Le cancer épithélial de l’ovaire est le cancer gynécologique le plus létal. La survie à 5 ans est de 30-40% chez les patientes atteintes d’une tumeur invasive (TOV), comparativement à 95% chez les patientes diagnostiquées pour une tumeur à faible potentiel de malignité ou borderline (LMP). Au laboratoire, l’analyse de l’expression des gènes de la micropuce à ADN U133 d’Affymetrix a révélé que la SERPINA1 est un gène dont l’expression varie entre les tumeurs LMP et TOV. La validation par Q-PCR nous a confirmé que cette antiprotéase est majoritairement surexprimée dans les tumeurs LMP, par rapport aux tumeurs bénignes (BOV) et aux tumeurs TOV. Nous avons donc surexprimé la SERPINA1 dans les lignées cellulaires invasives TOV 112D et TOV 1946 du cancer de l’ovaire et dérivé des clones stables. Les résultats obtenus nous indiquent que la surexpression de la SERPINA1 a un effet sur la capacité d’invasion et de migration cellulaire et non au niveau de la croissance cellulaire et la formation de structures tridimensionnelles. Les résultats issus de l’étude in vivo dans les souris SCID nous permettront de déterminer si la surexpression de la SERPINA1 a un effet sur la tumorigénèse ovarienne. Ainsi, la SERPINA1 demeure à notre avis un candidat d’intérêt pour tenter de mieux comprendre les différences biologiques entre les tumeurs LMP et TOV, ainsi que le rôle des protéases et de leurs inhibiteurs dans la progression tumorale du cancer de l’ovaire. / Epithelial ovarian cancer is the most lethal gynecologic cancer with a five-year survival rate of 30-40% in patients diagnosed with high-grade invasive disease (TOV). This is in stark contrast to the 95% five-year survival in patients diagnosed with low malignant potential (LMP) disease. It is therefore important to understand the biological differences between LMP and TOV. We have previously identified differential expression of SERPINA1 between serous LMP and TOV tumors through gene expression analysis using Affymetrix U133 DNA microarrays. Expression of this protease inhibitor in the majority of LMP tumors was confirmed and validated by Q-PCR. To study the effects of its overexpression on the invasive potential of ovarian cancer cell lines, SERPINA1 was cloned in the pcDNA3.1+ plasmid and stable clones were derived from two invasive ovarian cancer cell lines, TOV 112D and TOV 1946. Comparisons between clones and controls have shown no SERPINA1-dependent difference in cellular growth or spheroid formation. However, effects on cellular migration and invasion are observed in cells overexpressing SERPINA1. Results from an in vivo xenograft study in SCID mice will allow us to determine if SERPINA1 overexpression affects ovarian tumorigenesis. SERPINA1 remains an interesting candidate gene whose further characterization may lead to insights into its role, and the role of proteases and their inhibitors, in ovarian cancer disease progression.

Le cancer épithélial de l’ovaire est le cancer gynécologique le plus létal. La survie à 5 ans est de 30-40% chez les patientes atteintes d’une tumeur invasive(TOV), comparativement à 95% chez les patientes diagnostiquées pour une tumeur à faible potentiel de malignité (LMP). Au laboratoire, l’analyse de l’expression des gènes de la micropuce à ADN HuFL d’Affymetrix a révélé que Cks1 est un gène dont l’expression varie entre les tumeurs LMP et TOV. En effet, ce régulateur du cycle cellulaire est surexprimé dans les tumeurs TOV par rapport aux tumeurs LMP. Nous avons donc déplété Cks1 dans des lignées cellulaires tumorales invasives du cancer de l’ovaire dérivées au laboratoire, soit la TOV112D et la TOV1946, en utilisant des shRNAs sous le contrôle d’un répresseur inductible à la tétracycline. Puis, nous avons dérivé des clones stables inductibles à la tétracycline. Les résultats obtenus nous indiquent que la déplétion de Cks1 n’a pas d’effet sur la prolifération et la migration cellulaires, ni sur la formation de structures tridimensionnelles in vitro. Ainsi, nous pouvons conclure que Cks1 ne joue pas un rôle clé dans la progression tumorale par rapport aux paramètres testés. Or, des études supplémentaires seraient nécessaires pour expliquer les différences biologiques observées entre les deux