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Environmental factors, such as diet, can have a significant impact on the health of animals, influencing lifespan, development, and disease progression. The model organism Caenorhabditis elegans is a bacterivore whose development is characterized by an invariant pattern of cell division. This study investigated how C. elegans developmental rate is altered in response to 48 different bacterial diets. The bacterial species studied had a wide range of effects on developmental progression, with some bacteria dramatically decreasing developmental rate, while others caused developmental arrest in early larval stages. From these analyses, Staphylococcus species that caused very slow development of animals in the L1 stage were selected for further characterization. The slow developmental rate observed in these animals was rescued by supplementation with essential amino acids. Genetic analysis revealed that these effects were mediated through TOR signaling and were independent of insulin signaling. Loss of daf-15(raptor), a central component of TOR complex 1, inhibited the rescuing effects of the amino acid supplementation on developmental rate, suggesting that the slow development induced by Staphylococcus could be rescued by activating the TORC1 pathway. Autophagy, negatively regulated by TOR, is increased in worms fed Staphylococcus species, suggesting TOR activity is reduced, and supplementation through amino acids reduced autophagy. These data suggest that TOR signaling is reduced in C. elegans in response to certain bacterial diets, resulting in decreased developmental rate, and that this effect may be due to amino acid deficiency. / Thesis / Master of Science (MSc)

La protéine mTOR (mechanistic Target Of Rapamycin), lorsque dérégulée, favorise le développement tumoral par ses fonctions dans la prolifération et la survie cellulaire. Son activité est contrôlée principalement par les facteurs de sa voie d'activation canonique (PTEN/PI3K/AKT) qui sont souvent mutés dans les cancers. Cependant, certains cancers ne présentent pas d'altérations dans cette voie canonique bien que mTOR soit constitutivement active, suggérant ainsi un mécanisme différent. C'est le cas des gliomes de bas grade dont une grande partie présente des mutations hétérozygotes de l'enzyme Isocitrate déshydrogénase (IDH1 et IDH2) menant à un gain de fonction de celles-ci. En effet, l'α-cétoglutarate (αKG) produite par les formes sauvages sera rapidement transformé en 2-Hydroxyglutarate (2HG) par les formes mutées. De plus, ces gliomes présentent très tôt une activité accrue de mTOR et ce, de façon PTEN indépendante. Un criblage par ARN d'interférence ciblant des enzymes αKG dépendantes a permis l'identification de KDM4A, une lysine déméthylase, comme un nouveau régulateur de mTOR. La régulation de KDM4A sur mTOR n'est pas transcriptionnelle, mais semble due à son interaction avec DEPTOR. En effet, sa stabilité, en absence de KDM4A, est grandement diminuée, ce qui favorise l'activité de mTOR. Ainsi, l'implication de KDM4A sur l'activité de DEPTOR s'avère être un nouveau mode de régulation de la protéine mTOR. Nous avons également découvert que DEPTOR peut être phosphorylé sur sa tyrosine 289, ce qui favorise l'activité de mTOR. Cette tyrosine, située près de sérines connues pour réguler la dégradation de DEPTOR, permet une meilleure stabilité de la protéine. De plus, la phosphorylation favorise une réorganisation rapide du cytosquelette d'actine par l'activation de mTORC2. Nous avons par la suite montré qu'elle diminue l'affinité de DEPTOR pour mTOR amenant une activation accrue de cette voie. Un criblage avec différents inhibiteurs de tyrosines kinases de même qu'une analyse par spectrométrie de masse nous a permis d'identifier les kinases SYK (Spleen Tyrosine Kinase) et EPHB2 comme régulateurs de la phosphorylation tyrosine de DEPTOR. En effet, nous avons démontré que la phosphorylation de SYK sur DEPTOR était dépendante de l'activation de SYK par EPHB2. En plus de cette phosphorylation tyrosine, DEPTOR possède également de nombreuses autres modifications post-traductionnelles. En effet, il