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Orientador: Jörg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T01:34:02Z (GMT). No. of bitstreams: 1 Santos_MarcosTadeudos_D.pdf: 15943486 bytes, checksum: 39810fdf0ace76e5e8963354bdc460ca (MD5) Previous issue date: 2011 / Resumo: O objetivo deste projeto foi estudar genes ativados em células do estroma da medula óssea, induzidos pela co-cultura com blastos leucêmicos, na tentativa de uma melhor compreensão sobre o crostalk entre estas células no microambiente tumoral. Nós identificamos Stanniocalcina-1 (STC1) como um potencial marcador molecular do microambiente tumoral, uma vez que sua expressão foi aumentada cerca de 7 vezes em células do estroma co-cultivadas com blastos leucêmicos primários. STC1 humana é uma glicoproteína secretada e tem sido descrita participando em diferentes processos fisiológicos, incluindo a angiogênese, hipóxia e principalmente, a carcinogênese. Nós produzimos a proteína recombinante STC1 no sistema baculovírus e também anticorpos monoclonais, usados em um ensaio ELISA, que agora será testado como um novo kit de diagnóstico de leucemia por uma empresa brasileira. Além disso, identificamos novos parceiros de interação para STC1 através do sistema de duplo hibrido em levedura sendo que algumas destas interações foram confirmadas por GST-pull down. A região Nterminal foi identificada como sendo a região responsável pela interação de STC1 com seus parceiros. Estudos de localização sub-celular por microscopia, revelaram uma deposição ubíqua citoplasmática e puntiforme nuclear, lembrando corpúsculos nucleares relacionados a SUMOilação. Embora STC1 interaja com a proteína SUMO1 e tenha uma predição de alta probabilidade para ser SUMOilada, ensaios in vitro e in vivo não conseguiram detectar STC1 SUMOilada. No entanto, observamos que STC1 regula a SUMOilação de forma significativa em três outras proteínas. Essas descobertas sugerem um novo papel para STC1 no ciclo de SUMOilação, agindo como uma SUMO E3 ligase. Observamos também que STC1 possui um receptor na membrana plasmática em linhagem de células leucêmicas K562 e que a incubação de STC1 com outras células leucêmicas parece favorecer a proliferação destas células ao passo que estimula uma maior produção da própria STC1 intracelular em células do estroma. Juntos, todos estes resultados abrem novas pistas promissoras a serem exploradas no futuro, uma vez que todos os resultados mostram ligações interessantes com estudos funcionais anteriores em STC1 / Abstract: The aims of this project is to study upregulated genes on bone marrow stromal cells, induced by the co-culture with leukemic blasts, trying to have a better understand about the crosstalk between these cells in the tumor microenvironment. We identified Stanniocalcin-1 (STC1) as a putative molecular marker for the leukemic microenvironment, once its expression was increased around 7 times in stromal cells co-cultivated with primary leukemic blasts. Human STC1 is a secreted glycoprotein that has been implicated in different physiological process, including angiogenesis, hypoxia and mainly in carcinogenesis. We produced the recombinant protein STC1 in baculovirus system and monoclonal antibodies for an ELISA assay that now will be tested as a new leukemia diagnostic kit by a Brazilian company. Moreover, we identified new interacting protein partners for STC1 by yeast two hybrid system and some of these interactions were confirmed by GST-pull down assays. The N-terminal region was mapped to be the region that mediates the interaction between STC1 and its partners. Microscopic subcellular localization, revealed an ubiquitous cytoplasmic and dot-like nuclear deposition, resembling SUMOylation related nuclear bodies. Although STC1 interacts with SUMO-1 and has a high theoretical prediction score for a SUMOylation site, in vitro and in vivo assays could not detect STC1 SUMOylation. However, we found that STC1 significantly regulates the SUMOylation of three other proteins. These ??ndings suggest a new role for STC1 in SUMOylation cycle, acting as a SUMO E3 ligase. We either observe that STC1 has a plasmatic membrane receptor in K562 leukemic cell lines and the incubation of STC1 with other leukemic cells suggest a increase of proliferation of these cells and stimulates the production of more intracellular STC1 at stromal cells. Together, all of these findings open promising new avenues to be explored in future detailed studies, since they all show interesting connections with previous functional studies on STC1 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Au sein des cellules, les ARNms sont liés par de nombreuses protéines, générant ainsi des particules appelées mRNPs (Ribonucléoparticules de messagers). Leur formation est cotranscriptionnelle, et leur composition va réguler l’ensemble des étapes du métabolisme des ARNms : stabilité, maturation, export, localisation et traduction. Au vu de l’importance de ces mécanismes dans la physiologie cellulaire, le contenu protéique des mRNPs est finement régulé dans le temps et l’espace et fait l’objet de nombreux remodelages. Ces changements de composition dépendent notamment des hélicases, ainsi que des modifications post-traductionnelles ; cependant, ces mécanismes demeurent à caractériser de façon