types de tumeurs étudiées, et justifier cette variation observée de l’expression de Cks1. / Epithelial ovarian cancer is the most lethal gynecologic cancer with a five-year survival rate of only 30-40% in patients diagnosed with high-grade invasive disease (TOV). This contrasts with the 95% five-year survival in patients diagnosed with the low malignant potential (LMP)disease. Previously, we have identified differential expression of Cks1 between serous LMP and TOV tumors through gene expression analysis using Affymetrix HuFL DNA microarrays. Overexpression of this cell cycle regulator was observed in the TOV tumors, but not in the LMP samples. To study its role on the invasive potential of ovarian cancer cell lines, Cks1 was depleted in two tumoral invasive ovarian cancer cell lines established in our laboratory, TOV112D and TOV1946, using an inducible shRNA strategy. Then, tetracycline-inducible stable clones were derived and studied further. Comparisons between clones and controls have shown no Cks1-dependent effect on cellular growth, neither in migration capacity nor spheroid formation. Thus, we can conclude that Cks1 does not play a crucial role in the tested parameters for cancer progression, but further experiments could elucidate the biological differences observed between the two kinds of tumors studied.

Le cancer épithélial de l’ovaire est le plus létal des cancers gynécologiques. Les tumeurs de l’ovaire se divisent en différentes classes reflétant l’étendue de la maladie. Les tumeurs à faible potentiel de malignité présentent une survie relative à 5 ans de 90%, alors que pour les tumeurs invasives, la survie à 5 ans chute drastiquement à 35-40%. Au laboratoire, nous avons précédemment identifié la protéine Ran, un membre de la superfamille des GTPases Ras, comme marqueur fortement exprimé dans les cancers épithéliaux de l’ovaire de haut grade et de haut stade dont la surexpression est associée à un mauvais pronostic. Ran est déjà connue pour contribuer au transport nucléocytoplasmique et à la progression du cycle cellulaire, mais son rôle dans le cancer ovarien n’est pas bien défini. En utilisant une approche de shRNA inductibles à la tétracycline basée sur les lentivirus, nous avons montré que la diminution de l’expression de Ran dans des lignées cellulaires agressives du cancer de l’ovaire affecte drastiquement la prolifération cellulaire par l’induction d’une apoptose caspase-3 dépendante. Par un essai de tumeurs en xénogreffes, nous avons démontré que la déplétion de Ran résulte en une diminution de la tumorigenèse et que la formation éventuelle de tumeurs est associée à une sélection des cellules tumorales ayant la capacité de ré-exprimer la protéine Ran. Ces résultats suggèrent un rôle critique pour Ran dans la survie et la tumorigénicité des cellules du cancer ovarien, indiquant que Ran pourrait être une cible thérapeutique intéressante. / Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Malignant epithelial tumors can be divided into different classes reflecting the extent of the disease. Low malignant potential (LMP) tumors have a 5 years survival rate of 90-95%. For invasive cancers (TOVs), the survival rate drops dramatically to 35-40%. In the laboratory, we previously identified that Ran protein, a member of the Ras GTPase family, is highly expressed in high grade and high stage serous epithelial ovarian cancers, and that its over-expression is associated with a poor prognosis. Ran is known to contribute to both nucleocytoplasmic transport and cell cycle progression, but its role in ovarian cancer is not well defined. Using a lentivirus-based tetracycline inducible shRNA approach, we show that down-regulation of Ran expression in aggressive ovarian cancer cell lines drastically affects cellular proliferation by inducing a caspase-3 dependent apoptosis. Using a xenograft tumor assay, we demonstrate that depletion of Ran results in decreased tumorigenesis, and eventual tumor formation is associated with the selection of tumor cells able to re-express the Ran protein. These results suggest a critical role for Ran in ovarian cancer cell survival and tumorigenicity and suggest that this critical GTPase may be suitable as a therapeutic target.