peut être ubiquitinilé par des chaînes d'ubiquitine de type K48 promouvant sa dégradation par le protéasome, mais également par des chaînes d'ubiquitine K63 dont leur fonction est encore inconnue. L'absence de modification post-traductionnelles sur les 5 dernières lysines de DEPTOR augmente drastiquement la phosphorylation de la tyrosine 289 suggérant que la méthylation ou l'ubiquitination affecte cette modification. Nous avons également trouvé que DEPTOR pouvait être NEDDylée dans sa portion N-terminale au niveau de ses domaines DEP. Une analyse de spectrométrie de masse après des expériences de marquage de proximité par biotinilation a permis d'identifier de multiples enzymes pouvant potentiellement moduler ces modifications. Toutes ces modifications ouvrent la porte à de nouvelles avenues de régulation de l'activité de DEPTOR sur mTOR. L'implication de KDM4A sur l'activité de DEPTOR de même que la phosphorylation de la tyrosine 289 de DEPTOR se révèlent comme de nouveaux mécanismes régulant l'activité de mTOR pouvant expliquer l'augmentation de l'activité de mTOR dans les cancers où la voie canonique n'est pas affectée. Cela pourrait ouvrir la voie à de nouvelles avenues thérapeutiques qui, combinées à celles déjà existantes, permettra d'offrir des traitements prometteurs lorsque cette voie est dérégulée, notamment dans les gliomes de bas grade. / Dysregulated mTOR (mechanistic Target Of Rapamycin) is a potent tumor growth inducer known to promote cancer cell proliferation and survival. Its activity can be regulated by numerous factors composing the PTEN/PI3K/AKT canonical pathway, which are often mutated in cancer. However, in a subset of cancer showing a constitutively activated mTOR, there is no alteration within the canonical activation pathway, suggesting different activation mechanisms. Low-grade gliomas harbor mutation on Isocitrate dehydrogenase 1 and 2 (IDH1/2) conferring gain-of-function by the production of 2-Hydroxyglutarate (2HG) from α Ketoglutarate (αKG). This leads to a constitutive mTOR activation in a canonical independent manner. An RNAi screen led us to identify KDM4A, a αKG dependant lysine demethylase as a new regulator of mTOR activity. KDM4A interacts with DEPTOR, an endogenous inhibitor of mTOR and member of both mTOR complex. Depletion or inhibition of KDM4A by 2HG decreases DEPTOR stability and thereby increases mTOR activity. We also discovered a new post-translational modification (PTM) on DEPTOR, corresponding to a single phosphorylation event on tyrosine 289. While this modification increases DEPTOR stability, it also promotes its dissociation from mTORC1&2, leading to a rapid and sustain increase in mTORC1&2 activity. To identify the upstream signaling pathway(s) leading to tyrosine 289 phosphorylation, we performed mass spectrometry analysis, as well as a small drug screen of different tyrosine kinase inhibitors. Using these combined methods, we identify SYK (Spleen tyrosine kinase), whose expression levels correlate with levels of tyrosine 289 phosphorylation. We also found that SYK-induced phosphorylation of DEPTOR was regulated by the EPHB2 receptor. We have shown that DEPTOR harbors many other PTM like ubiquitination conjugated on lysine 48 promoting proteasomal degradation and ubiquitination conjugated on lysine 63 whose function is still unknown. The absence of PTM on the last 5 lysines of DEPTOR drastically increases the phosphorylation of tyrosine 289 suggesting that methylation and/or ubiquitination affect this modification. We also found that DEPTOR can be NEDDylated in its N-terminal part on its DEP domains. Mass spectrometry analysis after proximity biotinilation assays led us to discover multiple enzymes that could potentially modulate all these modifications. This open new insight on DEPTOR regulation and function on mTOR. Our findings uncovered new mechanisms regulating DEPTOR activity, which can explain the increased mTOR activity in cancer with unaffected PTEN/PI3K/AKT regulatory pathways. Better understanding of this mTOR/DEPTOR regulatory pathway could allow the development of a new therapeutic approach to inhibit mTOR associated cancer progression.

