Baksidestext The Tor network was originally designed as low-latency anonymity network.However, as the years progressed, Tor earned a reputation as also being a useful tool to circumvent Internet censorship. At times, the network counted 30,000 users only from China. Censors reacted by tightening their grip on the national communication infrastructure. In particular, they developed techniques to prevent people from being able to access the Tor network. This arms race now counts several iterations and no end is in sight. This thesis contributes to a censorship-resistant Tor network in two ways. First, it analyses how existing censorship systems work. In particular, the Great Firewall of China is analysed in order to obtain an understanding of its capabilities as well as to explore circumvention opportunities. Second, this thesis proposes practical countermeasures to circumvent Internet censorship. In particular, it presents a novel network protocol which is resistant to the Great Firewall's active probing attacks.

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L’initiation de la traduction est un processus complexe qui fait intervenir une douzaine de facteurs d’initiation. L’élément clef de ce mécanisme est le facteur eIF4E qui grâce à sa liaison avec la coiffe, recrute l’ensemble du complexe d’initiation au niveau de l’ARNm et permet l’assemblage du ribosome au voisinage du codon d’initiation. Chez Arabidopsis thaliana, il existe à coté de la protéine eIF4E, une isoforme : eIF(iso)4E. Ces deux protéines participent à l’initiation de la traduction. L’existence de ces deux protéines évoque un phénomène de redondance fonctionnelle qui est attestée par la létalité du double mutant alors que les simples mutants sont viables. Cependant, l’étude phénotypique de mutants pour les gènes eIF4E et eIF(iso)4E a permis de montrer que cette redondance est partielle et inégale. En effet, les mutants pour le gène eIF4E présentent un retard de croissance, un retard de floraison, une baisse de la fertilité, une sénescence précoce et une activité traductionnelle réduite. Inversement, le phénotype des plantes mutantes pour le gène eIF(iso)4E est comparable à celui du sauvage. Les mutations dans les gènes eIF4E et eIF(iso)4E induisent une hypersensibilité à la lumière Enfin, en présence d’un inhibiteur de TOR la croissance de la racine des plantes de la lignée mutante pour le gène eIF(iso)4E est moins inhibée que celle des plantes de la lignée sauvage. / More than 12 initiation factors are involved in eukaryotic translation initiation. The key step of this mechanism is the binding of eIF4E with the cap of the mRNA. This step allows the recruitment of the initiation complex and the assembly of the ribosome close to the start codon. Arabidopsis thaliana encodes a second eIF4E protein: eIF(iso)4E. Those two proteins perform translation initiation. The existence of those two proteins suggests that they may be functionally redundant. Double mutant lethality testifies for functional redundancy. However, phenotypic studies of mutant lines for gene eIF4E and eIF(iso)4E showed that redundancy is partial and unequal. Indeed, the eIF4E mutant lines exhibit growth delay in rosette and roots, bolting delay, impaired fertility and early senescence in leaves. Translational activity is also largely impaired. On the contrary, a mutant line for the eIF(iso)4E gene has the same phenotype as wild type line. Mutant lines for eIF4E and eIF(iso)4E are more sensitive to light and accumulate anthocyanins even in normal light. On the molecular level, the amounts of mRNA of genes that are involved in high light response and their association to polysomes increase. When plants are grown on media containing a TOR inhibitor, AZD-8055, plants of the eIF(iso)4E mutant line show less root growth inhibition compared to wild type and eIF4E mutant lines. This result suggests that eIF(iso)4E could be targeted by the TOR pathway.