Le mélanome malin est l’un des cancers les plus mortels dont l’incidence continue à augmenter chaque année avec peu de traitement efficace à long terme. Il est causé et initié principalement par l’exposition excessive aux rayons ultraviolets engendrant des photoproduits hautement génotoxiques. Il est bien connu que la cascade de signalisation PI3K/Akt joue un rôle crucial dans la régulation des processus qui sont généralement dérégulés durant le développement tumoral comme la prolifération, le contrôle du cycle cellulaire et l’apoptose. Néanmoins, l’implication de cette voie moléculaire dans la réponse aux dommages à l’ADN est peu caractérisée. Chez les mammifères, trois isoformes de la protéine kinase Akt ont été identifiées: Akt1, Akt2 et Akt3. Bien qu’elles soient très homologues en termes de séquence, plusieurs études ont montré que ces isoformes ont des fonctions biologiques distinctes, et nous suggérons qu’elles puissent contribuer différemment à la régulation de la réponse génotoxique. Les objectifs de ce projet étaient de: (i) évaluer l’activation d’Akt dans les cellules de mélanomes (ii) déterminer l’impact de l’inhibition de cette activité sur la régulation de la réponse cellulaire aux UV (iii) vérifier si la perte d’expression de l’un ou de l’autre des isoformes d’Akt peut réguler la réponse aux UV. Nous avons démontré qu’Akt est transitoirement hyperactivée par phosphorylation suite aux irradiations UV dans les lignées cellulaires de mélanomes. Afin de déterminer l'importance de cette activation dans la réponse cellulaire aux UV, notre approche était de diminuer (i) la phosphorylation d’Akt par l’usage d’inhibiteurs pharmacologiques ou (ii) l’expression de chaque isoforme d’Akt par l’approche des ARN interférents. Nous avons montré que l’inhibition de la phosphorylation d’Akt amène à l’augmentation du taux de l’apoptose induit par les UV d’une manière isoforme spécifique, alors qu’elle n’a aucun effet sur la régulation de la voie de réparation par excision de nucléotides (NER), qui est la seule voie humaine pour éliminer les dommages à l’ADN induits par les UV. En somme, notre étude constitue un nouvel aspect qui permet de mieux comprendre les mécanismes moléculaires du développement de mélanomes malins suites aux irradiations ultraviolettes. / Malignant melanoma is one of the deadliest cancers whose incidence continues to rise each year with a few effective long-term treatments. It is caused and initiated mainly by excessive exposure to ultraviolet radiation generating highly genotoxic DNA photoproducts. It is well known that the PI3K/Akt signaling cascade plays a crucial role in the regulation of processes commonly deregulated in tumor development such as proliferation, cell cycle control and apoptosis. Nevertheless, the nuclear involvement of this molecular pathway in the genotoxic response is poorly characterized. In mammals, three Akt kinase isoforms have been identified: Akt1, Akt2 and Akt3. Although these exhibit a high degree of homology, several studies have shown that they have distinct biological functions; therefore, we suggest that these isoforms may contribute differently to the regulation of genotoxic response. The objectives of this project were to: (i) evaluate Akt activation in UV-irradiated melanoma cells, (ii) determine the effect of the Akt phosphorylation inhibition on the regulation of the cellular response to UV, (iii) evaluate whether the loss of the expression of one or more of Akt isoforms can regulate the cellular response to UV. We demonstrated that Akt undergoes transient hyperactivation after UV treatment in melanoma cell lines. To determine the importance of this activation, our approach was to reduce (i) the phosphorylation of Akt by the use of pharmacological inhibitors or (ii) the expression of each individual Akt isoform using RNA interference. We have shown that inhibition of Akt phosphorylation leads to increased rates of UV-induced apoptosis in an isoform specific manner, while exerting no effect on regulation of nucleotide excision repair (NER), the only human pathway for eliminating UV-induced DNA damage. In summary, our study provides a better understanding of the molecular mechanisms of malignant melanoma development in response to UV.