I studien undersöks huruvida en grupp narkotikasäljare på den svenska hemsidan Flugsvamp drivs av andra motivationer än ett grundläggande vinstintresse. Sidan bygger på de teknologiska verktyg som utvecklades av den libertarianskt inspirerade Cypherpunkrörelsens arbete med stark kryptografi i slutet av 1990-talet. Dessa verktyg skyddar både brottslingar och frihetskämpar, men har främst associerats med de som använder anonymiteten i kriminella syften och således har verktygen fått ett dåligt rykte som riskerar äventyra kryptografins framtid. Genom semistrukturerade onlineintervjuer med fem säljare av skiftande karaktär samlade jag information gällande deras upplevelse av sidan, relationerna till kunder och administratörer, hur de rent moraliskt rationaliserade sin verksamhet och vilken påverkan de såg sig ha på samhället och kryptografin. En bild framträdde där majoriteten av säljarna uppvisade ansvarskänsla och eftertänksamhet genom att agera med transparens och professionalitet. Genom att utkonkurrera oärliga aktörer och med ett nära kollegialt samarbete med varandra använde dessa säljare sin roll för att utmana den traditionella majoritetsinställningen till narkotika och visa hur den ansvarsfulla digitala försäljningen i längden kan vara samhällsförändrande. En minoritet uppgav att de endast använde sidan för att tjäna extrapengar och egentligen tyckte att det var en rimlig idé för staten att förbjuda de kryptoanarkistiska verktygen då de främst anses användas i kriminella syften. Studien når fram till slutsatserna att samhället som helhet tjänar på att det narkotikarelaterade våldet försvinner från gatorna, visar på hur en inkonsekvent och konservativ lagstiftning gör sig mottaglig för starka motargument samt understryker att den framtida digitala mänskligheten kommer ha en betydande användning för de kryptoanarkistiska verktygen.

Made available in DSpace on 2014-12-17T14:13:52Z (GMT). No. of bitstreams: 1 ValdecyFOP_TESE.pdf: 1904907 bytes, checksum: d2ca008f2dd23c2884f4b0457e6705d9 (MD5) Previous issue date: 2014-05-09 / A analgesia p?s-operat?ria eficaz ? especialmente importante ap?s cirurgias tor?cicas, pois, al?m de aliviar a dor, facilita a retomada de atividades normais, incluindo a deambula??o, a respira??o e a tosse. Dessa forma, os objetivos deste estudo s?o: avaliar a efic?cia analg?sica da associa??o entre anestesia geral e raquianestesia com morfina e ropivaca?na mais esquema multimodal em rela??o ? anestesia geral e esquema multimodal em cirurgia de revasculariza??o do mioc?rdio; analisar a efic?cia analg?sica da inje??o subcut?nea de lidoca?na e analgesia multimodal na remo??o de tubos tor?cicos em cirurgia de revasculariza??o do mioc?rdio. A metodologia consiste em ensaio cl?nico randomizado, controlado, envolvendo 58 pacientes, de ambos os sexos, com idade m?dia de 59,8  8,9 anos, estado f?sico ASA II e III. Os participantes foram alocados em dois grupos, sendo o GI composto por indiv?duos submetidos ? anestesia geral combinada ? raquianestesia com morfina 400μg e 6 ml (30mg) a 8 ml (40mg) de ropivaca?na a 0,5% e analgesia multimodal; j? o GII foi composto por indiv?duos submetidos ? anestesia geral associada ? analgesia multimodal. Foi avaliada a dor, ao despertar, nas primeiras 24 horas, e ao realizar exerc?cio respirat?rio, ao retirar drenos de tor?cicos e o tempo para extuba??o. A an?lise estat?stica foi realizada pelos testes do Qui-quadrado e Teste t de Student e o teste de Fisher. O resultado obtido foi o seguinte: o GI apresentou menor intensidade de dor ao despertar (p= 0,001), nas primeiras 24 horas (p= 0,001) e durante a realiza??o dos exerc?cios respirat?rios (p= 0,004). Houve maior necessidade de analgesia complementar no grupo GII, com maior consumo de morfina (p= 0,05), e os efeitos colaterais leves, como n?useas (p= 0,001), v?mito (p= 0,002), prurido (p= 0,030), predominaram no GI. N?o houve diferen?a estatisticamente significante entre os grupos (P= 0,47), em rela??o ? intensidade de dor na remo??o dos drenos. Ap?s as observa??es feitas, o estudo sugere que a anestesia geral combinada ? raquianestesia com morfina associada ? ropivaca?na oferece melhor efeito analg?sico no p?s-operat?rio de cirurgia card?aca. Adicionalmente, o estudo sugere que o efeito analg?sico da inje??o subcut?nea de lidoca?na 1% associado ? analgesia multimodal n?o ? eficaz

