen als länger wahrgenommen werden und umgekehrt. In Kapitel 2 wurde festgestellt, dass Aufgabenmessungen einen signifikanten Einfluss auf die serielle Abhängigkeit in der Zeitwahrnehmung haben. Die Diskriminationsaufgabe zeigte konsistente sequenzielle Effekte, unabhängig davon, ob sie auf eine Zeit- oder eine Richtungsaufgabe folgte. Im Gegensatz dazu wies die Reproduktionsaufgabe einen stärkeren sequenziellen Effekt auf, wenn sie auf eine vorherige Zeitaufgabe folgte, im Vergleich zu einer vorherigen Richtungsaufgabe. Kapitel 3 zeigte einen Anziehungsbias in der Zeitreproduktion und einen Abstoßungsbias in der Richtungsschätzung. Der zeitliche Anziehungsbias war stärker ausgeprägt, wenn die vorherige Aufgabe ebenfalls zeitbezogen war, während die Richtungsabstoßung von der vorherigen Aufgabe unbeeinflusst blieb. Darüber hinaus verstärkten sich sowohl der Anziehungs- als auch der Abstoßungsbias durch den Post-Cue im Vergleich zum Pre-Cue. Diese Ergebnisse deuten darauf hin, dass entgegengesetzte sequenzielle Effekte im räumlichen und zeitlichen Bereich auf unterschiedliche Verarbeitungsstufen zurückgeführt werden können, die mit sensorischer Adaption und postperzeptuellen Prozessen zusammenhängen, welche das Arbeitsgedächtnis einbeziehen. In Kapitel 4 konnten wir auf neuronaler Ebene unsere Ergebnisse mit striato-thalamo-kortikalen Netzwerken und Performanzüberwachungsnetzwerken in Verbindung bringen, die jeweils für die Zeitwahrnehmung und die vorherigen Aufgaben verantwortlich sind. Insbesondere beobachteten wir, dass die Aktivität des Hippocampus direkt mit dem sequenziellen Bias sowohl für vorherige Aufgaben als auch für vorherige Zeitspannen verknüpft war. Diese hippocampale Aktivierung war besonders während der Enkodierungsphase nach passiven Beobachtungsdurchgängen ausgeprägt und führte zu einer Verringerung des sequenziellen Bias. Diese Erkenntnisse unterstreichen die Bedeutung postperzeptueller Stufen, die sensorische Repräsentationen mit Reaktionen verknüpfen, und heben die entscheidende Rolle von zeitbezogenen Netzwerken und Gedächtnisnetzwerken in der zeitlichen seriellen Abhängigkeit hervor
Characterisation of a skeletal muscle-specific myostatin over-expressing mouse model
Get PDFSarcopenia displays a progressive loss of muscle mass and performance, which extends above the level of physiological ageing processes. In ageing societies, this condition becomes increasingly significant. While patients may notice a strong impact on self-reliance, the socio-economic burden presents itself through rising costs for health care and increasing commitment of manpower. Unfortunately, no pharmacological treatments are yet available to prevent or treat this condition effectively.
To conduct further research on sarcopenia a reliable mouse model should be established. Myostatin (MSTN) or GDF-8, a member of transforming growth and differentiation factor (TGF) family, is a strong inhibitor of skeletal muscle mass. Physiologically, MSTN reduces skeletal muscle mass adapted to physical exercise. The absence of functional MSTN in animals with a natural loss-of-function mutation or in artificial models leads to a tremendous increase in muscle mass. The opposite (sarcopenic) phenotype could be hypothesised to result from Mstn gain-of-function. This makes MSTN a promising candidate for genetic modification to simulate sarcopenic conditions. Due to the strong monogenetic impact on morphology, the aim of this thesis was to analyse possible effects of Mstn over-expression on skeletal muscles. Hence a Mstn over-expressing mouse line was created and thoroughly analysed. Interestingly, the results showed no significant influence on overall bodyweight. However, a significant decrease in skeletal muscle weight of lower limb muscles could be found in homozygous Mstn over-expressing male mice. No significant changes in fibre area or fibre type composition were found, but a small tendency towards smaller myofibres in homozygous Mstn over-expressing male mice. In addition, no significant impact on grip strength was detected. Next generation sequencing (NGS) of Mstn over-expressing mice muscle revealed a pattern of up- and down-regulated genes that indicates a homeostatic regulatory function of MSTN.
In addition, the results of this work will provide the basis for further genetic modifications of the Mstn over-expressing mouse line toward an inducible gene modification to expand research opportunities by controlling the onset of over-expression.Sarkopenie beschreibt einen fortschreitenden Verlust an Muskelmasse und Leistungsfähigkeit, der über die physiologischen Alterungsprozesse hinausgeht. In alternden Gesellschaften gewinnt dieser Zustand zunehmend an Bedeutung. Während Patienten starke Einschränkungen ihrer Selbstständigkeit feststellen können, zeigt sich die sozioökonomische Belastung durch steigende Kosten für die Gesundheitsversorgung und eine zunehmende Beanspruchung von Arbeitskräften. Leider gibt es noch keinen pharmakologischen Therapieansatz, der diesem Zustand wirksam vorbeugen oder ihn behandeln könnte.
Für die weitere Erforschung der Sarkopenie sollte ein zuverlässiges Mausmodell etabliert werden. Myostatin (MSTN) oder GDF-8, ein Mitglied der Familie der transforming growth and differentiation factor (TGF), ist ein starker Inhibitor der Skelettmuskelmasse. Physiologisch reduziert es die Skelettmuskelmasse angepasst an den funktionellen Bedarf.
Das Fehlen von MSTN sowohl bei Tieren mit einer natürlichen Loss-of-Function Mutation als auch in künstlichen Tiermodellen führt zu einer enormen Zunahme der Muskelmasse. Es liegt auf der Hand, dass der gegenteilige (sarkopene) Phänotyp durch eine Überexpression von Mstn hervorgerufen werden könnte. Dies macht MSTN zu einem vielversprechenden Kandidaten für eine genetische Modifikation, um Sarkopenie zu simulieren. Aufgrund der starken monogenetischen Auswirkung auf die Morphologie war es das Ziel dieser Arbeit, die möglichen Auswirkungen einer Überexpression von Mstn auf die Skelettmuskulatur zu analysieren. Daher wurde eine Mstn-überexprimierende Mauslinie erstellt und gründlich analysiert. Interessanterweise zeigten die Ergebnisse keinen signifikanten Einfluss auf das Gesamtkörpergewicht. Allerdings konnte bei homozygoten Mstn-überexprimierenden männlichen Mäusen eine signifikante Abnahme des Skelettmuskelgewichts der Hinterbeinmuskulatur festgestellt werden. Es konnte kein signifikanter Einfluss auf die Größe der Muskelfasern oder der Fasertypzusammensetzung nachgewiesen werden, jedoch zeigte sich eine leichte Tendenz hin zu kleineren Muskelfasern in Mstn-überexprimierenden männlichen Mäusen. Ferner zeigte sich keine Einwirkung auf die Griffkraft der Mäuse. Die Analyse mittels Next-Generation-Sequencing (NGS) von Muskeln Mstn-überexprimierender Mäuse ergab ein Muster von hoch- und herunterregulierten Genen, welches auf eine homöostatische Regulationsfunktion von MSTN hinweist.
Darüber hinaus sollen die Ergebnisse dieser Arbeit die Grundlage für weitere genetische Modifikationen der Mstn-überexprimierenden Mauslinie in Richtung einer induzierbaren Genmodifikation bilden, um die Möglichkeiten der Forschung durch Kontrolle des Beginns der Überexpression zu erweitern