4-1BB (CD137) est un membre de la superfamille TNFR qui est impliqué dans la transmission des signaux de survie aux lymphocytes. TRAF1 est une protéine adaptatrice qui est recrutée par 4-1BB et autres TNFRs et est caractérisée par une expression très restreinte aux lymphocytes, cellules dendritiques et certaines cellules épithéliales. TRAF1 est nécessaire pour l’expansion et la survie des cellules T mémoire en présence d'agonistes anti-4-1BB in vivo. De plus, TRAF1 est requise en aval de 4-1BB pour activer (phosphoryler) la MAP kinase Erk impliquée dans la régulation de la molécule pro-apoptotique Bim. Suite à l’activation du récepteur 4-1BB, TRAF1 et ERK sont impliqués dans la phosphorylation de Bim et la modulation de son expression. L’activation et la régulation de TRAF1 et Bim ont un rôle important dans la survie des cellules T CD8 mémoires. Dans cette étude, nous avons utilisé une approche protéomique afin de pouvoir identifier de nouveaux partenaires de liaison de TRAF1. Utilisant cette stratégie, nous avons identifié que LSP1 (Leukocyte Specific Protein 1) est recruté dans le complexe de signalisation 4-1BB de manière TRAF1 dépendante. Une caractérisation plus poussée de l’interaction entre TRAF1 et LSP1 a montré que LSP1 lie la région unique N-terminal de TRAF1 de façon indépendante de la région conservée C-terminal. À l’instar des cellules T déficientes en TRAF1, les cellules T déficientes en LSP1 ne sont pas capables d’activer ERK en aval de 4-1BB et par conséquent ne peuvent pas réguler Bim. Ainsi, TRAF1 et LSP1 coopèrent en aval de 4-1BB dans le but d’activer ERK et réguler en aval les niveaux de Bim dans les cellules T CD8. Selon la littérature, le récepteur 4-1BB n’est pas exprimé à la surface des cellules B murines, mais le récepteur 4-1BB favorise la prolifération et la survie des cellules B humaines. Cependant, il est important d'étudier l'expression du récepteur 4-1BB dans les cellules B murines afin de disposer d'un modèle murin et de prédire la réponse clinique à la manipulation de 4-1BB. En utilisant différentes stimulations de cellules B murines primaires, nous avons identifié que le récepteur 4-1BB est exprimé à la surface des cellules B de souris suite à une stimulation avec le LPS (Lipopolysaccharides). Une caractérisation plus poussée a montré que le récepteur 4-1BB est induit dans les cellules B murines d'une manière dépendante de TLR4 (Toll Like Receptor 4). Collectivement, notre travail a démontré que la stimulation avec le LPS induit l’expression du récepteur 4-1BB à la surface des cellules B murines, menant ainsi à l'induction de TRAF1. De plus, TRAF1 et LSP1 coopèrent en aval de 4-1BB pour activer la signalisation de la Map kinase ERK dans les cellules B murines de manière similaire aux cellules T. Les cellules B déficientes en TRAF1 et les cellules B déficientes en LSP1 ne sont pas en mesure d'activer la voie ERK en aval de 4-1BB et montrent un niveau d’expression du récepteur significativement diminué comparé aux cellules B d’une souris WT. Ainsi, TRAF1 et LSP1 sont nécessaires pour une expression maximale du récepteur 4-1BB à la surface cellulaire de cellules B murines et coopèrent en aval de 4-1BB afin d'activer la cascade ERK dans les cellules B murines. / 4-1BB (CD137) is a member of the TNFR superfamily, which is involved in the transmission of survival signals in lymphocytes. TRAF1 is an adapter protein that is recruited by 4-1BB and other TNFRs and is characterized by a very restricted expression in lymphocytes, dendritic cells and some epithelial cells. TRAF1 is necessary for the expansion and survival of memory T cells in the presence of anti-4-1BB agonist in vivo. Also, TRAF1 is required downstream of 4-1BB to activate (phosphorylate) the MAP kinase ERK involved in the regulation of the proapoptotic molecule Bim. Upon activation of 4-1BB, TRAF1 and ERK are involved in the phosphorylation of Bim and modulation of its expression. The activation and regulation of TRAF1 and Bim have an important role in the survival of CD8 memory T cells. In this study, we used a proteomic approach in order to identify new TRAF1 binding partners. Using this strategy, we have identified that LSP1 (Leukocyte Specific Protein 1) is recruited to the 4-1BB signaling complex in a TRAF1-dependent manner. It has been shown that LSP1 is a target protein for signaling ERK / MAP kinase. Further characterization of the interaction between TRAF1 and LSP1 has shown that LSP1 binds the N-terminal