plus approfondie. Une modification post-traductionnelle susceptible de moduler ces remaniements depuis la levure S. cerevisiae jusqu’aux métazoaires est la sumoylation. En effet, la SUMO-protéase Ulp1/SENP2, une enzyme clé de la machinerie de sumoylation, est localisée au panier des pores nucléaires, à proximité d’une plateforme d’ancrage des mRNPs destinées à l’export. Par ailleurs, il a été rapporté chez la levure que des mutants affectant la localisation et la stabilité d’Ulp1 présentent des défauts d’export et de localisation des mRNPs. Au vu de ces données, le laboratoire s’est intéressé aux rôles potentiels de la sumoylation dans le métabolisme de ces particules d’ARNm. Dans ce but, un crible protéomique a été réalisé chez la levure S. cerevisiae afin de comparer la composition des mRNPs entre des cellules sauvages ou mutantes pour Ulp1. Ce crible a mis en évidence un rôle d’Ulp1 dans le recrutement de deux composants des mRNPs, le complexe THO et l’hnRNP Hek2. Le complexe THO est un facteur multiprotéique qui participe à la prévention de l’instabilité génique et contribue à la transcription des ARNms, à l’assemblage des mRNPs et à leur export. L’hnRNP Hek2 est une protéine aux rôles multiples, dont l’association à un ARNm est susceptible de moduler sa stabilité, sa traduction et/ou sa localisation. Des analyses biochimiques nous ont permis de mettre en évidence l’existence de formes sumoylées de la sous-unité Hpr1 du complexe THO ainsi que de l’hnRNP Hek2. Toutes deux sont Ulp1-dépendantes, et interviennent sur la partie C-terminale de ces protéines. Nous avons également mis en évidence que chacune de ces sumoylations contrôle le recrutement de son substrat au sein des mRNPs. L’analyse fonctionnelle d’un mutant affectant la sumoylation d’Hpr1 a identifié cette modification comme nécessaire au recrutement du complexe THO sur une population d’ARNms impliqués dans la résistance au stress acide, autrement dégradés par l’exosome. Ainsi, l’absence de sumoylation d’Hpr1 diminue fortement la viabilité cellulaire en conditions de stress, un phénotype supprimé par l’inactivation de l’exosome. L’étude des effets de la sumoylation d’Hek2 suggère une modulation par SUMO de certaines de ses fonctions, notamment dans la localisation cellulaire des ARNms. L’ensemble de ces données fournit donc les deux premiers exemples de régulation du métabolisme des mRNPs par des événements de sumoylation intervenant au niveau du pore nucléaire. / Within the cells, mRNAs are associated to proteins, thereby generating particles called mRNPs (messenger ribonucleoproteins). mRNPs form in a cotranscriptional manner and their composition defines the fate of mRNAs by modulating the different steps of their metabolism, including their stability, their processing, their export, their localisation and their translation. In view of the importance of such mechanisms for cell physiology, several mechanisms ensure a tight spatio-temporal control of mRNPs composition through multiple mRNP remodelling events. These changes in the protein content of mRNPs depend on helicases and post-translational modifications, but remain to be further investigated. Sumoylation is one of the modifications that could contribute to mRNPs remodelling from yeast (S. cerevisiae) to metazoans. Indeed, it has been reported that the SUMO-protease Ulp1/SENP2, a key enzyme of the sumoylation machinery, is localized at the basket of nuclear pore complexes, in close vicinity with mRNPs committed for export. This particular localization, together with the reported defects in mRNPs export and localisation of yeast mutants affecting Ulp1, prompted the lab to ask whether sumoylation could contribute to mRNP biogenesis. In order to investigate this hypothesis, our lab compared mRNPs composition between wild-type and ulp1 mutant S. cerevisiae yeast strains using a proteomic approach. This screen identified two mRNP components that depend on Ulp1 for their recruitment onto these particles: the THO complex and the hnRNP Hek2. The THO complex is a multi-subunit factor that prevents genome instability and contributes to transcription, mRNP assembly and export. Hek2 has multiple functions in mRNA stability, translation and/or localization. Using biochemical approaches, we have been able to visualize sumoylated versions of the Hpr1 subunit of the THO complex and of the hnRNP Hek2. In both cases, this modification depends on Ulp1 activity and occurs on the C-terminal part of the protein. We further showed that these sumoylation events control THO and Hek2 recruitment onto mRNPs. Functional analysis of a mutant impairing Hpr1 sumoylation revealed that this modification is required for proper recruitment of the THO complex onto a subset of mRNAs involved in acidic stress resistance, which are otherwise degraded by the exosome. Decreased Hpr1 sumoylation results in a strong reduction of viability in acid stress conditions, a phenotype that is rescued by inactivation of the exosome. The investigation of the role of Hek2 sumoylation in mRNPs metabolism suggests that this modification regulates some of Hek2 functions, especially in mRNA localisation. All together, these results provide the two first examples of mRNPs components whose functions are regulated by sumoylation events occurring at the level of nuclear pores.

