Chez les mammifères l’initiation de la traduction et, plus particulièrement, la formation du complexe eIF4F, est principalement régulée par la protéine kinase TOR (Target of rapamycin). Cette voie de régulation fait intervenir les protéines 4E-BP (eIF4E-binding proteins) dont l’activité est modulée par la phosphorylation par TOR. Sous leur forme non-phosphorylée, les 4E-BP se lient au facteur d’initiation eIF4E, empêchent son recrutement dans le complexe eIF4F et inhibent ainsi l’initiation de la traduction. Phosphorylées par TOR, les 4E-BP perdent leur affinité pour eIF4E et sont remplacées par eIF4G ce qui active la traduction. La régulation de l’initiation de la traduction par TOR via 4E-BP a été bien décrite dans plusieurs modèles eucaryotes, tels que la levure, les insectes et les mammifères, mais reste encore obscure chez les plantes. Les recherches réalisées au cours de ma thèse ont permis l’identification de deux protéines homologues de 4E-BP chez Arabidopsis. Ces protéines, que nous avons appelées ToRP1 et ToRP2 (TOR Regulatory Proteins), sont caractérisées par la présence d’un motif consensus indispensable pour la liaison à eIF4E, et qui existe chez les protéines 4E-BP des mammifères ainsi que chez eIF4G et eIFiso4G d’Arabidopsis. La protéine ToRP1 est capable d’interagir spécifiquement avec eIF4E, mais aussi avec TOR via son extrémité N-terminale en système double-hybride de levure. ToRP1 et ToRP2 ont également été caractérisées comme étant des cibles directement phosphorylées par TOR chez Arabidopsis. Deux sérines, en position 49 et 89 dans la protéine ToRP1, ont été identifiées comme des sites potentiels de cette phosphorylation. De plus, l’état de phosphorylation de ces sites affecte l’interaction avec eIF4E en système double-hybride de levure. Par ailleurs, des plants d’Arabidopsis déficients en ToRP1 et ToRP2 renforcent la traduction strictement coiffe-dépendante de l’ARNm CYCB1;1, alors que la surexpression de ToRP1 ou de ToRP2 réprime sa traduction. Ces résultats suggèrent donc que les protéines ToRP, identifiées chez Arabidopsis, sont de nouvelles cibles directes de TOR, qui, par leur phosphorylation, régule l’initiation de la traduction coiffe-dépendante. / The target of rapamycin (TOR) is an evolutionarily conserved kinase that is a critical sensor of nutritional and cellular energy and a major regulator of cell growth. TOR controls cap-dependent translation initiation, in particular the assembly of the eIF4F complex, by modulating the activity of eIF4E-binding proteins (4E-BPs). In their unphosphorylated state 4E-BP proteins sequester eIF4E and repress translation. Upon phosphorylation by TOR, 4E-BPs have a low affinity binding to eIF4E and are replaced by eIF4G thus activating translation initiation. 4E-BPs have been discovered in yeast and mammals but remain to be obscure in plants. Here, we identified and characterized two Arabidopsis proteins termed TOR Regulatory Proteins (ToRPs 1 and 2) that display some characteristics of mammalian 4E-BPs. ToRP1 and ToRP2 contain a canonical eIF4E-binding motif (4E-BM) found in mammalian 4E-BPs and Arabidopsis eIF4G and eIFiso4G. ToRP1 interacts with eIF4E, and, surprisingly, the N-terminal HEAT domain of TOR in the yeast two-hybrid system. ToRP1 and ToRP2 are highly phosphorylated at several phosphorylation sites in TOR-dependent manner in planta. Two of these phosphorylation sites have been identified as—S49 and S89—their phosphorylation status modulates ToRP1 binding to eIF4E in the yeast two-hybrid system. In plant protoplasts, ToRP2 can function as translation repressor of mRNAs that are strictly cap-dependent. Our results suggest that ToRPs can specifically bind the Arabidopsis cap-binding proteins (eIF4E/eIFiso4E) and regulate translation initiation under the control of TOR

No description available.

Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role.

Trypanosoma brucei est un parasite africain unicellulaire, responsable chez l’homme de la maladie du sommeil et chez les bovins de la Nagana. Il passe par différents stades lors de son cycle de vie, les deux principaux étant la forme sanguicole qui prolifère dans le sang des mammifères infectés, et la forme procyclique qui colonise le tube digestif du vecteur, la mouche glossine. Les trypanosomes sont extracellulaires, ils possèdent un flagelle qui leur permet de se mouvoir dans les différents milieux qu’ils infestent. La structure de celui-ci contient des éléments conservés au cours de l’évolution. Il constitue donc un excellent modèle de base pour en étudier l’architecture. D’autre part, le flagelle du parasite contient des structures propres à certains kinétoplastides, offrant ainsi une cible thérapeutique aux traitements anti-trypanosomiaux. Le flagelle est véritablement un organite plurifonctionnel nécessaire à la survie du parasite au sein des divers environnements qu’il rencontre lors de son cycle de développement. Outre son rôle moteur, il permet à la cellule d’échapper au système immunitaire de son hôte mammifère et de s’attacher à l’épithélium des glandes salivaires de l’insecte. Il est également requis pour le bon positionnement des organites, la morphogenèse et la division cellulaire. Enfin, il serait impliqué dans l’activité sensorielle du trypanosome. A ce jour, on ne connait quasiment rien des potentielles voies de « sensing ». Elles doivent pourtant exister, permettant l’appréhension de l’environnement, l’interaction avec les hôtes et la réception de signaux induisant la différenciation. Cet intérêt pour les voies de signalisation du parasite a abouti à l’étude des composantes de la voie TOR. TOR-Target of Rapamycin est un contrôleur central de la croissance cellulaire qu’il régule en fonction de différents stimuli externes. Il a été démontré depuis que chez T.brucei aussi, TOR régulerait la croissance temporelle et spatiale de la cellule. La kinase TOR est inhibée par sa liaison avec le complexe rapamycine-FKBP12. Nous avons identifié cette peptidyl-prolyl cis-trans isomérase chez le parasite : TbFKBP12. Elle y serait localisée au niveau du cytosquelette/flagelle. Contrairement à ce qui est observé chez la levure S.cerevisiae, l’isomérase est essentielle chez le trypanosome. Son invalidation par RNAi bloque la cytocinèse des parasites sanguicoles et provoque l’apparition d’axes de clivage internes à la cellule. Chez les formes procycliques par contre, la disparition de la protéine entraîne un défaut sévère de motilité du flagelle qui se traduit par une immobilisation partielle du parasite. TbFKBP12 est donc impliquée dans l’homéostasie du flagelle chez le trypanosome africain, organite nécessaire à la motilité et à la division cellulaire.