unique region independently of the conserved C-terminal of TRAF1. Like the T cells deficient in TRAF1, T cells deficient in LSP1 are not capable of activating ERK downstream of 4-1BB and therefore cannot regulate Bim levels in T cells. Thus, TRAF1 and LSP1 cooperate downstream of 4-1BB in order to activate ERK and regulate Bim levels in murine CD8 T cells. According to the literature, the 4-1BB receptor is not expressed on the surface of murine CD19+ B cells, but 4-1BB activation promotes the proliferation and survival of human CD19+B cells. However, it is important to study the expression of 4-1BB receptor in murine B cells to have a murine model and predict the clinical response to the manipulation of 4-1BB. Using different stimulation of primary murine CD19+B cells, we have found that the 4-1BB receptor is expressed on the surface of murine B cells in response to LPS (lipopolysaccharide) stimulation. Further characterization showed that the 4-1BB was induced in murine CD19+B cells in a TLR4-dependent manner (Toll like Receptor 4). Collectively, our work has shown that the stimulation with LPS induces the expression of 4-1BB on the surface of murine B cells leading to the induction of TRAF1. Also, TRAF1 and LSP1 cooperate downstream of 4-1BB to activate the signaling of the Map kinase ERK in murine B cells similarly to T cells. Similarly, as for the T cells, TRAF1-/- B cells or LSP1-/- B cells are not able to activate the ERK pathway downstream of 4-1BB. In addition, the B cells deficient in either TRAF1 or LSP1 show a level of expression of the 4-1BB receptor significantly decreased compared to B cells from a WT mouse. Thus, TRAF1 and LSP1 are required for maximal expression of the 4-1BB receptor on the cell surface of murine B cells and cooperate downstream of 4-1BB to activate the ERK cascade in the murine B cells.

Malignant lymphomas are tumors arising by clonal proliferation of lymphocytes stopped at a specific stage of differentiation. All tumor cells arising from the original clone thus share the same characteristics and that can be used in their detection. Finding a suitable molecular marker of tumor cells is an essential step not only to disease diagnosis, but also for monitoring of minimal residual disease. Minimal residual disease is defined as the subclinical disease level, which malignant cells are not detectable for conventional cytological methods during the therapy. These residual cells can cause relapse. The main goals of the diploma thesis are a detection and analysis of immunoglobulin genes rearrangement and chromosomal translocation t(11; 14) in the MTC region, and a development and optimization of RQ-PCR system for detection of minimal residual disease. Quantification of clonal rearrangement or chromosomal translocation allows the detection of minimal residual disease level in patients with malignant lymphomas. Clonal immunoglobulin genes rearrangement or characteristic chromosomal translocation were analyzed in 19 patients with malignant lymphomas. There were analyzed individual gene segments, N-region and combination variability in immunoglobulin genes rearrangement. There was developed...

In all of the developed countries, malignant neoplasms are, along with cardiovascular diseases, among the most frequent causes of death. This tendency persists in the European Union countries for many years. The goal of this diploma thesis is to analyze the development of mortality caused by selected malignant neoplasms in the European Union countries from the year 1996 until 2010. The analysis is divided into 4 sections. Aside from standard demographic methods of evaluating the mortality rates by means of age-adjusted death rates calculation, statistical methods are used in this thesis as well. Primary methods used are Joinpoint regression, analysis of the course of specific death rates according to age groups and cluster analysis. In the observed period of time, mortality caused by stomach, cervical, and within the male population also respiratory tract malignant neoplasms declines. On the contrary, within the female population, the death rate caused by respiratory tract malignant neoplasms increases. Malignant skin melanoma represent a great future danger concerning the male population, and malignant pancreatic neoplasms concerning both genders. Over the observed time period, malignant skin melanoma death rate almost doubled in some of the countries. Within the European Union, the variations...