La régulation de l’expression des gènes joue un rôle fondamental dans la réponse et l’adaptation des cellules à leur environnement. L'expression des gènes peut être régulée à plusieurs étapes distinctes, mais un niveau de contrôle critique est l’initiation de la transcription. Celle-ci implique le recrutement séquentiel de nombreux régulateurs différents, dont les complexes co-activateurs. De nombreuses études ont démontré et caractérisé leurs fonctions dans la transcription. Cependant, il est moins bien compris comment les co-activateurs sont directement régulés par les conditions environnementales. Des travaux précédents de mon laboratoire de thèse ont montré, dans la levure fissipare Schizosaccharomyces pombe, que le complexe co-activateur SAGA contrôle l’expression des gènes en réponse aux nutriments et contribue ainsi à l’équilibre entre la prolifération cellulaire et la différenciation sexuelle. L’objectif de mon travail de thèse a été de comprendre comment le complexe SAGA répond à la disponibilité en nutriments et régule l’expression des gènes de différenciation. Pour cela, j’ai combiné des approches de génétique, de biochimie et de protéomique quantitative. Des analyses d’interactions génétiques m’ont permis de montrer que SAGA, par l’intermédiaire de sa sous-unité acétyltransférase Gcn5, contrôle l’équilibre entre prolifération et différenciation en aval des voies de signalisation TORC1 et TORC2. Puis, des études biochimiques ont établi que les voies de signalisation TORC1 et TORC2 contrôlent SAGA via la phosphorylation différentielle d’une sous-unité architecturale du complexe, nommée Taf12. En effet, lorsque les nutriments sont présents, TORC1 active la phosphatase PP2A, via la kinase Greatwall, pour déphosphoryler Taf12. Au contraire, la carence en nutriments active la voie de signalisation TORC2-AKT, qui permet la phosphorylation de Taf12, afin de moduler l’intensité de la réponse de différenciation. Nous avons également identifié d’autres sous-unités de SAGA qui sont différentiellement phosphorylées en fonction du niveau en nutriments et qui pourraient donc également contribuer à la régulation de SAGA. Notamment, nous avons observé que les sous-unités Ada3 et Sgf29, impliquées dans la régulation de l’activité de Gcn5, sont également phosphorylées dans les conditions carencées en nutriments. Enfin, j’ai observé que TORC2 et Gcn5 contrôlent la transition G2/M de façon synergique, suggérant que SAGA et les voies de signalisation des kinases TOR interagissent fonctionnellement dans le contrôle d’autres processus. Mon travail révèle que SAGA est une cible directe des voies de signalisation qui détectent les nutriments et établit un nouveau mécanisme par lequel TORC1 et TORC2 convergent pour contrôler l’expression génique et le destin cellulaire / The regulation of gene expression plays a fundamental role in the ability of cells to respond to external changes. One critical level of regulation is transcription, which is controlled by large complexes with many distinct activities. Little is known about how these activities integrate developmental or environmental signals to regulate transcription. We are using S. pombe as a model system to address this issue, in the context of cell fate control by nutrient availability. Previous work in the lab has established that, in this yeast, the SAGA co-activator complex controls whether cells proliferate or not in response to nutrients. Following up on these observations, we determined which nutrient-sensing signaling pathways regulate SAGA activities. A comprehensive genetic approach demonstrated that SAGA functions downstream of the TOR kinase-containing complexes, TORC1 and TORC2. In parallel, quantitative mass spectrometry analysis of the SAGA complex revealed that the Taf12 subunit is differentially phosphorylated, depending on nutrient levels. In agreement with our genetic analyses, Taf12 phosphorylation depends on the PP2A phosphatase, which we found is activated by TORC1 when nutrients are present. Conversely, upon nutrient starvation, TORC2 is activated allowing the AKT kinase to phosphorylate Taf12. We are now testing the in vivo roles of these modifications as well as their impact on SAGA functions at nutrient-regulated promoters. Altogether, our results contribute to a better understanding of the control of transcription by signal transduction pathways.