While advances to the Internet have enabled users to easily interact and exchange information online, they have also created several opportunities for adversaries to prey on users’ private information. Whether the motivation for data collection is commercial, where service providers sell data for marketers, or political, where a government censors, blocks and tracks its people, or even personal, for cyberstalking purposes, there is no doubt that the consequences of personal information leaks can be severe. Low-latency anonymity networks have thus emerged as a solution to allow people to surf the Internet without the fear of revealing their identities or locations. In order to provide anonymity to users, anonymity networks route users’ traffic through several intermediate relays, which causes unavoidable extra delays. However, although these networks have been originally designed to support interactive applications, due to a variety of design weaknesses, these networks offer anonymity at the expense of further intolerable performance costs, which disincentivize users from adopting these systems. In this thesis, we seek to improve the network performance of low-latency anonymity networks while maintaining the anonymity guarantees they provide to users today. As an experimentation platform, we use Tor, the most widely used privacy-preserving network that empowers people with low-latency anonymous online access. Since its introduction in 2003, Tor has successfully evolved to support hundreds of thousands of users using thousands of volunteer-operated routers run all around the world. Incidents of sudden increases in Tor’s usage, coinciding with global political events, confirm the importance of the Tor network for Internet users today. We identify four key contributors to the performance problems in low-latency anonymity networks, exemplified by Tor, that significantly impact the experience of low-latency application users. We first consider the lack of resources problem due to the resource-constrained routers, and propose multipath routing and traffic splitting to increase throughput and improve load balancing. Second, we explore the poor quality of service problem, which is exacerbated by the existence of bandwidth-consuming greedy applications in the network. We propose online traffic classification as a means of enabling quality of service for every traffic class. Next, we investigate the poor transport design problem and propose a new transport layer design for anonymous communication networks which addresses the drawbacks of previous proposals. Finally, we address the problem of the lack of congestion control by proposing an ATM-style credit-based hop-by-hop flow control algorithm which caps the queue sizes and allows all relays to react to congestion in the network. Our experimental results confirm the significant performance benefits that can be obtained using our privacy-preserving approaches.

Tor is a network designed for low-latency anonymous communications. Tor clients form circuits through relays that are listed in a public directory, and then relay their encrypted traffic through these circuits. This indirection makes it difficult for a local adversary to determine with whom a particular Tor user is communicating. Tor may also be used to circumvent regional Internet censorship, since the final hop of a user's connection can be in a different country. In response, some local adversaries restrict access to Tor by blocking each of the publicly listed relays. To deal with such an adversary, Tor uses bridges, which are unlisted relays that can be used as alternative entry points into the Tor network. Unfortunately, issues with Tor's bridge implementation make it easy to discover large numbers of bridges. This makes bridges easy to block. Also, an adversary that hoards this information may use it to determine when each bridge is online over time. If a bridge operator also browses with Tor on the same machine, this information may be sufficient to deanonymize him. We present BridgeSPA as a method to mitigate these issues. A client using BridgeSPA relies on innocuous single packet authorization (SPA) to present a time-limited key to a bridge. Before this authorization takes place, the bridge will not reveal whether it is online. We have implemented BridgeSPA as a working proof-of-concept for GNU/Linux systems. The implementation is available under a free licence. We have integrated our implementation to work in an OpenWRT environment. This enables BridgeSPA support for any client behind a deployed BridgeSPA OpenWRT router, no matter which operating system they are running.

Tropospheric ozone columns (TCOs) derived from differences between the Dutch-Finnish Aura Ozone Monitoring Instrument (OMI) measurements of the total atmospheric ozone column and the Aura Microwave Limb Sounder (MLS) measurements of stratospheric ozone columns are discussed. Because the measurements by these two instruments are not spatially coincident, interpolation techniques, with emphasis on mapping the stratospheric columns in space and time using the relationships between lower stratospheric ozone and potential vorticity (PV) and geopotential heights (Z), are evaluated at mid-latitudes. It is shown that this PV mapping procedure produces somewhat better agreement in comparisons with ozonesonde measurements, particularly in winter, than does simple linear interpolation of the MLS stratospheric columns or the use of typical coincidence criteria at mid-latitudes. The OMI/MLS derived tropospheric columns are calculated to be 4 Dobson units (DU) smaller than the sonde measured columns at mid-latitudes. Standard deviations between the derived tropospheric columns and those measured by ozonesondes are 9 DU (30%) annually but they are just 6 DU (15%) in summer. Uncertainties in the interpolated MLS stratospheric columns are likely to be the primary cause of these standard deviations. An important advantage of the PV mapping approach is that it works well when MLS data are missing (e.g., when an orbit of measurements is missing). In the comparisons against ozonesonde measurements, it provides up to twice as many comparisons compared to the other techniques. The OMI/MLS derived tropospheric ozone columns have been compared with corresponding columns based