The skin has a system that can detect light in a fashion similar to the retina. Although its presence was initially reported almost 20 years ago, only in 2011 functional studies started to be reported. The biological clock of the skin has also been reported in the beginning of the century, but its function and relevance still remain unexplored. Thus, this Ph.D. project was designed to explore the functionality of both systems in melanocytes, and whether the disruption of these systems is associated with the development of melanoma cancer. Using in vitro, in vivo, and bioinformatics approaches, we have shown that: 1) the biological clock of malignant melanocytes is more responsive to visible light, UVA radiation, estradiol, and temperature compared to normal cells; 2) UVA radiation is detected by melanopsin (OPN4) and rhodopsin (OPN2), which triggers a cGMP related cascade that leads to immediate pigment darkening (IPD) in normal and malignant melanocytes; 3) in addition to detecting UVA radiation, OPN4 also senses thermal energy, which activates the biological clock of both normal and malignant melanocytes; 4) regarding the biological clock, we have provided several layers of evidence that proves that in melanoma a chronodisruption scenario is established compared to healthy skin and/or normal pigment cells; 5) in vivo tumor samples display a low amplitude circadian rhythm of clock gene expression and an ultradian oscillatory profile in melanin content; 6) a non-metastatic melanoma leads to a systemic chronodisruption, which we suggest that could favor the metastatic process; 7) in human melanoma, we demonstrated the role of BMAL1 as a prognostic marker and a putative marker of immune therapy success. Taken altogether, these results significantly contributed to the literature as it brought to light new and interesting targets and processes, which will be explored in future projects / A pele possui um sistema que pode detectar luz de forma análoga à retina. Embora a presença deste sistema tenha sido inicialmente descrita quase há 20 anos, apenas no ano de 2011 estudos funcionais começaram a ser relatados. Sabe-se que o relógio biológico da pele também foi identificado no início do século, mas sua função e relevância ainda continuam pouco exploradas. Diante deste cenário, este projeto de doutorado foi desenhado para investigar a funcionalidade de ambos os sistemas em melanócitos e se perturbação dos mesmos estaria associada com o desenvolvimento de melanoma. Através do uso de abordagens in vitro, in vivo e de bioinformática, nós demonstramos que: 1) o relógio biológico de melanócitos malignos é mais responsivo à luz visível, radiação UVA, estradiol e temperatura comparado ao de células normais; 2) a radiação UVA é detectada por melanopsina (OPN4) e rodopsina (OPN2), que ativam uma via de sinalização dependente de GMPc, levando ao processo de pigmentação imediata (IPD) em melanócitos normais e malignos; 3) além de detecção de radiação UVA, a OPN4 também detecta energia térmica que, por sua vez, ativa o relógio biológico de melanócitos normais e malignos; 4) relativo ao relógio biológico, provamos por diferentes abordagens que, no melanoma, um cenário de cronoruputura está estabelecido em comparação a pele saudável e/ou melanócitos; 5) tumores in vivo apresentam um ritmo circadiano de baixa amplitude na expressão dos genes de relógio e um ritmo ultradiano oscilatório no conteúdo de melanina; 6) um melanoma não metastático leva a um quadro sistêmico de cronoruptura, o qual sugerimos favorecer o processo de metástase; 7) em melanoma humano, demonstramos o papel do gene BMAL11 como marcador de prognóstico e um possível indicador de sucesso de imunoterapias. Portanto, este projeto contribuiu de forma significante para a literatura científica uma vez que trouxe à luz novos e interessantes alvos terapêuticos e processos, os quais serão explorados em projetos futuros

