O papilomavírus humano (HPV) é o principal agente etiológico do câncer cervical e anogenital, sendo o HPV16 e o HPV18 os vírus de alto risco. Estudos recentes evidenciaram que além da transmissão sexual do HPV, há outras formas de contágio. Entretanto, a dificuldade na obtenção de quantidades viáveis do tipo selvagem ou mutante do HPV tem limitado em muito os estudos de diversos aspectos da biologia do papilomavírus. Este estudo investigou a possibilidade de o HPV infectar células leucocitárias do sangue periférico humano. Concluímos que as VLPs L1L2 do HPV16 podem utilizar a via endocítica do ferro mediada por clatrina, através do complexo VLPs-Transferrina-Receptor de Transferrina, permanecendo de forma latente em leucócitos. Esta porta de entrada oportunista poderia explicar a propagação crescente e alarmante deste agravo à saúde humana, motivo de preocupação nos sistemas mundiais de saúde pública. Este trabalho demonstrou pela primeira vez a internalização de VLPs L1L2 do HPV16 em leucócitos do sangue periférico humano. / Human papillomavirus (HPV) is the primary etiologic agent of anogenital and cervical cancer, caused mainly by the high-risk HPV16 and HPV18 viruses. Recent studies revealed that besides the sexual transmission of HPV, there are other forms of contagion. However, the difficulty in obtaining quantities of viable wild-type or mutant of HPV constitutes a limiting factor in the studies of various aspects of the biology of human papillomavirus. This study investigated the possibility of HPV infect the cells of human peripheral blood leukocytes. We conclude that the VLPs L1L2 of HPV16 may use the iron endocytic pathway clathrin-mediated through the complex VLPs-Transferrin-Transferrin Receptor and remained so latent in leukocytes. This port of entry opportunist could explain the growing and alarming spread of this disease to human health, cause for concern in the global public health. This study showed for the first time the internalization of VLPs L1L2 of HPV16 in human peripheral blood leukocytes.

Im ersten Teil meiner Arbeit habe ich den molekularen Mechanismus beschrieben, mit dem endogene neuronale Vorläuferzellen antitumorigen gegen Gliomstammzellen wirken. Unsere Forschungsgruppe hat in bereits veröffentlichten Arbeiten gezeigt, dass neuronale Vorläuferzellen zu experimentellen Gehirntumoren migrieren und Tumorzelltod induzieren können. In der nun vorliegenden Arbeit zeige ich, dass die neuronalen Vorläuferzellen nicht nur benefiziell gegen die Hauptpopulation der Tumorzellen wirken, sondern darüber hinaus auch die kleinere Population der sehr aggressiven Tumorstammzellen – mittels Sekretion von BMP7 – supprimieren. Insgesamt zeigt meine Arbeit, dass neuronale Vorläuferzellen die Pathogenität der Gliomstammzellen unterdrücken. Im zweiten Teil meiner Arbeit habe ich einen zellautonomen Mechanismus untersucht, der Gliomzellen in vitro und in vivo vermehrt expandieren lässt. Meine Ergebnisse zeigen, dass die Familie der ets-Transkriptionsfaktoren Gliomzellen zur Proliferation anregen, indem sie die Expresion eines Eisentransporters (dem Transferrin-Rezeptor-1) induzieren und damit die intrazelluläre Akkumulation von Eisenionen begünstigen. Die Veränderung des Redox-Gleichgewichts in den Gliomzellen regt die Tumore zu verstärkter Sekretion von Glutamat an. Dadurch werden die Gliome sehr zytotoxisch und induzieren Zelltod in den Zellen des tumorumgebenden Parenchyms. Das untergegangene Nervengewebe schafft damit den Platz, den der Tumor zur Expansion braucht. Insgesamt zeigt meine Arbeit, dass die ets1-induzierte CD71 Expression nicht nur das Tumorwachstum befördert, sondern auch den Platz zum Tumorwachstum schafft. / In my first part,Gliomas cells with stem-like properties (GSCs) control tumor growth and recurrence. Here, I showed that endogenous neural precursor cells (NPCs) perform an anti-tumor response by specifically targeting GSCs: In vitro, NPCs predominantly expressed BMP7; BMP7 was constitutively released from neurospheres and induced canonical BMP-signaling in GSCs. Exposure of human and murine GSCs to neurosphere-derived BMP7 increased GSC differentiation, attenuated GSC-marker expression, GSC self-renewal and the ability for tumor initiation.This anti-tumor response of NPCs protect the brain from gliomas by releasing BMP7, which acts as a paracrine tumor suppressor that represses proliferation, self-renewal and tumor-initiation of GSCs. In the 2nd part, Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, I showed that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and increases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused NMDA mediated reduction of neuron cell mass. Overall my results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass.