on the Tropospheric Emission Spectrometer (TES) measurements, and Regional chEmical trAnsport Model (REAM) simulations. The variability of tropospheric ozone columns has been examined for spring and summer 2005 over North America and the surrounding oceans. Comparisons of monthly mean distributions show good agreements between OMI/MLS tropospheric ozone columns, REAM columns, and TES columns. Two six-day periods in March have been selected to study the periodic TCO enhancements in two regions, around the Baja peninsula (Mexico) and over the West Coast of California. Thirteen-day back trajectories and daily maps of carbon monoxide (CO) and ozone from GEOS-CHEM and OMI/MLS have been used to investigate the influence of cross-Pacific transport. It is concluded that in the first period of the case study, the high ozone concentrations in mid and lower troposphere over the West Coast of California have been under the influence of cross-Pacific transport. Meteorological fields indicate that the high ozone concentrations in the upper troposphere over the West Coast of California and the high TCOs over the Baja peninsula are associated with stratospheric intrusions through a deep Rossby wave breaking event. The correlations between REAM TCOs and surface ozone from Environmental Protection Agency ground network measurements indicate that the TCO enhancement over the West Coast is associated with an increase of surface ozone. The correlations of REAM TCOs with geopotential height, wind fields, and tropopause height during the case study period suggest that TCO enhancement is best characterized in springtime by decreases of geopotential height on the 500 mb surface.

Tor is a network designed for low-latency anonymous communications. Tor clients form circuits through relays that are listed in a public directory, and then relay their encrypted traffic through these circuits. This indirection makes it difficult for a local adversary to determine with whom a particular Tor user is communicating. Tor may also be used to circumvent regional Internet censorship, since the final hop of a user's connection can be in a different country. In response, some local adversaries restrict access to Tor by blocking each of the publicly listed relays. To deal with such an adversary, Tor uses bridges, which are unlisted relays that can be used as alternative entry points into the Tor network. Unfortunately, issues with Tor's bridge implementation make it easy to discover large numbers of bridges. This makes bridges easy to block. Also, an adversary that hoards this information may use it to determine when each bridge is online over time. If a bridge operator also browses with Tor on the same machine, this information may be sufficient to deanonymize him. We present BridgeSPA as a method to mitigate these issues. A client using BridgeSPA relies on innocuous single packet authorization (SPA) to present a time-limited key to a bridge. Before this authorization takes place, the bridge will not reveal whether it is online. We have implemented BridgeSPA as a working proof-of-concept for GNU/Linux systems. The implementation is available under a free licence. We have integrated our implementation to work in an OpenWRT environment. This enables BridgeSPA support for any client behind a deployed BridgeSPA OpenWRT router, no matter which operating system they are running.

While advances to the Internet have enabled users to easily interact and exchange information online, they have also created several opportunities for adversaries to prey on users’ private information. Whether the motivation for data collection is commercial, where service providers sell data for marketers, or political, where a government censors, blocks and tracks its people, or even personal, for cyberstalking purposes, there is no doubt that the consequences of personal information leaks can be severe. Low-latency anonymity networks have thus emerged as a solution to allow people to surf the Internet without the fear of revealing their identities or locations. In order to provide anonymity to users, anonymity networks route users’ traffic through several intermediate relays, which causes unavoidable extra delays. However, although these networks have been originally designed to support interactive applications, due to a variety of design weaknesses, these networks offer anonymity at the expense of further intolerable performance costs, which disincentivize users from adopting these systems. In this thesis, we seek to improve the network performance of low-latency anonymity networks while maintaining the anonymity guarantees they provide to users today. As an experimentation platform, we use Tor, the most widely used privacy-preserving network that empowers people with low-latency anonymous online access. Since its introduction in 2003, Tor has successfully evolved to support hundreds of thousands of users using thousands of volunteer-operated routers run all around the world. Incidents of sudden increases in Tor’s usage, coinciding with global political events, confirm the importance of the Tor network for Internet users today. We identify four key contributors to the performance problems in low-latency anonymity networks, exemplified by Tor, that significantly impact the experience of low-latency application users. We first consider the lack of resources problem due to the resource-constrained routers, and propose multipath routing and traffic splitting to increase throughput and improve load balancing. Second, we explore the poor quality of service problem, which is exacerbated by the existence of bandwidth-consuming greedy applications in the network. We propose online traffic classification as a means of enabling quality of service for every traffic class. Next, we investigate the poor transport design problem and propose a new transport layer design for anonymous communication networks which addresses the drawbacks of previous proposals. Finally, we address the problem of the lack of congestion control by proposing an ATM-style credit-based hop-by-hop flow control algorithm which caps the queue sizes and allows all relays to react to congestion in the network. Our experimental results confirm the significant performance benefits that can be obtained using our privacy-preserving approaches.

