Les dystrophies rétiniennes héréditaires (DRH) sont des maladies qui conduisent à une perte de la vision au cours de leur évolution. Les premiers essais cliniques utilisant la thérapie génique pour traiter ces maladies ont été réalisés et apportent des résultats encourageants. En amont de telles études, les essais précliniques s'effectuent le plus souvent sur modèle animal. Cependant, pour un certain nombre de DRH, il n'existe pas de modèle animal approprié ce qui compromet l'arrivée d'un traitement à un stade clinique. C'est le cas de la Choroïdérémie, qui représente 2% des DRH. La choroïdérémie est caractérisée par une perte de la vision nocturne dès la petite enfance et conduit à la cécité autour des 40-50 ans. Son diagnostic précoce et son évolution lente résultent en une grande fenêtre thérapeutique qui fait de la choroïdérémie une bonne candidate pour la thérapie génique. Sur le plan génétique, la maladie est causée par une mutation dans le gène CHM qui est localisé sur le chromosome X et code pour la Rab Escort Protein 1 (REP1). Cette protéine est impliquée dans le processus de prénylation de petites protéines GTPases, les protéines Rab. Afin de pallier au manque de modèle animal, nous avons généré au cours de ce travail de thèse, un modèle cellulaire humain de la choroïdérémie pour évaluer l'efficacité d'un protocole de thérapie génique sur le tissu réellement atteint in vivo. Pour cela, nous avons reprogrammé des fibroblastes de patient CHM-/y en cellules souches pluripotentes induites (iPS), que nous avons ensuite différenciées en Epithélium Pigmentaire Rétinien (EPR). Nous avons caractérisé cet EPR, montrant que c'est une couche monocellulaire polarisée possédant une morphologie et une expression de marqueurs caractéristiques. De plus, ce tissu est fonctionnel, sur le plan du transport de fluide et de la phagocytose, et possède le même phénotype biochimique que celui observé chez les patients. Dans un but de thérapie génique et afin d'évaluer le vecteur viral le plus efficace sur nos cellules, j'ai testé un panel de 5 sérotypes d'AAV et démontré que l'AAV2/5 est le plus efficient pour transduire un EPR dérivé de cellules iPS humaines. J'ai ensuite utilisé un AAV2/5-CAG-CHM afin d'évaluer l'efficacité fonctionnelle du vecteur et j'ai pu montrer qu'outre une expression correcte du transgène, le traitement de cellules de patients déficientes pour REP1 avec ce vecteur permet de restaurer une activité normale de prénylation. Nous avons donc démontré la supériorité d'efficacité de transduction de l'AAV2/5 dans des cellules d'EPR humain et soulignons le potentiel d'un modèle d'EPR pathologique dérivé de cellules iPS pour apporter une preuve de concept de thérapie génique en absence d'un modèle animal approprié. / Inherited retinal dystrophies (IRDs) lead to a progressive vision loss. The first clinical trials using gene transfer to treat such diseases have been performed with positive results. Prior to clinical trials, preclinical studies are usually performed on animal models. However, for many IRDs, appropriate animal models do not exist, which compromises their progress towards a clinical trial. An example of an IRD that lacks an appropriate model is choroideremia, which represents 2% of IRD patients. It is characterized by night blindness in childhood, followed by progressive loss of the visual field resulting in blindness by 40–50 years of age. Its early diagnosis and slow evolution result in a large therapeutic window making choroideremia a good candidate for gene therapy. Genetically, the disease is caused by a mutation in the CHM gene located on the X chromosome and encoding the Rab Escort Protein 1 (REP1). This protein is involved in the prenylation of small GTPases, the Rab proteins. To palliate the lack of an animal model, we generated a human cellular model of choroideremia in order to evaluate the efficacy of a gene therapy approach in the tissue that is affected in vivo.Towards this aim, we reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPScs), which we differentiated into retinal pigment epithelium (RPE). We characterized the iPSc-derived RPE that is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. In terms of gene therapy and to evaluate the most efficient viral vector, I assayed a panel of 5 adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transduce the iPSc-derived RPE. I then transduced the iPSc-derived RPE of a choroideremia patient with an AAV2/5-CAG-CHM and demonstrated that this vector is able to restore a normal prenylation function to the cells.To conclude, I demonstrated the superiority of the transduction efficiency of AAV2/5 in the iPSc-derived RPE and highlight the potential of a diseased RPE model derived from iPS cells to provide a proof of concept of gene therapy in the absence of a suitable animal model.