Metazoans have evolved conserved mechanisms to promote cell survival under low oxygen tensions by initiating a transcriptional cascade centered on the action of Hypoxia Inducible transcription Factors (HIFs). In aerobic conditions, HIFs are inactivated by ubiquitin-proteasome-mediated degradation of their a subunit, which is dependent on prolyl hydroxylation by 2-oxoglutarate (2-OG) and Fe(II)-dependent prolyl hydroxylases (PHDs). In hypoxia, HIF-$\alpha$ is no longer hydroxylated and is therefore stabilised, activating a global transcriptional response to ensure cell survival. Interestingly, HIFs can also be activated in aerobic conditions, however the mechanisms of this oxygen-independent regulation are poorly understood. Here, I have explored the role of the vacuolar H+-ATPase (V-ATPase), the major proton pump for acidifying intracellular vesicles and facilitating lysosomal degradation, in regulating HIF-$\alpha$ turnover. Unbiased forward genetic screens in near-haploid human cells identified that disruption of the V-ATPase leads to activation of HIFs in aerobic conditions. Rather than preventing the lysosomal degradation of HIF-$\alpha$, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-$\alpha$ isoforms by altering the intracellular iron pool and preventing HIF-$\alpha$ prolyl hydroxylation. In parallel, I characterised two putative mammalian V-ATPase assembly proteins, TMEM199 and CCDC115, identified by the forward genetic screen and subsequent mass spectrometry analysis. I confirmed that both TMEM199 and CCDC115 are required for V-ATPase function, and established assays to determine how TMEM199 and CCDC115 associate with components of the core V-ATPase complex. Lastly, to measure how V-ATPase activity leads to changes in the labile iron pool, I developed an endogenous iron reporter using CRISPR-Cas9 knock-in technology. This approach confirmed that iron homeostasis is impaired during V-ATPase inhibition, and demonstrated that exogenous ferric iron can restore the labile iron pool in a transferrin-independent manner. Together my studies highlight a crucial link between V-ATPase activity, iron homeostasis, and the hypoxic response pathway.

L’érythropoïèse est le processus de différentiation d’un progéniteur érythroïde multipotent en globules rouges. La différentiation érythroïde est essentiellement contrôlée par le récepteur à l’érythropoïétine (EPOR). Nous avons montré que le récepteur à la transferrine de type 2 (TFR2) est un membre important du complexe formé par l’EPOR. Le TFR2 présente, comme l’EPOR une expression restreinte qui dépend du type cellulaire. Ainsi son expression n’a pu être détectée que dans le foie, l’érythron et l’intestin grêle. Le rôle du TFR2 a été exploré dans les hépatocytes et il a été montré qu’il joue le rôle d’un senseur de fer dans cette lignée et de ce fait contribue à l’homéostasie du fer. Nous avons déterminé le rôle du TFR2 dans les érythroblastes et montré que TFR2 est une protéine escorte de l’EPOR qui contribue à l’érythropoïèse in vitro et in vivo. De plus, nos travaux montrent que le TFR2 est requis pour la production de GDF15 (Growth Differentiation Factor 15) dans les érythroblastes. D’autre part nous avons démontré que la production de GDF15 est augmentée par l’EPO, la déplétion intracellulaire en fer et l’activité transactivatrice de P53. L’inhibition de l’expression de P53, réalisée au cours de l’étude de son rôle dans la production de GDF15, a révélé son implication dans l’érythropoïèse normale. Nous avons mis en évidence l’existence de plusieurs formes du TFR2. Deux d’entre elles résultent de l’utilisation de sites distincts d’initiation de la traduction. Ces deux isoformes sont régulée différemment au cours de la maturation des érythroblastes. La troisième isoforme, appelée TFR2 soluble (sTFR2), est relargée dans le plasma suite au clivage du TFR2. Nous avons montré que la production du sTFR2 est inhibée en présence du