A hipertensão essencial tem alta prevalência mundial, bem como, causas genéticas e ambientais. Na busca de correlações genéticas para a hipertensão, foi descrito um potencial papel do DARC (Duffy Antigen Receptor of Chemokines) como receptor de Interleucina-8 em endotélio e que essa interação poderia contribuir para a patogênese da pré-eclampsia. O DARC está expresso em vários tecidos além da linhagem eritróide, em especial nas células endoteliais. A glicoproteína DARC carreia determinantes antigênicos e também é receptora para Plasmodium vivax, tendo relevância biológica significante. Esse estudo teve como objetivo estudar a freqüência fenotípica e genotípica do Sistema de Grupo Sangüíneo Duffy comparando pacientes com hipertensão maligna com doadores de sangue normotensos. Foram estudadas 43 amostras de sangue de pacientes com diagnóstico de hipertensão maligna da Unidade de Hipertensão do Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. O grupo controle foi constituído por 100 amostras de doadores de sangue da Fundação Pró-Sangue/Hemocentro de São Paulo. Em todas as amostras foi realizada a fenotipagem Duffy, a genotipagem DUFFY e a dosagem de IL-8 sérica. A fenotipagem foi realizada pela técnica em tubo. Na genotipagem DUFFY, foram estudadas as mutações 125G>A, 265C>T, 29 G>A e -33T>C pela técnica de PCR-RFLP. Na análise da freqüência alélica encontramos que o alelo FYB-33 foi o mais observado no grupo de pacientes com hipertensão maligna com diferença estatisticamente significante (p= 0,0191). Conseguimos demonstrar uma correlação entre níveis elevados de IL-8 em pacientes com hipertensão maligna e genótipo FYB- 33/FYB-33 (p=0,003). Também observamos níveis elevados de IL-8 nos pacientes com hipertensão maligna quando comparados com o grupo controle (doadores de sangue), p<0,001. Esses resultados indicam que a IL-8 tem papel potencial na fisiopatologia da hipertensão maligna por apresentar um efeito regulatório inibitório em pacientes Duffy negativo. / Essential hypertension has a high prevalence worldwide and the has genetic and environment causes. Searching genetics correlation for hypertension, a potential role of DARC (Duffy Antigen Receptor of Chemokines) was described as an Interleukine-8 receptor in endothelium and that this interaction might contribute for the pathogenesis of pre-eclampsia. DARC is expressed in many tissues beyond the erythrocyte lineage, in special endothelium cells. DARC glycoprotein carries antigens determinants and is also Plasmodium vivax receptor, with a significantly biological relevance. This study had as objective to verify the phenotypic and genetic frequencies of the Duffy Blood Group System in patients with malignant hypertension and, to compare them with norm tension blood donors. Forty three patients from the Hypertension Service of the InCor of Clinical Hospital of School of Medicine of the University of São Paulo and 100 blood donors from Fundação Pró-Sangue/Hemocentro de São Paulo were studied. Duffy phenotyping and genotyping and IL-8 serum dosage was performed in all samples. Phenotyping tests were performed by tube technique. We studied the 125 G>A, 265 C>T, 298 G>A and -33 T>C mutations by PCR RFLP genotyping. The FYB-33 allele was the most observed in the malignant hypertension patients group with p= 0.0191. We have shown a correlation in high between high levels of IL-8 in patients with malignant hypertension and FYB-33/FYB-33 genotype (p=0,003). We observed also high levels of IL-8 in patients with malignant hypertension when the control group (blood donors) was compared, p<0.001. This results indicate that IL-8 has a potential role in malignant hypertension physiopathology due to an regulatory inhibitory effect in Duffy negative patients.