As advancement and usage of data communication has increased exponentially on a global scale, with a devastating exposure to attacks and varying security threats to home offices as well as to large enterprises, there is always a need for enhanced network protection. The IT department of the company OneDefence, located in western Sweden, was chosen for the thesis and based on the stated information from the organisation, aims were set on how to improve their network defence capabilities. The aim of this thesis is to list ten countries posing the most serious IT threats, and to limit the attack surface of OneDefence’s IT network as much as possible while still providing the necessary services to users abroad. After researching the countries, a prototype was set up to mimic OneDefence’s topology of interest and test attacks were conducted as detailed in the Methodology chapter. The results of the investigations showed the countries posing most serious cyber threats included China, Russia and North Korea among others which were statistically calculated based on the total number of recognised cyberwarfare attacks. The results obtained from the different DoS attacks in the prototype showed that an IPS should be at the heart of an organisation's network defence for combating these intrusions, as well as potentially other types. With the help of a prototype built based on the organisation's topology, several attacks were somewhat successfully mitigated with the equipment used on hand, with only a low percentage of packets allowed to pass through the security unit. Lastly, to explore further enhancements of defence capabilities of OneDefence, a comparison between different products and devices were performed. This resulted in products from the Fortinet brand such as FortiGate NGFW and UTM capabilities as they are offering several advantages compared to competitors. / Då stora framsteg och användning av datakommunikation har ökat exponentiellt på en global skala, med en förödande exponering av attacker och säkerhetshot mot hemanvändare såväl som stora företag, finns detalltid ett behov av förbättrad nätverksskydd. IT-avdelningen hos företaget OneDefence, valdes för att utföra examensprojektet och baserade sig på organisationens angivna information för att förbättra deras nätverksförsvar. Syftet med denna rapport är att sammanställa en lista på tio länder som utgör de allvarligaste IT-hoten i världen, samt begränsa attackytan för organisationens nätverk så mycket som möjligt medan man behåller alla nödvändiga tjänster till användare utomlands. Efter att ha undersökt länderna, anordnades en prototyp för att efterlikna delar av OneDefences topologi av intresse och testattacker utfördes enligt metodologikapitlet. Resultaten av utredningarna visade att från de länder som utfört de allra allvarliga cyberhoten inkluderade bland annat Kina, Ryssland och Nordkorea, som har beräknats statistiskt baserat på antalet igenkända cyberwarfare attacker. Resultaten från de olika DoS-attackerna visade att en IPS bör vara kärnan i en organisations nätverksförsvar för att kunna bekämpa dessa intrång, samt potentiellt andra typer. Med hjälp av den prototyp som byggdes baserad på organisationens topologi, blockerades flera attacker rätt framgångsrikt, med en låg procentandel av paketen som gick genom säkerhetsenheten. Slutligen utforskades ytterligare förbättringar av försvarsförmågan hos organisationen genom att jämföra olika produkter och enheter. Detta resulterade i produkter från Fortinet-varumärket såsom FortiGate NGFW med UTM förmåga, då de erbjuder flera fördelar jämfört med konkurrenter.

Arbete är en betydelsefull del av både ett socialt och ekonomiskt oberoende liv för alla människor i yrkesverksam ålder. Individer med funktionsnedsättning har generellt svårare att hitta, få och behålla ett arbete på en öppen, konkurrensutsatt arbetsmarknad. I denna kvalitativa studie har vi valt att fokusera på individer med schizofreni och genom ett systemteoretiskt perspektiv undersöka vad som möjliggör och hindrar individer med schizofreni att få en plats på arbetsmarknaden. Metoden vi har använt i denna studie är en scoping study enligt Arksey och O’Malley. Vi valde att utgå från systemteori, stigmatisering och interaktionsordning vid analysering av resultatet för att få en helhetsbild av de främjande och hindrande faktorerna på olika systemnivåer. Frågeställningarna avser vad som hindrar eller främjar målgruppens tillträde på arbetsmarknaden utifrån individernas livssituation, professionellas arbetssätt samt insatsernas effektivitet. Inför analysen identifierades fem teman, vilket sattes ihop till fyra under processens gång. Resultaten visar på att det finns behov av individualisera arbetet för att professionella ska kunna skapa individuella förutsättningar och inte se gruppen som homogen. Detta kan ske genom specialiserade kunskaper inom området för att främja samverkan mellan professionella. Resultaten synliggör problemen som finns på olika systemnivåer inom den svenska kontexten. Vi anser att vi besvarat forskningsfrågorna samt att våra resultat stämmer överens med övrig forskning som tagits upp i arbetet. Vidare kunskap behövs inom området som