Orientador: Gabriel Forato Anhê / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T19:01:58Z (GMT). No. of bitstreams: 1 Araujo_ThiagoMatosFerreirade_D.pdf: 1239554 bytes, checksum: 87d5fc958173ca83a217021c9d866455 (MD5) Previous issue date: 2014 / Resumo: A obesidade é caracterizada pela deposição ectópica de gordura no fígado. Este acúmulo de gordura hepática (NAFLD) pode gerar consequências graves, como a hepatite não alcoólica (NASH), fator de ricos para carcino hepatocelular (HCC). A morte de hepatócitos, evento chave na evolução da NAFLD para NASH, é causada pelo excesso de nutrientes e é dependente do estresse de retículo endoplasmático (RE). O estresse no RE resulta no acúmulo de proteínas não processadas desencadeia a "unfolded protein response" (UPR), podendo gerar apoptose. A mTOR é formada basicamente por dois complexos: mTOR1 e mTOR2; ambos são sensíveis a nutrientes, a insulina e a rapamicina. O complexo mTOR2/Rictor catalisa a fosforilação da AKT, aumentando a sinalização da insulina. Deste modo, o objetivo deste trabalho foi avaliar a relação entre ativação da mTOR, do estresse de RE e da apoptose em hepatócito expostos a ácidos graxos livres. Observamos que a apoptose causada pelo palmitato ativa o estresse de RE de maneira tempo dependente. Não observamos alterações na fosforilação de proteínas alvo específicas para o complexo mTOR1. No entanto, a fosforilação geral da mTOR foi estimulada pelo palmitato. Altas doses de rapamicina inibiram a apoptose e do estresse de RE causado pelo palmitato, sugerindo a participação do complexo mTOR2. Estes resultados ainda foram confirmados pelo silenciamento gênico da Rictor. A fosforilação em serina 473 da AKT apresenta um caráter transitório, elevando-se em tempos que precedem morte e o estresse de RE, e diminuindo em tempos prolongados concomitantemente à apoptose. A inibição da AKT pelo "AKT inhibitor" gerou diminuição da apoptose, do estresse de RE e da incorporação lipídica na linhagem de hepatoma. Estes dados sugerem que a AKT, como alvo preferencial da mTOR2 é necessária para geração de morte e da UPR. A glicose (33.3mM) gera morte as células HepG2 e esta é inibida com baixas doses de rapamicina, mostrando possível atividade via mTOR1 nesta resposta. De outro modo, a frutose (4.5mM) que também desencadeia apoptose das células de hepatoma, tem seu efeito inibido por doses maiores de rapamicina, indicando atividade mTOR2 neste processo. No entanto, a possibilidade de diferentes monossacarídeos recrutarem complexos diferentes de mTOR para desencadear apoptose ainda precisa ser melhor explorada / Abstract: Obesity is characterized by fat ectopic deposition in liver. This hepatic fat accumulation our non-alcoholic fat liver disease (NAFLD) can have serious consequences such as non-alcoholic hepatitis (NASH), that is a factor to liver cancer. The cell death of hepatocytes is an important event in the development to NAFLD to NASH, all that are caused by excess nutrients and dependent of endoplasmic reticulum (ER) stress. The ER stress is caused by accumulation of unfolded proteins triggers the unfolded protein response (UPR), which mau cause apoptosis. mTOR is basically formed by two complexes: mTOR1 and mTOR2, both are sensitive to nutrients, insulin and rapamycin. The mTOR2/Rictor complex catalyse AKT phosphorylation increasing the insulin pathway. All together, the aim of this study was evaluate the relationship between mTOR, ER stress and apoptosis in liver cells exposed to free fatty acids. We observed that apoptosis caused by palmitate activates ER stress in a manner dependent on time. We din¿t observed changes in phosphorylation of specific target proteins to mTOR1 complex. However, a general phosphorylation of mTOR was stimulated by palmitate. High doses of rapamycin inhibited apoptosis and ER stress caused by palmitate, suggesting the participation of the mTOR2 complex. These results were further confirmed by gene silencing of Rictor. The AKT phospholylation in serine 473 has a transitional character, rising in times that preceding cell death and ER stress, and decreasing concomitantly apoptosis in prolonged times. Inhibition of AKT by AKT inhibitor caused a decrease in apoptosis, ER stress and lipid incorporation in hepatoma cell line. These data suggest that AKT, preferential targets of mTOR2 is required for generation death and UPR. Glucose (33.3mM) generates HepG2 cell death and this is inhibited by low doses on rapamycin, showing possible mTOR1 activity. Otherwise, fructose (4.5mM) also triggers apoptosis of hepatoma cells; its effect is inhibited by higher doses of rapamycin, indicating mTOR2 activity in this process. However, the possibility of different monosaccharide recruit different complexes of mTOR to trigger apoptosis should be further explored / Doutorado / Farmacologia / Doutor em Farmacologia