O carcinoma epidermóide oral (CEO) tem alta incidência e mortalidade no mundo, sendo frequentes casos de recidivas em função da resistência aos tratamentos convencionais. Essa resistência tem sido associada à presença de células tronco tumorais (CTT). A terapia fotodinâmica (PDT) tem sido utilizada nos casos de desordens orais potencialmente malignas (DOPM) e CEOs superficiais, com relativo índice de sucesso. Contudo, são relatados casos de recidivas, que sugerem certa resistência à PDT. O objetivo do presente trabalho foi avaliar se células de DOPM desenvolvem resistência à PDT mediada pelo ácido 5-aminolevulínico (5-ALA), tal qual ocorre com células de CEO expostas ao mesmo tratamento, bem como verificar se as células displásicas e neoplásicas orais exibem um fenótipo sugestivo de células tronco tumoral. Foram cultivadas células epiteliais displásicas orais (linhagem DOK) e células de CEO de língua (linhagem SCC9), divididas nos seguintes grupos experimentais: Grupo Controle - células sem tratamento; Grupo ALA - células tratadas com 5-ALA, com concentração e tempo de incubação igual ao do grupo PDT; Grupo LED - células tratadas com LED, com dosimetria igual ao do grupo PDT; Grupo PDT - células tratadas com PDT mediada pelo 5-ALA, subdivididas em duas gerações: geração 1 - células expostas a somente um ciclo de PDT; geração 4 - células expostas a quatro ciclos de PDT e que exibiram resistência (maior viabilidade) à dose inicial aplicada para a geração 1. Para esses grupos, foram realizados ensaios de formação de colônias e de invasão, bem como citometria de fluxo para verificar a expressão de CD44, ESA e p75NTR. Observou-se que todos os grupos expostos à PDT contendo células da linhagem DOK exibiram resistência à dose inicial de PDT, porém em intensidade menor àquela observada para a linhagem SCC9. As células resistentes à PDT de ambas as linhagens exibiram maior potencial de formação de colônias, mas somente a primeira geração de células exibiu maior potencial de invasão. Na análise de citometria de fluxo, observou-se que as células resistentes à PDT de ambas as linhagens exibiram superexpressão de CD44high/ESAhigh. A linhagem SCC9 exibiu ainda maior frequência de células CD44high/ESAlow e CD44high/p75NTR+, sugerindo a presença de uma subpopulação com alto potencial de invasão e resistente aos tratamentos convencionais. Não houve diferenças significativas na frequência de células ESAhigh/p75NTR+ em ambas as linhagens. Concluiu-se que as células displásicas orais desenvolvem resistência à PDT com intensidade menor se comparada às células SCC9. Em ambas as linhagens, as células resistentes exibiram um fenótipo sugestivo de células tronco tumorais. Esse fato deve ser considerado ao se instituir a PDT mediada pelo 5-ALA em lesões displásicas e neoplásicas orais que demandam múltiplos ciclos de tratamento. / Oral squamous cell carcinoma (SCC) has a high incidence and mortality in the world. There are frequent cases of relapses due to its resistance to conventional treatments. This resistance has been associated with the presence of cancer stem cells (CSC). Photodynamic therapy (PDT) has been used in cases of potentially malignant oral lesions (PMOL) and superficial SCC\'s, with a relative success rate. However, relapses are reported, which suggest some resistance to PDT. The aim of the present study was to evaluate whether PMOL cells develop resistance to PDT mediated by the 5- aminolevulinic acid (5-ALA), as occurs with SCC cells exposed to the same treatment, as well as to verify if dysplastic and oral neoplastic cells exhibit a suggestive phenotype of CSC. Oral dysplastic epithelial cells (DOK lineage) and CSC cells of the tongue (SCC9 lineage) were divided into the following experimental groups: Control Group - cells with no treatment; Group ALA - 5-ALA treated cells, with concentration and incubation time equal to that of the PDT group; LED group - LED light treated cells, with dosimetry equal to that of the PDT group; PDT group - cells treated with PDT mediated by the 5-ALA, subdivided into two generations: Generation 1 - cells exposed to only one PDT cycle; Generation 4 - cells exposed to four PDT cycles and exhibiting resistance (greater viability) to the initial dose applied for Generation 1. Colony forming and invasion assays, as well as flow cytometry to verify the expression of CD44, ESA and p75NTR were performed to the experimental groups. It was observed that all groups exposed to PDT containing DOK lineage cells exhibited resistance to the initial dose of PDT, but at a lower intensity than those observed for the SCC9 lineage. PDTresistant cells from both lineages exhibited increased potential for colony formation, but only the first generation of cells exhibited increased invasion potential. In flow cytometric analysis, PDT-resistant cells from both lineages exhibited overexpression of CD44high / ESAhigh. SCC9 lineage also exhibited higher frequency of CD44high / ESAlow and CD44high / p75NTR+ cells, suggesting the presence of a subpopulation with high invasion potential and resistant to conventional treatments. There were no significant differences in the frequency of ESAhigh / p75NTR+ cells in both lineages. It was concluded that the oral dysplastic cells develop resistance to PDT, with lower intensity when compared to SCC9 cells. In both lineages, the resistant cells exhibited a phenotype suggestive of CSC. This fact should be considered when establishing PDT mediated by the 5-ALA in oral dysplastic and neoplastic lesions that require multiple cycles of treatment.