berör den arbetsinriktade forskningen för att få fram lösningar på hur man kan stötta fler individer med schizofreni ut på arbetsmarknaden. Specifikt avser våra kunskapsluckor ett glapp i den forskning som berör implementering av IPS i befintliga återhämtningsinrikade verksamheter, arbetsgivares perspektiv på att anställa individer med psykiska funktionsnedsättningar samt hur de ekonomiska klyftorna för individerna kan förändras eller förbättras. / Work is an important part of both a socially and financially independent life for all people of working age. Individuals with disabilities generally find it more difficult to find, get and keep a job in an open, competitive labor market. In this qualitative study, we have chosen to focus on individuals with schizophrenia and, through a systems theory perspective, examine what enables and hinders individuals with schizophrenia a place in the labor market. The method we have used in this study is a scoping study according to Arksey and O’Malley. We chose to start from systems theory, stigma and interaction order when analyzing the results to get an overall picture of the promoting and hindering factors at different system levels. The questions refer to what hinders or promotes the target group's access to the labor market based on the individuals' life situation, the working methods of professionals and the effectiveness of the efforts. Prior to the analysis, five themes were identified, which was put together into four during the process. The results show that there is a need to individualize the work so that professionals can create individual conditions and not see the group as homogeneous. The results make visible the problems that exist at different system levels within the Swedish context. We believe that we have answered the research questions and that our results are in line with other research that has been included in the work. Further knowledge is needed in the area that concerns work-oriented research in order to find solutions on how to support more individuals with schizophrenia into the labor market. Specifically, our knowledge gaps refer to a gap in the research that concerns the implementation of IPS in existing recovery-oriented businesses, employers' perspectives on employing individuals with mental disabilities and how the financial gaps for individuals can be changed or improved.

European forests have experienced vitality loss and dieback due to increasing disturbances, temperatures, droughts, and forest management. Around 160 million cubic meters of woody biomass in Germany were damaged from 2018 to 2020. Protected areas can offer insights into the natural resilience of European forests after disturbances. However, they face conflicts such as mass outbreaks and the spread of insect pests that can threaten biodiversity and natural habitats. The European spruce bark beetle (Ips typographus) is among Europe’s most important and severe forest pest species and can act as an ecological disturbance. Due to ongoing climate change, no weakening is expected in the future. Forest management can directly influence important drivers of disturbance regimes. However, the response times are likely slow, requiring effective and adaptive risk-assessing management. Simulation models can play an important and influential role in such management as decision-making tools or for analysis of important drivers. Such models have been successfully applied in fishery management, disease control, and forest fire management. While there are many models published about Ips typographus, there is still no simulation tool that individually describes trees and beetles with their properties and decisions, tests the effectiveness of management measures, and uses spatially explicit data for natural landscapes. This approach would allow for investigating infestation patterns and development in natural landscapes based on individual beetle and tree traits. It could also be used to study the impact of climate change and forest protection management on all spatial scales - from individual beetles to entire landscapes. The main goal of the thesis is to predict the risk of bark beetle infestations and outbreaks in a national park region in Germany under different climate change and management scenarios as an emergent outcome of individual traits of the European spruce bark beetle and its host tree. To achieve this goal, a combination and improvement of existing models on the life history of Ips typographus and its host tree Picea abies is done to simulate infestations on an individual level. The results are then scaled to the landscape level using a Markov chain metamodel for 20 years into the future. It is shown how (i) management scenarios consisting of different sanitation felling intensities, and (ii) climate change scenarios representing increasing numbers of yearly beetle generations interact and affect the infestation spread. Chapter 2 discusses the creation, testing, and implementation of a new simulation model for the dispersal and infestation of Ips typographus in a natural European landscape. The IPS-SPREADS (Infestation Pattern Simulation Supporting PREdisposition Assessment