L’Insulin Degrading Enzyme (IDE) est une protéase dont les mécanismes de fonctionnements ne sont pas encore complètement élucidés.Dans ce but, nous avons identifié un homologue d’IDE chez la levure Schizosaccharomyces pombe (S. pombe) : iph (Insulinase Pombe Homologue), et mis en évidence un lien entre Iph et la voie TOR (Target of Rapamycin) lors d’un stressprotéotoxique.La voie TOR comme les voies de vieillissement et de réponse au stress sont régulées par la présence de nutriments dans le milieu. Dans cette optique, nous nous sommes intéressés chez S. Pombe à la durée de vie chronologique qui, dans une souche sauvage, augmente lors d’une restriction en glucose.Cependant, les levures invalidées pour iph perdent cette capacité, et présentent une DVC identique indépendamment de la concentration du glucose dans le milieu.L’ensemble de ces résultats a permis de démontrer que la protéine Iph régule négativement la voie TOR, qui elle même favorise la survie lors d’un stress protéotoxique et le vieillissement des cellules. / Insulin Degrading Enzyme (IDE) is a 110 kDa protease whose function is not completely elucidated.To this aim, we have identified a homologue of IDE in the yeast Schizosaccharomyces pombe (S. pombe) : iph (Insulinase Pombe Homologue), and we have pointed out a link between Iph and the TOR (Target of Rapamycin) pathway during proteotoxic stress.The TOR pathway- like the pathways of ageing and the stress response- are regulated by the presence of nutrients in the environment.Knowing this, we were interested in S. Pombe chronological life span that increases in the case of glucose restriction in the wildtype strain. However, the yeast cells deleted for iph loose this capacity and show a lifespan chronology that is identical independently of the glucose concentration in the environment.Taking together these results show that the protein Iph regulates negatively the TOR pathway, which by oneself favours the survival during proteotoxic stress and ageing of the cells.

Les plantes sont des organismes statiques et tributaires des ressources minérales présentes dans leur rhizosphère. La remobilisation des nutriments est un processus qui permet une économie nutritionnelle et un recyclage de macro- et micro - nutriments qui sont le plus souvent limitants. Le rôle du démantèlement des chloroplastes au cours de ce processus est très important pour le recyclage de l’azote, puisque ceux-ci contiennent la majeure partie des protéines foliaires. Bien que les protéines chloroplastiques soient une source essentielle pour le recyclage de l’azote foliaire, leur mécanisme de dégradation est mal connu. L’autophagie, a été proposée comme mécanisme participant au recyclage des nutriments, notamment en situation de carence ou de limitation en azote. L’autophagie, processus cellulaire de dégradation, représente un mécanisme de survie et d’adaptation, par le recyclage et l’élimination des protéines et organelles altérés.La détermination des flux d’azote, entre la rosette et les graines par l’utilisation du marquage à l’isotope stable 15N chez des mutants d’autophagie, nous a permis de montrer que l’autophagie est nécessaire à la remobilisation de l’azote. L’analyse fonctionnelle des mutants d’autophagie a permis de mettre en évidence de profondes perturbations métaboliques résultant dans l’élévation du rapport C/N. Les modifications métaboliques observées montre que les mutants d’autophagie ne présentent pas les signatures métaboliques habituellement retrouvées chez les plantes adaptées à la limitation en azote minéral, qu’ils accumulent au contraire les composés azotés et sont pauvres en ressources carbonées. Les investigations ont également révélé que l’autophagie est sélective envers certaines protéines. L’activité autophagique a été évaluée en fonction de différents niveaux d’expression d’AtTOR et à la suite de l’inhibition de son activité kinase. Ces résultats ont montré qu’AtTOR, senseur du statut nutritionnel, est un régulateur négatif de l’autophagie. L’autophagie est une étape clef du recyclage nutritionnel en réponse à une situation de stress telle que la limitation en azote. / Plants are static organisms dependent on minerals resources available in the rhizosphere. Nutrient recycling is a process allowing a nutritional economy and recycling of macro- and micro- nutrients, which are often limiting. The role of chloroplast dismantling during this process is very important for nitrogen recycling because chloroplasts contain the major part of foliar proteins. Albeit chloroplastic proteins are an essential source for foliar nitrogen recycling, their degradation process is not well understood. Autophagy has been proposed to participate in nutrients recycling, notably in nitrogen starvation or limitation. Autophagy, a cellular degradation process, represents a survival and an adaptation mechanism by recycling and eliminating defectives proteins and organelles.Based on nitrogen fluxes determination between the rosette and the seeds by using 15N labeling in autophagy (atg) mutants, the study has shown that autophagy is necessary for nitrogen remobilization. The functional analysis of atg mutants revealed deep metabolic perturbations resulting in elevated C/N ratio, marker of plant physiology status. The observed metabolic modifications are not the hallmarks of an adaptation to nitrogen limitation. Autophagy mutants indeed accumulate nitrogen compounds and present low carbohydrate contents. The investigations also revealed that autophagy is selective towards some proteins. Autophagic ativity has been evaluated function of different AtTOR expression levels and following AtTOR activity inhibition. Results have shown that AtTOR, a sensor of the nutritional status, is a negative regulator of autophagy. Autophagy is a key step for nitrogen recycling in response to stress situation like nitrogen limitation.