ligand de TFR2, la transferrine saturée en fer (holoTF) alors que le TFR2 est stabilisé dans ces mêmes conditions. Les rôles spécifiques des trois formes du TFR2 doivent encore être élucidés. / Erythropoiesis is the differentiation process of a multipotent erythroid progenitor into red blood cells. Erythroid differentiation is primarily controlled by the erythropoietin receptor (EPOR). We showed that the Transferrin receptor 2 (TFR2) is an important member of the EPOR complex. TFR2 has like EPOR a lineage-restricted expression and can solely be detected in the liver, erythron and small intestine. TFR2 function has been explored in hepatocytes where it plays the role of an iron sensor and contributes to iron homeostasis. We determined the role of TFR2 in erythroblasts and showed that TFR2 is an escort protein for EPOR that contributes to optimal erythropoiesis in vitro and in vivo. Moreover we evidenced that TFR2 is absolutely required for the production of Growth differentiation factor 15 (GDF15) in erythroblasts. We further demonstrated that GDF15 production is increased by EPO levels, by intracellular iron depletion as well as by P53 trans-activation activity. The inhibition of P53 expression, realized for the study of its role in GDF15 production, revealed its implication in normal erythropoiesis. We evidenced that TFR2 is expressed under several forms, two of which result from the utilization of distinct translational initiation sites. These two isoforms are differently regulated during erythroid maturation. The third form called soluble TFR2 (sTFR2) is released in the plasma after TFR2 cleavage. We showed that sTFR2 production is inhibited in the presence of TFR2 ligand, iron loaded transferrin (holoTF) whereas cell surface TFR2 expression is stabilized by holoTF. The specific roles of the three forms of TFR2 expressed by erythroblasts remain to be elucidated.

O papilomavírus humano (HPV) é o principal agente etiológico do câncer cervical e anogenital, sendo o HPV16 e o HPV18 os vírus de alto risco. Estudos recentes evidenciaram que além da transmissão sexual do HPV, há outras formas de contágio. Entretanto, a dificuldade na obtenção de quantidades viáveis do tipo selvagem ou mutante do HPV tem limitado em muito os estudos de diversos aspectos da biologia do papilomavírus. Este estudo investigou a possibilidade de o HPV infectar células leucocitárias do sangue periférico humano. Concluímos que as VLPs L1L2 do HPV16 podem utilizar a via endocítica do ferro mediada por clatrina, através do complexo VLPs-Transferrina-Receptor de Transferrina, permanecendo de forma latente em leucócitos. Esta porta de entrada oportunista poderia explicar a propagação crescente e alarmante deste agravo à saúde humana, motivo de preocupação nos sistemas mundiais de saúde pública. Este trabalho demonstrou pela primeira vez a internalização de VLPs L1L2 do HPV16 em leucócitos do sangue periférico humano. / Human papillomavirus (HPV) is the primary etiologic agent of anogenital and cervical cancer, caused mainly by the high-risk HPV16 and HPV18 viruses. Recent studies revealed that besides the sexual transmission of HPV, there are other forms of contagion. However, the difficulty in obtaining quantities of viable wild-type or mutant of HPV constitutes a limiting factor in the studies of various aspects of the biology of human papillomavirus. This study investigated the possibility of HPV infect the cells of human peripheral blood leukocytes. We conclude that the VLPs L1L2 of HPV16 may use the iron endocytic pathway clathrin-mediated through the complex VLPs-Transferrin-Transferrin Receptor and remained so latent in leukocytes. This port of entry opportunist could explain the growing and alarming spread of this disease to human health, cause for concern in the global public health. This study showed for the first time the internalization of VLPs L1L2 of HPV16 in human peripheral blood leukocytes.