The consequences of traffic congestion include increased travel time, fuel consumption, and the number of crashes. Studies suggest that most traffic delays are due to nonrecurring traffic congestion. Adaptive traffic control using real-time data is effective in dealing with nonrecurring traffic congestion. Many adaptive traffic control algorithms used today are deterministic and prone to human error and limitation. Reinforcement learning allows the development of an optimal traffic control policy in an unsupervised manner. We have implemented a reinforcement learning algorithm that only requires information about the number of vehicles and the mean speed of each incoming road to streamline traffic in a four-way intersection. The reinforcement learning algorithm is evaluated against a deterministic algorithm and a fixed-time control schedule. Furthermore, it was tested whether reinforcement learning can be trained to prioritize emergency vehicles while maintaining good traffic flow. The reinforcement learning algorithm obtains a lower average time in the system than the deterministic algorithm in eight out of nine experiments. Moreover, the reinforcement learning algorithm achieves a lower average time in the system than the fixed-time schedule in all experiments. At best, the reinforcement learning algorithm performs 13% better than the deterministic algorithm and 39% better than the fixed-time schedule. Moreover, the reinforcement learning algorithm could prioritize emergency vehicles while maintaining good traffic flow.

O presente trabalho de doutorado tem por objetivo expor e comentar a tese do prazer (voluptas, em latim, edoné, em grego) como o sumo bem (summum bonum), conforme a defesa do humanista romano Lorenzo Valla (1407-1457), inscrita no seu diálogo De Voluptate (Do Prazer), publicado pela primeira vez em 1431. Nesta obra, que foi alvo de inúmeras polêmicas entre pensadores humanistas e escolásticos de seu tempo, a Europa do Renascimento, Valla propõe um debate entre um representante estoico e um epicurista acerca da questão sobre o fim último que dirige nossas ações, dito também o sumo bem. Como representante do partido da virtude ou do honesto (o honestum ou a honestas), o estoico, Valla escolhe ninguém menos do que um dos maiores representantes do republicanismo italiano do renascimento, o chanceler de Florença Leonardo Bruni (1370-1444). Do lado oposto, como representante do partido do prazer (a voluptas), isto é, o epicurista, ele escolhe ninguém menos do que o autor do primeiro volume de epigramas satíricos da Renascença, o poeta panormitano Antonio Beccadelli (1394-1471). Através desse debate, Valla procura rebater as críticas feitas por escolásticos e humanistas ao modelo de vida epicurista, pejorativamente chamado por seus detratores de vida dos agricultores ou rústicos, por oposição ao modelo de vida estoico do homem de negócios públicos, compromissado com deveres (officia) muito elevados, pois que visam a proteção e glória de sua cidade. / The purpose of this dissertation is to expose and comment on the thesis of pleasure (voluptas, in Latin, edoné, in Greek) as the highest good (summum bonum), according to the Roman humanist Lorenzo Valla\' defense (1407-1457) inscribed in his dialogue De Voluptate (On Pleasure), published for the first time in 1431. In this work, which was the subject of numerous controversies between humanists and other scholastics of his time in Renaissance Europe, Valla proposes a debate between a Stoic representative and an Epicurean on the question about the ultimate end that directs our actions, also said the highest good. As a representative of the party of virtue or of the honest (honestum or honestas), that is to say, the Stoic, he chooses no one but one of the greatest representatives of the Italian republicanism of the Renaissance, the chancellor of Florence, Leonardo Bruni (1370-1444). On the opposite side, as the representative of the pleasure party (voluptas), that is, the Epicurean, he chooses no one but the author of the first volume of satirical epigrams of the Renaissance, the panormitan poet, Antonio Beccadelli (1394-1471). Through this debate, Valla seeks to counter scholastic and humanist criticisms of the \"epicurean\" model of life, pejoratively called life of \"farmers or rustics\" by his detractors, as opposed to the \"stoic\" model of public business man, committed to very high duties (officia), as they seek the protection and glory to his city.