Orientadores: Sandra Martha Gomes Dias, Marília Meira Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T09:41:29Z (GMT). No. of bitstreams: 1 Ascencao_CarollineFernandaRodrigues_M.pdf: 4713312 bytes, checksum: b65183d96535d66661af745a562f2d58 (MD5) Previous issue date: 2014 / Resumo: A proliferação celular comanda os processos de embriogênese e de crescimento do organismo, sendo essencial para a correta função de vários tecidos adultos. Apesar de ser importante para a homeostase do organismo, a sua desregulação compõe a força motriz do desenvolvimento tumoral. Somente nos últimos vinte anos começou a ser evidenciada a relação entre as vias de tradução de sinais estimuladas por fatores de crescimento e a reorganização da atividade metabólica, a qual precisa priorizar a biossíntese e o aumento da biomassa, processos essenciais para a divisão celular. Em células tumorais, o consumo de glutamina é aumentando concomitante ao aumento da atividade de glutaminase. Três isoenzimas de glutaminase são expressas na maioria dos tecidos (liver-type glutaminase, kidney-type glutaminase e glutaminase C), todavia pouco se sabe sobre a necessidade específica de cada uma delas para o metabolismo tumoral. Vários artigos recentes têm definido o papel da glutaminólise, ou metabolismo da glutamina e seus subprodutos, na ativação da mTOR. Neste sentido é uma hipótese válida imaginar que mTOR possa contra-regular glutaminase. Desta maneira, resolvemos investigar se mTOR atua na regulação da atividade de glutaminase. Para tanto, realizamos knockdown estável de PTEN em células MDA-MB 231 e verificamos que não o mesmo afetou os níveis protéicos de GAC e KGA, assim como não houve mudança na localização subcelular das isoformas. Cinética enzimática da fração mitocondrial desta linhagem revelou que o knockdown de PTEN levou à uma diminuição do KM da enzima sem alteração de Vmax. De acordo, o tratamento com rapamicina, inibidor da mTOR, elevou o KM para os níveis detectados nas células controles. A atividade de glutaminase de lisado total de MDA-MB 231, NIH 3T3, IMR90 e BJ5TA foi afetada pelo tratamento com rapamicina conforme julgado por ensaios de dose e tempo resposta. Mais, ensaios de privação de glicose, glutamina e de fatores de crescimento levaram à inibição de mTOR e concomitante redução da atividade de glutaminase. Somado a isso, o knockdown estável de TSC2 em MDA-MB 231 e BJ5TA, assim como o knockout de TSC2 em MEF, promoveu superestimulação de mTOR e foi capaz de aumentar a atividade de glutaminase. Dosagem de atividade de glutaminase de células MDA-MB 231 com knockdown de GAC, KGA ou GAC/KGA tratadas com rapamicina indicaram que mTOR possa agir em ambas as isoformas. Curioso foi que apenas células shGAC e shGAC/KGA apresentaram redução da fosforilação de S6K em Thr389 indicando que GAC ou o metabolismo de glutamina via esta isoforma, possa contra-regular mTOR. Em adição, na comparação entre PC3 e DU145, verificamos que DU145 apresentou maior expressão de GAC, maior consumo de glutamina, maior dependência de glutamina em seu crescimento, maior sensibilidade ao inibidor de glutaminase, BPTES, e por fim, se mostrou mais responsiva à metformina, ativador indireto de AMPK. A ativação de AMPK por metformina, um conhecido sensor de estresse energético, mostrou diminuir a atividade de glutaminase em célula de tumor de próstata, DU145, indicando uma potencial ação de AMPK na atividade de glutaminase / Abstract: Cell proliferation is crucial for embryogenesis and organism growth, being also essential for the proper function of several adult tissues. Although important for the homeostasis of the organism, its deregulation composes the driving force of tumor development. In the past twenty years the relationship between the processes of signal translation stimulated by growth factors and the reorganization of metabolic activity has become more evident. Growing cells need to prioritize the biosynthesis and biomass increase, processes essential for cell division. In tumor cells, the glutamine consumption is increased concurrently with the increasing in the glutaminase activity. Three glutaminase isoenzymes are expressed in most tissues (liver- type glutaminase, kidney -type glutaminase and glutaminase C), but not much