No description available.

Introdução. A Anemia das Doenças Crônicas (ADC) é uma desordem comum em idosos, freqüentemente multifatorial, e exacerbada por citocinas pró-inflamatórias. Nesta população, o diagnóstico de anemia por deficiência de ferro (ADF) é difícil utilizando-se os testes laboratoriais convencionais, devido à prevalência destes estados crônicos. Espécies reativas de oxigênio (ERO) são produzidas por fagócitos durante o burst oxidativo em defesa do hospedeiro, mas são também implicadas como agentes deletérios em um grande número de desordens inflamatórias. Objetivos. Verificar a eficiência do receptor de transferrina sérico (sTfR) e índice sTfR-log ferritina (sTfR-F) no diagnóstico da ADF e ADC; determinar os efeitos das anemias e de estados inflamatórios crônicos sem anemia no burst oxidativo, fagocitose, produção de óxido nítrico (NO) por monócitos, e produção de ácido hipocloroso (HOCl) por neutrófilos. Métodos. Participaram do estudo cinqüenta e três indivíduos (42 mulheres e 11 homens) idosos recrutados do departamento de Cardio-Geriatria da Rede Pública de Saúde de Ribeirão Preto-SP. A proteína C reativa (PCR), utilizada como marcador inflamatório foi analisada pela metodologia ultra-sensível. O status férrico foi estabelecido pelos níveis do sTfR (enzimaimunoensaio, kit Quantikine soluble transferrin receptor, R&D Systems, USA), ferritina sérica (ensaio quimioluminescente, kit Ferritin Immulite®- DPC, England) e índice sTfR-F. Foram compostos os seguintes grupos, com base nos valores de normalidade dos parâmetros PCR, concentração de hemoglobina, ferritina e sTfR: controle (n=15), ADC (n=12), inflamação (n=08), anemia inexplicável (n=06) e grupo ADF (n=12). Nenhum paciente apresentou função renal prejudicada, hemoglobinopatias, diabetes mellitus descompensada, doenças infecciosas ou malignas. Estudou-se o efeito da ADC, ADF, anemia inexplicável e inflamação no burst oxidativo, fagocitose de neutrófilos e monócitos por citometria de fluxo, produção de HOCl por neutrófilos purificados e de NO produzido em cultura de monócitos em meio RPMI 1640, estimulados por lipopolissacáride (LPS). Resultados. Os níveis de sTfR foram capazes de diagnosticar ADF em sete (58,34%) dos doze pacientes do grupo ADF. A intensidade de fluorescência (IF) do burst oxidativo de neutrófilos, que reflete a atividade celular, no grupo ADF foi significativamente menor que a do grupo controle (p<0,05), mas a IF de monócitos não foi estatisticamente diferente. As percentagens de monócitos e neutrófilos realizando burst oxidativo e fagocitose dos grupos ADF, anemia inexplicável e inflamação não foram estatisticamente diferentes do grupo controle. As percentagens de monócitos e neutrófilos realizando fagocitose do grupo ADC foram estatisticamente maiores que as do grupo controle (p<0,05). A produção de HOCl nos grupos ADC e inflamação foram estatisticamente menores comparadas ao grupo controle (p<0,05), enquanto houve uma superprodução de NO por monócitos no grupo ADC (p<0,05). Conclusão. No presente estudo, o sTfR foi uma ferramenta diagnóstica adicional à ferritina no diagnóstico da ADF, mas o valor do índice sTfR-F não aumentou a utilidade diagnóstica do sTfR em indivíduos idosos. NO parece modular a produção de HOCl, provavelmente por regular a atividade enzimática da mieloperoxidase (MPO). Estes resultados mostraram o papel importante do ferro na ADC e ADF em manter a resposta imunológica de fagócitos durante o processo de envelhecimento. / Background: Anemia of Chronic Disease (ACD) is a very common disorder in elderly and it is often multifatorial, exacerbated by pro-inflammatory cytokines. In these people, the diagnostic of iron deficiency anemia (IDA) or iron deficiency is difficult in these patients using the conventional iron status tests, because of the prevalence of these chronic states. Reactive oxygen species (ROS) are produced by phagocytic cells during the oxidative burst in defense of the host, but