MDM2 and MDMX are two major negative regulators of tumor suppressor p53. Both MDM2 and MDMX can inactivate p53 and play important roles in mouse embryonic development in a p53 dependent manner. MDM2 possesses ubiquitin E3 ligase activity and mediates self-ubiquitination as well as ubiquitination and degradation of p53 by proteasome. We identify MDMX as another ubiquitin E3 ligase substrate of MDM2. MDM2 promotes the ubiquitination and degradation of MDMX through proteasome pathway. The RING domains of both MDMX and MDM2 are required and sufficient for MDM2-mediated MDMX ubiquitination. ARF overexpression, DNA damage or MDM2 overexpression can all stimulate MDMX ubiquitination and degradation. We present evidence that MDMX is also sumoylated. The sumoylation sites on MDMX are identified. ARF N-terminus is required for stimulating both MDMX ubiquitination and sumoylation. We also demonstrate that MDMX binds to ARF in an MDM2-dependent fashion.

PML et les corps nucléaires (CN) sont impliqués dans la défense antivirale. En effet, notre équipe a montré que la surexpression de PMLIII confère la résistance au virus de la stomatite vésiculaire, au virus de l'influenza, au virus foamy mais pas au virus de l'encéphalomyocardite (EMCV). J'ai montré dans mon travail de thèse que l'EMCV contrecarre le pouvoir antiviral de PMLIII en induisant sa dégradation par un processus dépendant du protéasome et de SUMO. Cependant, les cellules de souris invalidées pour PML sont plus sensibles à l'infection par l'EMCV que les cellules issues de souris parentales. Pour déterminer l'isoforme de PML responsable de cet effet antiviral, j'ai analysé l'effet des sept isoformes de PML (PMLI-VII) et j'ai montré que seule l'expression en stable de PMLIV confère la résistance à l'EMCV en séquestrant la polymérase virale 3Dpol au sein des CN PML. De plus la déplétion de PMLIV augmente la production de l'EMCV dans les cellules traitées par l'interféron. Ces données indiquent le mécanisme par lequel PML confère la résistance à l'EMCV et révèlent que PML est l'une des protéines médiatrices des effets anti-EMCV de l'interféron.

Idrottsparken roundabout in Norrkoping is located in the more dense part of the city.Congestion occurs in peak hours causing queue and extended travel time. This thesis aims to provide alternative model to reduce queue and travel time. Types ofobservation data are flow, length of queue, and travel time that are observed during peakhours in the morning and afternoon. Calibration process is done by minimising root meansquare error of queue, travel time, and combination both of them between observation andcalibrated model. SUMO version 0.14.0 is used to perform the microsimulation. There are two proposed alternatives, namely Scenario 1: the additional lane for right turnfrom East leg to North and from North leg to West and Scenario 2: restriction of heavy goodsvehicles passing Kungsgatan which is located in Northern leg of Idrottsparken roundaboutduring peak hours. For Scenario 1, the results from SUMO will be compared with AIMSUNin terms of queue and travel time. The result of microsimulation shows that parameters that have big influence in the calibrationprocess for SUMO are driver imperfection and driver’s reaction time, while for AIMSUN isdriver’s reaction time and maximum acceleration. From analysis found that the model of thecurrent situation at Idrottsparken can be represented by model simulation which usingcombination between root mean square error of queue and travel time in calibration andvalidation process. Moreover, scenario 2 is the best alternative for SUMO because itproduces the decrease of queue and travel time almost in all legs at morning and afternoonpeak hour without accompanied by increase significant value of them in the other legs. Thecomparison between SUMO and AIMSUN shows that, in general, the AIMSUN has higherchanges value in terms of queue and travel time due to the limited precision in SUMO forroundabout modelling.

Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer predisposition. Cell lines derived from FA patient exhibit chromosomal instability and sensitivity to DNA interstand crosslinkers (ICLs) like mitomycin (MMC). The key event in Fanconi anemia pathway is the regulated ubiquitination and deubiquitination of FANCD2 and FANCI. Upon DNA damage, FANCD2 and FANCI are monoubiquitinated by FA core complex. They then move into the chromatin and serve as the landing site for downstream players, like FANCP/SLX4 and FAN1. USP1, the deubiquitinating enzyme (DUB), removes ubiquitin from FANCD-Ub/FANCI-Ub, and this step is required for the integrity of FA pathway. This dissertation addresses how USP1 is regulated in the cell. In Chapter 2, we discovered UAF1/WDR48 as a critical binding partner for USP1, by activating its enzymatic activity in vitro and in vivo. We then generated DT40 knockout cell lines of USP1 and UAF1. We showed that USP1/UAF1 complex is functionally required for homologous recombination (HR). Interestingly, PCNA-Ub is also a substrate for USP1. We discovered that hELG1, through its binding to USP1/UAF1 complex, regulates the deubiquitination of PCNA-Ub and translesion DNA synthesis (TLS). Then in Chapter 3, we discovered a tandem repeat of SUMO-like domains (SLD1 and SLD2) in the C terminus of UAF1. SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 of UAF1 or mutation of the SIM of FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination. The SLD2 sequence of UAF1 also binds to a SIM on hELG1, and targets the USP1/UAF1 complex to its PCNA-Ub substrate. We proposed the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates HR and TLS. Originating from USP1/UAF1 complex, we worked out a general mechanism of DUB regulation by WD40 proteins, which involved in two more DUBs, USP12 and USP46 (discussed in Chapter 4 and Appendix A). Lastly in Chapter 5, through bioinformatic analysis we identified a series of novel proteins containing ubiquitin-binding zinc fingers (UBZ). We then focused on SNM1A and FAAP20/C1orf86, and characterized their function in DNA crosslink repair.

Activated islet specific T cells are central to the destructive autoimmune response observed in type 1 diabetes (T1D). Not surprisingly, intense focus is placed on understanding how autoreactive T cell responses arise and contribute to disease pathology in the hope of using this information to develop novel therapeutic strategies for treatment of T1D. Here we investigate the mechanisms underlying defective c-Maf binding to the IL-4 promoter in T cells from diabetes prone mice and identify the mechanisms responsible for suppression of T cells by the inhibitory receptor Ly49A. It is not clear why development of protective Th2 cells is poor in T1D. c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. Here we demonstrate that, despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblots demonstrate that c-Maf can be modified at lysine 33 by small ubiquitin-like modifier-1 (SUMO-1). Sumoylation is facilitated by direct interaction with the E2 conjugating enzyme Ubc9 and increases following T cell stimulation. In addition, c-Maf physically interacts with p65/RelA. This interaction is dependent on the DNA binding domain of c-Maf and phosphorylation of p65 at serine 536. In transfected cells, overexpression of SUMO-1 or p65 decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation (ChIP) assays and enhances c-Maf localization into promyelocytic leukemia nuclear bodies (PML-NBs) or nucleoli, respectively. Sumoylation of c-Maf and phosphorylation of p65 are increased in NOD CD4 cells compared to CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation and interaction with p65 contribute to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene. Islet specific CD4 cells expressing inhibitory receptors may be a useful therapeutic tool for treating T1D. Engagement of transgenic Ly49A inhibits CD4 cell activation and delays onset of T1D in mice. However, in vitro studies suggest the inhibitory effect of Ly49A is incomplete. Here we report that following simultaneous T cell receptor (TCR) and Ly49A engagement, phosphorylation of Zap70, Erk1/2 and c-Jun were significantly diminished. Kinetic studies indicated that Ly49A did not simply delay activation but had a long-lasting effect. In contrast, when only costimulatory signals were provided through CD28, Ly49A engagement did not block p38 MapK or Akt phosphorylation. Likewise, expression of the downstream targets Bcl-xl and Baff were unaffected. Together these data suggest that engagement of Ly49A selectively inhibits signals downstream of the TCR but spares those unique to CD28. These results suggest that when considering its use as an immunotherapy, the potency of inhibitory receptors such as Ly49A may be further improved by pairing them with costimulatory blockade. Take together, these studies suggest that abnormal post-translational regulation of c-Maf function is a novel marker of altered T cell function in T1D and use of inhibitory receptors such as Ly49A may be optimized combining this approach with other complementary therapies.