CARVALHO, L. L. Inibição do mTOR agrava a mucosite intestinal induzida por irinotecano. 2016. 118 f. Dissertação (Mestrado em Farmacologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-02-06T15:50:42Z No. of bitstreams: 1 2016_dis_llcarvalho.pdf: 2848340 bytes, checksum: aaf346e9f648dc008eef1d64977af996 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-02-06T15:50:51Z (GMT) No. of bitstreams: 1 2016_dis_llcarvalho.pdf: 2848340 bytes, checksum: aaf346e9f648dc008eef1d64977af996 (MD5) / Made available in DSpace on 2017-02-06T15:50:51Z (GMT). No. of bitstreams: 1 2016_dis_llcarvalho.pdf: 2848340 bytes, checksum: aaf346e9f648dc008eef1d64977af996 (MD5) Previous issue date: 2016-10-21 / INTRODUCTION. Colorectal cancer (CRC) is one of the most prevalent neoplasms worldwide, being one of the leading causes of death from cancer. In Brazil, it is the third most frequent cancer in men and second in women. In the last decades new drugs incorporated into the clinical protocols for the treatment of mCRM have provided a significant increase in the survival of patients with this type of malignancy. Among these drugs, irinotecan occupies a prominent place and is commonly used as the first line in the treatment of metastatic RCC. However, since all chemotherapy treatments have a burden, a quarter of patients receiving irinotecan have a severe intestinal mucositis as a more noticeable side effect. Deregulation of cell signaling pathways is certainly one of the main reasons for the development of cancer. The mammalian rapamycin target protein (mTOR) is responsible for cell growth and the PI3K / Akt / mTOR axis is often dysregulated in several types of cancer. Thus, this work focused on investigating the role of mTOR during experimental intestinal mucositis induced by irinotecan. METHODS. C57BL / 6 male mice (n = 6 / group) were given intraperitoneally with saline or irinotecan (75 mg / kg or 45 mg / kg) for four consecutive days and treated with rapamycin or everolimus (mTOR inhibitors, 1.5 mg / Kg and 3 mg / kg, respectively) followed by irinotecan administration. Periodically the body weight variation and scores were evaluated for the degree of diarrhea. After euthanasia of the animals, ileus samples were collected for determination of myeloperoxidase activity (MPO), histopathology and morphometric analysis and cytokine levels measurement [KC (human IL-8 analogue chemokine), IL-1β, TGF-β And IFN-γ]. Gene expression of the mRNA of the PI3K / Akt / mTOR pathway proteins was performed by qRT-PCR. Blood was also collected from the animals to evaluate the total white blood cell count. The ANOVA / Bonferroni or Kruskal-Wallis / Dunn tests were used for statistical analysis. P <0.05 was accepted as significant. The present work was approved by the ethics committee of UFC (protocol number 100/14). RESULTS. Animals injected with irinotecan 75 mg / kg showed an increase in the gene expression of PI3K, Akt and mTOR mRNA evidencing the participation of the pathway in iatrogeny. Irinotecan 75 mg / kg significantly reduced body weight, total leukocyte count, villus / crypt ratio and promoted a characteristic intestinal lesion when compared to the saline control group. In addition, inhibition of mTOR with rapamycin or everolimus generally aggravated most of these parameters, such as weight loss, diarrhea, histopathology, neutrophil infiltration, and inflammatory