DetailS) model is an innovative combination of existing models on beetle phenology (PHENIPS), spruce tree predisposition (PAS - predisposition assessment systems), and precise mechanistic dispersal flight in an artificial forest (IPS - Infestation Pattern Simulation). The model is used to study the effectiveness of sanitation felling in five different research areas at the border of a national park in Germany under varying annual beetle generation numbers. Chapter 3 explores the various purposes and types of metamodels used in agentbased modeling and attempts to identify and rank the most suited or efficient model types for each application. This assessment was accomplished through a systematic literature review of 27 scientific publications containing 40 different metamodel applications in an individual-based modeling context. A standardized rating instruction was created and used by the candidate and a group of other Ph.D. students and postdoctoral researchers to objectively rate different metamodel applications, such as upscaling agent-based models or conducting sensitivity analyses. In Chapter 4 of the dissertation, the IPS-SPREADS simulation model is used to predict the risk of bark beetle infestations in a national park region for 20 years into the future. A metamodel approach based on Markov chains is used to scale the computationally demanding IPS-SPREADS model to larger temporal and spatial scales. The metamodel is calibrated and validated using data on infested trees from 2015 to 2017. The model is then used to assess the effectiveness of sanitation felling in preventing the spread of a bark beetle outbreak from the national park to adjacent forests until 2038. This work adds to a series of previous studies investigating several aspects of the Norway spruce (Picea abies) and European spruce bark beetle (Ips typographus) system, such as risk assessment, beetle development, beetle dispersal, and infestation patterns, the effectiveness of forest protection measures against the beetle spread or the impact of bark beetle disturbance on the forest as well as its interaction with other disturbances. The results of the studies presented as thesis chapters 2 to 4 indicate that regardless of the number of yearly beetle generations and the spatial or temporal scale, a sanitation felling intensity of 80 % and above seems to mitigate further mass outbreak propagation. It is also shown that habitat inter-connectivity and individual traits of beetles and host trees substantially affect the infestation patterns. In addition, commonly applied metamodel types and application purposes in an individual-based modeling context are revealed. The most promising model variant varies in regard to the chosen application aim. Further research directions based on the work presented in this thesis incorporate investigating spatial configurations of mixed forest stands and their effect on the dispersal and infestation risk of the European spruce bark beetle. Furthermore, it is discussed how applying and integrating open-access GIS data, such as the European Unions Copernicus program, could improve model validity and applicability. The effects and implications for implementing and analyzing further management measures, such as buffer zones around protected areas, terrestrial detection based on visual clues, and dispensers with anti-attractants in the proposed model framework, are discussed. / Europas Wälder haben aufgrund von zunehmenden Störungen, Temperaturen, Dürren und vergangener Managemententscheidungen an Vitalität verloren und zeigen großflächige Absterbeerscheinungen. Allein in Deutschland betrug das Schadvolumen von 2018 bis 2020 rund 160 Millionen Kubikmeter. Schutzgebiete können Einblicke in die natürliche Widerstandsfähigkeit der Wälder nach Störungen gewähren, bieten aber auch Potenzial für Konflikte wie Massenvermehrungen und Ausbreitung von Schädlingen, die die biologische Vielfalt und die natürlichen Lebensräume bedrohen können. Der Große Achtzähnige Fichtenborkenkäfer (Ips typographus) gehört zu den wichtigsten und schwerwiegendsten Waldschädlingen in Europa. Aufgrund des anhaltenden Klimawandels ist keine Gefährdungsabnahme der Waldbeständen zu erwarten. Während die Forstwirtschaft wichtige Treiber von Störungsregimen direkt oder indirekt beeinflussen kann, sind die Reaktionszeiten sehr langsam und verlangen ein wirksames und adaptives Risikomanagement. Simulationsmodelle können eine wichtige und effektive Rolle für das Management als Entscheidungshilfen oder zur Analyse wichtiger Treiber spielen. Beispiele für erfolgreiche Anwendungen im Entscheidungsprozess stellen Bereiche wie das Fischereimanagement, die Bekämpfung von Epidemien und das Waldbrandmanagement dar. Obwohl es bereits einige Simulationsmodelle für Ips typographus gibt, wurde bisher kein Ansatz untersucht, bei dem sowohl die Bäume als auch die Käfer als Individuen mit Eigenschaften, Wahrnehmung und Interaktionen abgebildet werden. Mit solch einem Modell könnte untersucht werden, wie sich die individuellen Eigenschaften der Käfer und der Bäume auf die Befallsmuster und die Wirksamkeit von Managementmaßnahmen in Abhängigkeit der betrachteten räumlichen oder