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T15:05:51Z (GMT). No. of bitstreams: 1 Almeida_GustavoPereirade_M.pdf: 7665145 bytes, checksum: 3fef8dc5d333834f8117015fea3b10ef (MD5) Previous issue date: 2012 / Resumo: A produção de energia por meio de fontes renováveis é uma exigência atual para se atingir uma economia sustentável. Os organismos fotossintetizantes surgem nesse contexto como ferramentas importantes na produção de compostos carbônicos ricos em energia, com destaque para microalgas em que tais compostos podem atingir até 80% do peso seco. Entretanto, um fator ainda desfavorável para sua utilização é o seu baixo rendimento na produção de biomassa. A espécie Chlamydomonas reinhardtii, por exemplo, é capaz de duplicar apenas algumas vezes durante 24 horas. As vias que controlam o crescimento celular, portanto, são alvos promissores para modificação genética. Dentre essas vias, à via de sinalização sensível à rapamicina aparece como um controlador central. Com o intuito de entender melhor como esse controle é exercido ao nível da expressão gênica global, foi utilizado a ferramenta de sequenciamento de RNA em larga escala para obtenção dos transcriptomas de culturas (sincronizadas) sob inibição dessa via e na condição controle, em oito momentos ao longo de um ciclo celular de 24h. O controle exercido por essa via sobre o metabolismo e sobre o ciclo celular foi o foco das análises. Foi encontrado que a inibição da via da TOR é capaz de gerar uma resposta de direcionamento parcial do metabolismo para a produção de TAG em detrimento de moléculas complexas como proteínas. Esse direcionamento foi considerado parcial devido à ocorrência concomitante de reações catabólicas. Outros dados obtidos sugerem que a via da TOR, além de regular o metabolismo de uma maneira geral e diversas funções celulares, também exerce influência sobre o progresso do ciclo celular e sua inibição resulta no atraso do desenvolvimento das fases do ciclo. Diversos fatores reguladores da transcrição envolvidos no desenvolvimento, no crescimento e na regulação do ciclo celular, foram encontrados diferencialmente expressos e constituem possíveis genes chave no controle do crescimento. Eles representam alvos em potencial para modificação genética com intuito de otimizar as taxas de crescimento na primeira etapa do sistema de produção. Na busca de alternativas aos processos atuais de indução do acúmulo de cadeias carbônicas, os efeitos da combinação rapamicina e via da TOR representam uma abordagem interessante para pesquisas futuras para viabilização da utilização de microalgas como fonte de energia. Este estudo possibilitou um melhor entendimento da atuação da via da TOR no crescimento e progresso do ciclo celular em C. reinhardtii ao nível de expressão gênica / Abstract: The energy production through renewable sources is an actual demand for achieving a sustainable economy. In this context, photosynthesizing organisms come to light as important tools for the production of energy-rich carbonic compounds, especially the microalgae, in which these compounds can reach up to 80% of the dry weight. However, an unfavorable factor for its utilization is the low yield of biomass production. The species Chlamydomonas reinhardtii, for instance, is capable of achieving only some duplication after 24 hours. The pathways that control cell growth are therefore promising targets for genetic modification. Among them, the rapamycin-sensitive signaling pathway emerges as a central controller. With the aim of better understanding how this control is fulfilled by the means of global gene expression, the high throughput RNA sequencing technology was used. With it, the synchronized cultures transcriptome under the inhibition of this pathway and in the control condition, of eight points during a cellular cycle of 24 hours, were obtained. The metabolism and the cell cycle control by the TOR pathway was the main focus of the analysis. It was found that the inhibition of this pathway is capable to partially draw the metabolism towards TAG production to the detriment of producing more complex chains as proteins. This directing was considered partial due to the concomitant occurrence of catabolic reactions. Other data suggested that the TOR pathway, apart from the metabolism regulation in a general way and regulation of many other cellular functions, also influence the cell cycle progression and its inhibition retards the development of cell phases. Several transcription