is known about the necessity of each isoform for the tumor metabolism. Several recent papers have defined the role of glutaminolysis or glutamine metabolism in mTOR activation. So it is a valid hypothesis to speculate that mTOR can counter-regulate glutaminase. Thus, we decided to investigate whether mTOR can control glutaminase activity. To this end, we have made MDA - MB 231 cells stably knocked down for PTEN and verified no alteration in KGA and GAC protein levels, as well as there was no change on their subcellular location. Enzyme kinetics of the MDA-MB 231 mitochondrial fraction revealed that PTEN knockdown led to a decrease in the KM of the enzyme without changing Vmax. Accordingly, the treatment with rapamycin (mTOR inhibitor), led to an increase in KM back to the level detected in control cells. The glutaminase activity of MDA - MB 231, NIH 3T3, IMR90 and BJ5TA total cellular lysates was also affected by rapamycin treatment in a dose- and time-response fashion. Moreover, glucose, glutamine and growth factors deprivation promoted mTOR inhibition and concomitant reduction on glutaminase activity. Glutaminase activity of MDA-MB 231 cells knocked down for GAC, KGA or GAC/KGA and treated with rapamycin indicated that mTOR can regulate both isoforms. Curiously, it was only on GAC or GAC/KGA knocked down cells that we observed a decrease in S6K Thr 389 phosphorylation, which could indicate that GAC or the GAC dependent-glutamine metabolism is a specific mTOR counter-regulator. Accordling, stable TSC2 knockdown in MDA-MB 231 and BJ5TA, as well as TCS2 knockout in MEF cells, promoted overstimulation of mTOR and increasing on glutaminase activity. Moreover, a comparison between PC3 and DU145 revealed that DU145 has higher GAC expression, greater consumption of glutamine, is more dependent on glutamine for its growth, more sensitive to the inhibitor of glutaminase, BPTES, and more responsive to metformin, an indirect AMPK activator. The activation of AMPK by metformin, a known energy stress sensor, led to a decreased glutaminase activity in the prostate tumor cell line DU145 indicating a potential role of AMPK on glutaminase activity / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular

Les triacylglycérol (TAG) est un métabolite hautement énergétique qui peut être facilement converti en biodiesel. Les TAG peuvent être produits à partir de plantes et de microalgues. L'étude des voies de signalisation de l'énergie peut offrir de nouvelles stratégies pour améliorer l'accumulation de biomasse et de TAG sans compromettre la croissance. Dans cette thèse, j'ai étudié le rôle de deux voies principales de signalisation énergétique: la voie du de guanosine ppGpp (guanosine penta(tétra) phosphate) dans le chloroplaste et la voie de TOR (Target of rapamycin) dans le cytosol. J'ai choisi de travailler sur la mousse Physcomitrella patens, un modèle d'eucaryote photosynthétique en raison de sa position évolutive entre les algues et les plantes vasculaires. Pour étudier le rôle du ppGpp, nous avons créé des lignées transgéniques exprimant de manière inductible une ppGpp synthase et sur-accumulant le ppGpp. J'ai trouvé que l'induction de SYN provoque une forte inhibition de la capacité photosynthétique en raison de l'inhibition de l'expression des protéines clés codées par les chloroplastes et aussi une réorganisation des membranes thylakoïdes. Pour l’étude de TOR nous avons traité la mousse avec des inhibiteurs de TOR et montré que cela provoque l’inhibition de la croissance de manière dose dépendante et l’accumulation de TAG. L’utilisation des marqueurs lipidiques a révélé la perte de petites vésicules associées à la croissance et l'accumulation de plus grandes structures de corps lipidiques. Des études supplémentaires permettront de développer des stratégies pour améliorer la production de biocarburants chez les organismes photosynthétiques. / Triacylglycerol (TAG) is a highly energetic metabolite that can be easily converted into biodiesel. TAG can be produced from both plants and microalgae. However, plants have low TAG yields in their dominant vegetative tissues. Microalgae can accumulate high amounts of TAG, but only under stress, leading to growth inhibition and limiting yield. The study and