zeitlichen Skala auswirken. Das Hauptziel der vorliegenden Dissertation besteht daher darin, das Risiko von Borkenkäferbefall und von Ausbrüchen unter verschiedenen Klimawandel- und Managementszenarien als emergentes Ergebnis individueller Eigenschaften des Großen Achtzähnigen Fichtenborkenkäfers und seines Wirtsbaums zu analysieren. Dies geschieht durch die Kombination, Verbesserung und Anwendung bestehender Modelle zur Lebensgeschichte von Ips typographus und dessen Wirtsbaum Picea abies am Beispiel einer Nationalparkregion in Deutschland. Die Ergebnisse des Individuenbasierten Modells werden anschließend auf die Landschaftsebene und für 20 Jahre in die Zukunft skaliert, indem ein auf Markov-Ketten basierendes Metamodel kreiert und validiert wird. Es wird gezeigt, wie (i) Managementszenarien bestehend aus verschiedenen Entnahmeintensitäten befallener Bäume sowie (ii) Klimawandelszenarien bestehend aus einer Zunahme der jährlichen Käfergenerationen interagieren und die Befallsausbreitung beeinflussen. In Kapitel 2 der Dissertation wird die Entwicklung, Überprüfung und Implementierung eines neuen Simulationsmodells für die Ausbreitung und den Befall von Ips typographus in einer realen Landschaft Europas diskutiert. Das IPS-SPREADS (Infestation Pattern Simulation Supporting REdisposition Assessment DetailS) Modell ist eine innovative Kombination bestehender Modelle zur Phänologie des Käfers (PHENIPS), der Prädisposition des Wirtsbaumes gegenüber Borkenkäferbefall (PAS - Prädispositionsabschätzsysteme) und der Individuen-basierten Simulation des Ausbreitungsflugs der Käfer in einem künstlichen Wald (IPS - Infestation Pattern Simulation). Das neue Modell IPS-SPREADS wird dann verwendet, um die Wirksamkeit der Entnahme befallener Bäume in fünf verschiedenen Probeflächen des Nationalparks Sächsische Schweiz während zunehmender jährlicher Käfergenerationen zu untersuchen. Darüber hinaus wird der Einfluss individueller Eigenschaften auf das Befallsgeschehen und auf die Wirksamkeit der Entnahme befallener Bäume analysiert. Kapitel 3 widmet sich der Anwendungsgebiete und Typen von Metamodellen, die für Agenten-basierten Modelle verwendet werden, und versucht, die vielversprechendsten Modelltypen je Anwendung zu identifizieren. Dies geschieht mittels einer systematischen Literaturanalyse, die 27 wissenschaftliche Veröffentlichungen mit insgesamt 40 verschiedenen Metamodellanwendungen für Individuen-basierte Modelle untersucht. Ein standardisiertes Bewertungsverfahen wird erstellt und vom Kandidaten zusammen mit einer Gruppe von Doktoranden und Postdoktoranden angewendet, um die Metamodelle im Bezug auf ihr Anwendungsziel objektiv zu bewerten. In Kapitel 4 der Dissertation wird das entwickelte IPS-SPREADS Modell verwendet, um das Risiko von Borkenkäferbefall in einer Nationalparkregion für 20 Jahre in die Zukunft vorherzusagen. Dafür wird ein Metamodell basierend auf Markov-Ketten entwickelt, welches das rechenintensive IPS-SPREADS Modell auf größere zeitliche und räumliche Skalen hebt. Dieses Metamodell wird mittels Befallsdaten aus der Nationalparkregion für die Jahre 2015 bis 2017 validiert. Im Anschluss wird das Metamodell dann verwendet, um die Wirksamkeit der Entnahme befallener Bäume für die Ausbreitungsbekämpfung eines Borkenkäfermassenbefalls vom Nationalpark in die angrenzenden Wälder während eines 20-jährigen Zeitraums zu untersuchen. Die vorliegende Arbeit baut auf einer Reihe von Studien auf, die verschiedene Aspekte des Systems von Großem Achtzähnigen Fichtenborkenkäfer (Ips typographus) und Gemeiner Fichte (Picea abies) untersuchen: Prädispositionsbewertung, Käferentwicklung, Ausbreitungs- und Befallsmuster sowie die Wirksamkeit von Waldschutzmaßnahmen gegen die Befallsausbreitung. Die Ergebnisse der als Kapitel zwei bis vier vorgestellten Studien zeigen, dass unabhängig von der Anzahl der jährlichen Käfergenerationen und der räumlichen oder zeitlichen Skala eine Entnahmeintensität befallener Bäume von 80 % und mehr die Ausbreitung eines Massenbefalls zu mindern scheint. Es wird auch gezeigt, dass die Habitatvernetzung und die individuellen Eigenschaften der Käfer und Wirtsbäume die Befallsmuster beeinflussen. Darüber hinaus werden häufig angewandte Metamodelltypen und Anwendungszwecke in einem Individuenbasierten Modellierungskontext zusammengetragen und die vielversprechendste Modellvariante je Anwendungsgebiet extrahiert. Zum Schluss werden weitere Forschungsrichtungen basierend auf den vorgestellten Studien diskutiert, wie zum Beispiel die Untersuchung der räumlichen Konfiguration von Mischwäldern und deren Auswirkungen auf das Befallsgeschehen des Großen Achtzähnigen Fichtenborkenkäfers. Des Weiteren wird diskutiert, wie die Anwendung und Integration von frei verfügbaren GIS-Daten, wie beispielsweise des Copernicus Programms der Europäischen Union, die Validität und Anwendbarkeit der entwickelten Modelle verbessern können. Am Ende werden potenzielle Implikationen für die Implementierung und Analyse weiterer Managementmaßnahmen wie Pufferzonen um Schutzgebiete, terrestrische Erkennung von Borkenkäferbefall sowie Dispenser mit Anti-Aggregationspheromonen im Rahmen der vorgestellten Modellstudien diskutiert.