mediators such as TGF-β and IFN-γ. CONCLUSION. Inhibition of mTOR aggravates irinotecan-induced intestinal mucositis probably by increasing the pro-inflammatory response via TGF-β and IFN-γ with important involvement of Paneth's Cells. Financial support: CNPq. / INTRODUÇÃO. O câncer colorretal (CCR) é uma das neoplasias mais prevalentes em todo o mundo, sendo uma das principais causas de óbito por câncer. No Brasil, apresenta-se como o terceiro câncer mais frequente nos homens e segundo nas mulheres. Nas últimas décadas novas drogas incorporadas à protocolos clínicos para o tratamento do CCRm tem proporcionado um aumento significativo na sobrevida de pacientes com esse tipo de malignidade. Dentre estas drogas o irinotecano ocupa lugar de destaque e é comumente utilizado como primeira linha no tratamento do CCR metastático. Entretanto, como todo tratamento quimioterápico há um ônus, um quarto dos pacientes em tratamento com irinotecano apresentam uma grave mucosite intestinal como efeito colateral mais notório. A desregulação de vias de sinalização celular certamente é uma das principais razões para o desenvolvimento do câncer. A proteína alvo da rapamicina em mamíferos (mTOR) é responsável pelo crescimento celular e o eixo compreendido por PI3K/Akt/mTOR está frequentemente desregulada em vários tipos de câncer. Dessa forma, esse trabalho focou em investigar o papel do mTOR durante a mucosite intestinal experimental induzida pelo irinotecano. MÉTODOS. Camundongos machos C57BL/6 (n=6/grupo) foram administrados intraperitonialmente com salina ou irinotecano (75 mg/kg ou 45 mg/kg) durante quatro dias consecutivos e tratados com rapamicina ou everolimo (inibidores de mTOR, 1,5 mg/kg e 3 mg/kg, respectivamente) seguidos da administração de irinotecano. Periodicamente foram avaliados a variação de peso corpóreo e escores para avaliação do grau de diarreia. Após a eutanásia dos animais, amostras de íleo foram coletadas para determinação da atividade de mieloperoxidase (MPO), histopatologia e análise morfométrica e dosagem dos níveis de citocinas [KC (quimiocina análoga da IL-8 humana), IL-1β, TGF-β e IFN-γ]. A expressão gênica do RNAm das proteínas da via PI3K/Akt/mTOR foi realizada por qRT-PCR. Também foi colhido sangue dos animais para avaliação da contagem total de leucócitos. Os testes de ANOVA/Bonferroni ou Kruskal-Wallis/Dunn foram usados para as análises estatísticas. P<0,05 foi aceito como significativo. O presente trabalho foi aprovado pelo comitê de ética da UFC (número de protocolo Nº100/14). RESULTADOS. Os animais injetados com irinotecano 75 mg/kg apresentaram um aumento na expressão gênica do RNAm de PI3K, Akt e mTOR evidenciando a participação da via na iatrogenia. O irinotecano 75 mg/kg reduziu significativamente o peso corpóreo, contagem total de leucócitos, razão vilo/cripta e promoveu uma lesão intestinal que lhe é característico quando comparado ao grupo controle salina. Além disso, de forma geral, a inibição do mTOR com rapamicina ou everolimo agravou a maioria desses parâmetros, tais como perda de peso, diarreia, histopatologia, infiltração de neutrófilos e mediadores inflamatórios como TGF-β e IFN-γ. CONCLUSÃO. A inibição do mTOR agrava a mucosite intestinal induzida por irinotecano provavelmente pelo aumento da resposta pro-inflamatória via TGF-β e IFN-γ com participação importante das Células de Paneth. Apoio financeiro: CNPq.