manipulation of stress and energy signaling pathways can offer new strategies to improve biomass and TAG accumulation without compromising growth. In this thesis, I studied the role of two major energy signaling pathways: the guanosine penta(tetra) phosphate (ppGpp) pathway in the chloroplast and the target of rapamycin (TOR) pathway in the cytosol. I chose to work on the moss Physcomitrella patens which is an interesting model of photosynthetic eukaryote because of its evolutionary position between algae and vascular plants. To study the role of ppGpp we created transgenic lines that inducibly express a ppGpp synthase and over-accumulate ppGpp. I found that ppGpp accumulation causes a strong inhibition of photosynthetic capacity due to the inhibition of the expression of key chloroplast encoded proteins, and also reorganization of the thylakoid membrane system into super grana. For the TOR pathway, we treated P. patens protonema with active site TOR inhibitors and showed that this cause growth inhibition in a dose dependent manner and is accompanied by TAG accumulation. The use of lipid dyes reveals a shift from small growth associated vesicles to a larger oil body structures after treatment. Further studies will allow the development of new strategies for improving biofuel production in photosynthetic organisms.

Friction modification within the wheel-rail interface is an important way of achieving ecologically friendly transportation of both persons and goods. This thesis aims to develop a new TOR lubricant, which will be able to maintain suitable frictional conditions while securing minimal adhesion required for traction. All measurements were carried out on tribometer MTM in the ball-on-disc configuration. In the first step, individual components were examined. More complex compositions were measured after that and the three best of them were compared with commercial TOR lubricants. The results show a good ability of developed compositions to maintain required adhesion, reduce wear, and all of them had resistivity against over-lubrication. Lastly, the process of lubricant verification before its application in real traffic was discussed.

A recently proposed defense for the anonymity network Tor uses preload lists of domains to determine what should be cached in the Domain Name System (DNS) caches of Tor relays. The defense protects against attacks that infer what is cached in Tor relays. By having domains continuously cached (preloaded), the cache will become independent of which websites have been visited. The current preload lists contain useless domains and have room for improvement. The objective of this project is to answer the question of "How can we generate better preload lists?" and to provide improved methods for generating preload lists, with the ultimate goal of generating better preload lists that the Tor Project can benefit from.  We further developed existing tools to use web crawling to find more useful domains, as well as implementing filtering to remove useless domains from the preload lists. The results of this project showed promising results, as the useless domains decreased by an average of around 57% and more useful domains were found. / Ett nyligen föreslaget försvar för anonymitetsnätverket Tor använder preload listor av domäner för att avgöra vad som ska cachelagras i domännamnssystemets (DNS) cacher för Tor reläer. Försvaret skyddar mot attacker som avgör vad som cachelagras i Tor relärer. Genom att ha domäner kontinuerligt cachade (förladdade), blir cachen oberoende av vilka websidor som har besökts. De nuvarande preload listorna innehåller värdelösa domäner och har utrymme för förbättring. Syftet med detta projekt är att svara på frågan "Hur kan vi generera bättre preload listor?" och att bidra med förbättrade metoder för att generera preload listor, med det ultimata målet att generera bättre preload listor som Torprojektet kan dra nytta av. Vi vidareutvecklade befintliga verktyg till att använda webbkrypning för att hitta mer användbara domäner, samt implementerade filtrering av värdelösa domäner från preload listorna. Detta projekt visade lovande resultat, då de värdelösa domänerna minskade med i genomsnitt ungefär 57% och mer användbara domäner hittades.