A striking feature of Parkinson's disease (PD) is that the distal axonal terminals of neurons degenerate prior to the soma, a process referred to as 'dying-back'. Another hallmark of the disease is the pathological accumulation of abnormal protein aggregates in soma and axons. Lysosomes, a critical component of the protein quality control machinery, have thus been thought to be altered in PD. LRRK2 G2019S, a gain-of-kinase-function mutation, is one of PD's most common known causative mutations, and LRRK2-specific small molecule inhibitors have been developed as possible therapeutics. However, LRRK2 G2019S is incompletely penetrant, and its role in axonal degeneration is unclear. LRRK2 phosphorylates a subset of Rab GTPases, including Rab10. Since Rab GTPases are mediators of organelle trafficking, we speculated that LRRK2 G2019S affects the transport of organelles, such as lysosomes, thereby contributing to early PD pathogenesis. Using neural progenitor cell-derived neurons from two LRRK2 G2019S-PD patients; we developed a model of axonal trafficking of lysosomes to characterize the impact of mutant LRRK2 on lysosomal trafficking. In comparison to their isogenic gene-corrected controls, we observed a subtle reduction in mutant axonal lysosomal speed, which could indicate that mutant LRRK2 mildly disrupts retrograde lysosomal transport. We also observed that this trafficking phenotype was only partially rescued by LRRK2 kinase inhibitors, which could indicate the importance of other factors regulating axonal transport. Consistent with this idea, we found that mutant LRRK2 was associated with increased co-localization of phosphorylated Rab10 on a small subset of distal axonal lysosomes. Furthermore, the over-expression of Rab10 only mildly affected lysosomal trafficking in axons. Interestingly, damaging the lysosomal membrane increased LRRK2-dependent Rab10 phosphorylation, leading us to speculate that membrane damage in the axon might induce LRRK2 activity. Since lysosomes have been shown to mediate plasma membrane repair, we speculated that membrane damage might exacerbate LRRK2-dependent phenotypes in distal axons. Axotomy was used to test this idea, and we observed an inconsistent delay in the regrowth of mutant axons after axotomy. Moreover, we identified an association between mutant LRRK2 and the transient increase in lysosomes at the injury site, indicating that LRRK2 G2019S might potentially affect damage-prone distal axons. Since the LRRK2 G2019S-associated phenotypes observed in our assays were relatively mild in one isogenic pair, we were curious about the clinical and genetic phenotypes of the patients from whom the somatic cells for neural progenitor cell generation were sourced. Interestingly, we observed that clinical features of PD, including age-of-onset, motor symptoms, cognitive impairment, and the level of cerebrospinal fluid biomarkers, were heterogeneous between the two patients. Additionally, genetic analysis of specific PD risk-associated loci in MAPT and SNCA revealed that one patient was more at risk of developing PD than the other, indicating influence from genetic factors in addition to LRRK2 G2019S. These factors might affect the axonal phenotypes observed in our assays. Overall, we have developed assays to investigate the effects of LRRK2 G2019S on axonal lysosomes. These assays can potentially be a useful tool to better understand early pathogenesis in heterogeneous PD patients and test targeted therapeutics that can be successful over an eclectic cohort of PD patients, all of whom are diagnosed based on deteriorating motor symptoms.:TABLE OF CONTENTS I LIST OF FIGURES IV LIST OF TABLES VI ABBREVIATIONS VII 1 INTRODUCTION 1 1.1 Neurodegenerative diseases 1 1.2 Parkinson’s disease 2 1.2.1 General Features 2 1.2.2 Phenomenon of “dying back” in PD 6 1.2.3 Contribution of axonal architecture and function to “dying back” 7 1.2.4 Etiology of PD 10 1.2.4.1 Environmental factors 10 1.2.4.2 Genetic factors linked to axonal function 11 1.3 Lysosomes 12 1.3.1 Composition and biogenesis of lysosomes 13 1.3.2 Lysosomes as digestive centers 15 1.3.3 Lysosomes as secretory organelles 18 1.3.4 Lysosomes in PD 20 1.3.4.1 Genetic PD factors linked to lysosomal function 21 1.4 Leucine-rich repeat kinase 2 (LRRK2) 22 1.4.1 LRRK2 domain organization and function 22 1.4.2 Clinical features of PD patients with LRRK2 mutations (LRRK2-PD) 24 1.4.3 LRRK2 