regulators involved in development, growth and cell cycle regulation were found out to be differentially expressed and are likely to constitute key genes in growth control. They represent potential targets for genetic modification aiming the optimization of growth rate in the first step of the production system. In the search for alternatives to the current process of inducing carbon chain accumulation, the effects of the combination between rapamycin and TOR pathway represent an interesting approach for future research intending to turn the utilization of microalgae as an energy source into a feasible option. This study enabled a better understanding of the role of the TOR pathway in growth and cell cycle progression of C. reinhardtii at the level of gene expression / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Top-of- rail lubricants are an effective way to control adhesion, reduce contact wear and noise in rail transport. However, despite the widespread use of these lubricants, there is currently no standard defining how to test and evaluate their performance. This work aims to develop a methodology for testing top-of-rail lubricants in a laboratory environment using a tribometer in a ball-on-disk configuration. At first, important operational parameters of experiments are analyzed and experimentally tested. These are mainly wear-in and run-in, method of application, roughness and geometric parameters of contact bodies. The result of this work is a testing methodology that guarantees good repeatability and reliability of results. In the last part of this work, the methodology is verified using commercial top-of-rail lubricants.

This diploma thesis deals with ways of using Tor anonymization network both from the point of view of the use in the positive sense and the use in the negative sense, the abuse. The aim of this work is to identify different ways of using the network and to quantify network abuse based on analyzed abuse reports. First, the basic concepts are defined and the development of the Internet and identification technologies in its environment are briefly described. Then, the concept of Onion routing, based on which the Tor network works, together with more technical details about the functioning of the network, is introduced. Last but not least, the Tor Browser is described as the most common tool for using the Tor network along with the types of users who use it. The last chapter of the theoretical part introduces the problem of network abuse. The practical part is primarily solved in the form of a quantitative analysis of abuse, due to their volume of almost 3 million. Quantitative analysis is done using the statistical language R and basic mining data, text mining and statistical methods. The analyzed data are related to several large nodes of the Tor network and contains several years of history. Although the total number of complaints increases almost exponentially over time, complaints about malicious...

More than two-and-a-half million people currently use the Tor network to communicate anonymously via the Internet and gain access to online media that are not accessible using standard Internet technology. This sphere of communication can be described as the hidden web. In part because this phenomenon is very recent, the subject has scarcely been studied in the social sciences. It is therefore the purpose of this paper to answer four fundamental questions: What is the hidden web? What characterises the communication sphere of the hidden web in contrast to the “normal Internet”? Which reasons can be identified to explain the development of the hidden web as a new communication sphere? And, finally, what is the social significance of the hidden web? / Über zweieinhalb Millionen Menschen nutzen gegenwärtig das Tor Network, um anonym über das Internet zu kommunizieren und Zugriff auf Online-Medien zu erhalten, die mit gewöhnlicher Internettechnik nicht nutzbar ist. Diese Kommunikationssphäre kann als Hidden Web bezeichnet werden. Unter anderem weil es sich um ein sehr junges Phänomen handelt, liegen bisher nahezu keine sozialwissenschaftlichen Erkenntnisse zu dem Thema vor. Dementsprechend werden hier vier grundlegende Fragen beantwortet: Was ist das Hidden Web? Welche Eigenschaften weist die Kommunikationssphäre des Hidden Web im Vergleich zum „normalen“ Internet auf? Welche Gründen lassen sich identifizieren, die die Entstehung des Hidden Web als neue Kommunikationssphäre erklären können? Und welche gesellschaftliche Bedeutung kommt dem Hidden Web schließlich zu?