animal models 24 1.4.4 LRRK2 induced pluripotent stem cell (iPSC)-based models 25 1.4.5 Animal and iPSC-based models demonstrate a role for LRRK2 in the endo-lysosomal system 27 1.4.6 LRRK2 kinase inhibitors 30 2 AIMS OF THE THESIS 32 3 MATERIALS AND METHODS 33 3.1 Materials 33 3.1.1 Chemicals 33 3.1.2 Purchased kits 34 3.1.3 Plasmids 34 3.1.4 Antibodies 35 3.1.5 Dyes 36 3.1.6 Primers and oligonucleotides 36 3.1.7 Cell culture media and reagents 37 3.1.8 Small molecules 38 3.1.9 Compounds 38 3.1.10 Cell culture media 39 3.1.11 Human Neural Progenitor Cell (NPC) lines 40 3.2 Methods 41 3.2.1 Ethics statement 41 3.2.2 Licenses 41 3.2.3 Information about iPSC and NPC line generation 41 3.2.4 Preparation of cell culture coated plates 41 3.2.5 Maintenance of NPCs 42 3.2.6 Differentiation of NPCs to neurons 42 3.2.7 Preparation of microfluidic chambers 43 3.2.8 Seeding neurons as single cells 44 3.2.9 HEK293T cell culture 45 3.2.10 Treatment of neurons with compounds 45 3.2.11 Genomic DNA isolation 46 3.2.12 Polymerase-Chain Reaction (PCR) 46 3.2.13 Agarose gel electrophoresis 46 3.2.14 Plasmid DNA isolation 46 3.2.15 Lentiviral vector production 47 3.2.16 Lentiviral infection of human neurons 48 3.2.17 Protein isolation and quantification 48 3.2.18 Capillary electrophoresis 49 3.2.19 Axotomy 49 3.2.20 Immunostaining 50 3.2.21 Live cell imaging 51 3.2.22 Quantification of axonal trafficking using kymographs 52 3.2.23 Quantification of axonal trafficking using an object based method 53 3.2.24 Apotome imaging and quantification 54 3.2.25 Confocal imaging and quantification 54 3.2.26 Clinical and biomarker data collection 55 4 RESULTS 57 4.1 Establishing an axonal lysosomal trafficking assay 57 4.1.1 NPCs from LRRK2 G2019S patients and their respective isogenic controls differentiate into neurons 57 4.1.2 Axons can be spatially separated from soma and dendrites 60 4.1.3 Setting up the axonal trafficking assay 62 4.2 Axonal lysosomal trafficking assay detects LRRK2 G2019S associated changes in lysosome movement 65 4.3 Axonal lysosomal trafficking assay detects partial rescue by a small molecule LRRK2 inhibitor 71 4.4 LRRK2 G2019S is associated with an increase in the proportion of lysosomes co-localizing with phosphorylated Rab10 76 4.5 Rab10 over-expression mildly affects lysosomal trafficking in axons 78 4.6 Lysosomal membrane damage increases LRRK2-mediated Rab10 phosphorylation 81 4.7 LRRK2 G2019S is not associated with consistent effects on long-term axonal regrowth after axotomy 82 4.8 LRRK2 G2019S is associated with transient accumulation of lysosomes at the injury site after axotomy 86 4.9 Assessment of clinical, biomarker and genetic data from the LRRK2 G2019S patient donors 88 5 DISCUSSION 92 6 APPENDIX 101 7 SUMMARY 104 8 ZUSSAMENFASSUNG 106 9 BIBLIOGRAPHY 108 10 ACKNOWLEDGEMENTS 136 11 DECLARATIONS 138

Intrusion Prevention Systems (IPSs) have long been proposed as a defense against attacks that propagate too fast for any manual response to be useful. While purely-network-based IPSs have the advantage of being easy to install and manage, research have shown that this class of systems are vulnerable to evasion [70, 65], and can be tricked into filtering normal traffic and create more harm than good [12, 13]. Based on these researches, we believe information about how the attacked hosts process the malicious input is essential to an effective and reliable IPS. In existing IPSs, honeypots are usually used to collect such information. The collected information will then be analyzed to generate countermeasures against the observed attack. Unfortunately, techniques that allow the honeypots in a network to be identified ([5, 71]) can render these IPSs useless. In particular, attacks can be designed to avoid targeting the identified honeypots. As a result, the IPSs will have no information about the attacks, and thus no countermeasure will ever be generated. The use of honeypots is also creating other practical issues which limit the usefulness/feasibility of many host-based IPSs. We propose to solve these problems by duplicating the detection and analysis capability on every protected system; i.e., turning every host into a honeypot. / text

A great need exists for an abundant, easily accessible source of patient-specific cells that will function for use in regenerative medicine. One promising source is the adult stem cell derived from adipose tissue (ASCs). Isolated from waste lipoaspiration, these cells could serve as a readily available source for the regeneration of damaged tissues. To further define the biology of ASCs, we have isolated multiple cell strains from different adipose tissue sources, indicating wide-spread distribution in the body. We find that a widely used set of cell surface markers fail to distinguish ASCs from normal fibroblasts. However, our ASC isolations are multipotent while fibroblasts show no differentiation potential. In further contrast to fibroblasts, these cells also show expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG. Together, our data suggest that while the cell surface profile of ASCs do not distinguish them from normal fibroblasts and their lack of telomerase shows their limited proliferation capacity, the expression of genes closely linked to pluripotency and their differentiation capacity clearly define ASCs as multipotent stem cells. iPS cells are another promising cell type for tissue regeneration, due to their expression of hTERT and their capacity to differentiate into all three germ layers. Interestingly, telomerase is activated during the induction process, accomplished by the exogenous expression of four genes in normal, non-hTERT-expressing fibroblasts. To elucidate the mechanisms behind this activation, we examined the overexpression of these four factors in BJ fibroblasts and ASCs, which resulted in undetectable hTERT expression. We then demonstrated a lack of an acetylated histone H3K9 with the opposing di-methylation, indicative of a closed chromatin state at the hTERT promoter. Subsequent treatment of cells with TSA alone showed an upregulation of hTERT mRNA without telomerase activity. However, telomerase activity was found when ASCs, but not BJs were treated with TSA and all four factors, indicating differential regulation of hTERT in cells of similar mesenchymal origins. Our data suggest that while hTERT’s expression is universally dependent on the presence of a relaxed chromatin state and sufficient transactivating factors, other cell to cell differences can prevent its expression.

Includes bibliographical references

This paper explores the business implications of Integrated Product and Service Offerings (IPSOs). The objective is to show examples of the business implications of IPSOs from a supplier’s perspective, and to suggest specifications for supporting methods needed for such an industrial company. The paper is largely based on empirical case studies of 120 Swedish manufacturing companies of all sizes. Results from the case studies show that both small and large companies that conduct the transition towards IPSOs face several important strategic challenges, some of them associated with high risk.

Le contrôle traductionnel est une étape critique de la régulation de l'expression des gènes impliqués dans le développement embryonnaire et de nombreux processus cellulaires. Au cours de l'initiation de la traduction chez les eucaryotes, le facteur eIF4E (eukaryotic Initiation Factor 4E) fixe la coiffe des ARNm et recrute la protéine eIF4G pour former le complexe d'initiation. L' interaction eIF4E/eIF4G est une étape clé de la régulation traductionnelle, faisant du site de liaison à eIF4E un lieu de compétition entre eIF4G, le répresseur général de la traduction 4E-BP et d'autres régulateurs appelés 4E-IPs (4E-interacting partners), qui partagent avec eIF4G un motif consensus de liaison à eIF4E. Longtemps considérée comme une protéine désordonnée, nous avons montré au cours de cette thèse que 4E-BP adopte en réalité une conformation repliée lorsqu'il se lie à eIF4E, impliquant une surface d'interaction plus importante. Ces résultats ont apporté un nouveau regard sur les interactions qui s'établissent entre eIF4E et ses partenaires, et ont fourni des informations cruciales pour l'établissement de nouvelles thérapies, notamment celles développées dans le cadre des cancers. Mon travail de thèse a également permis l'établissement d'un criblage de nouvelles 4E-IPs, basé sur une approche alliant analyses structurales, bioinformatiques et biochimiques. Parmi les 4E-IPs détectées, nous avons caractérisé la protéine Angel1, membre d'une famille de déadénylases. Ces résultats ont ouvert de nombreuses perspectives pour la compréhension du métabolisme des ARNm mais aussi celle des régulations traductionnelles spécifiques prenant pour cible moléculaire eIF4E