it has also been implicated as a very harmful agent in an increasing number of inflammatory-mediated disorders. Objectives: to elucidate the use of serum transferrin receptor (sTfR) and sTfR-log ferritin index (TfR-F index) to diagnose IDA and ACD; to determinate the effects of anemia and chronic inflammatory states without anemia in some function of oxidative stress, such as oxidative burst, phagocytosis, nitric oxide (NO) production by monocytes and the hypochlorous acid (HOCl) production by purified neutrophils. Methods: Fifty three (42 women and 11 men) elderly subjects recruited from geriatric department of healthy public system of Ribeirão Preto city were selected. The high sensitivity C-Reactive Protein (CRP) was analyzed as an inflammatory marker. The iron status was confirmed by sTfR (enzyme-linked immunosorbent assay, ELISA, kit Quantikine soluble transferrin receptor R&D Systems, USA), serum ferritin levels (chemiluminescence assay, kit Ferritin Immulite®- DPC, England) and TfR-F index. The groups were established according to the criteria CRP assay, hemoglobin analysis, ferritin and sTfR: control (n =15), ACD (n =12), inflammation (n =08), unexplained anemia (n=06), and IDA (n=12). Patients with impaired kidney function, hemoglobinopathy, decompensate diabetes mellitus or infectious and malignancies diseases were excluded. We also studied the effect of ACD, IDA, unexplained anemia and inflammation on oxidative burst, phogocytosis of neutrophils and monocytes by flow cytometry and HOCl generation of neutrophils and NO produced by the monocytic cell culture, cultured in RPMI 1640 medium, stimulated by lipopolysaccharide (LPS). Results: Our results showed that sTfR could diagnose IDA in seven (58.34%) of twelve patients in IDA group. Oxidative burst fluorescence intensity of neutrophil in IDA group was lower statistically compared to control group, p<0.05. Oxidative burst fluorescence intensity of monocytes did not differ significantly. The percentages of monocytes and neutrophils expressing oxidative burst and the phagocytosis did not differ significantly amongst control, IDA, unexplained anemia and inflammatory groups. However, the percentages of neutrophils and monocytes expressing phagocytosis in ACD group were statistically higher when compared to control group (p<0.05). The HOCl generation in ACD, and inflammation groups were lower significant statistically than control group, p<0.05, while there was an overproduction of NO by monocyte in ACD group (p<0.05). Conclusion: In the present study, the sTfR was an additional diagnostic tool to ferritin for the diagnosis of IDA, but the value of the sTfR-F index did not increase the utility diagnostic of sTfR in elderly patients. NO seems modulate the HOCl production, perhaps by regulates the myeloperoxidase (MPO) enzyme activity. These results showed the important role of iron in ACD and IDA to maintaining phagocyte immune defense of organism during the aging process.

No description available.

Las hormonas recombinantes eritropoyetina (EPO) y hormona de crecimiento (GH), prácticamente iguales a las endógenas y de corta vida media en circulación, son de difícil detección directa en el control antidopaje. Se determinaron los valores poblacionales de los biomarcadores indirectos EPO, receptor soluble de la transferrina, insulin-like growth factor-I (IGF-I) y procolágeno tipo III péptido (P-III-P), en poblaciones seleccionadas de deportistas, y el efecto del ejercicio y los distintos tipos de entrenamiento sobre su concentración sérica. La comparación de resultados obtenidos mediante distintos ensayos demostró la necesidad de una validación exhaustiva previa a su utilización. A excepción del P-III-P, los biomarcadores séricos propuestos para la detección de rhEPO y rhGH no se encuentran directamente afectados por el nivel atlético, el ejercicio o la distinta carga de entrenamiento realizada a lo largo de la temporada deportiva. La edad es la principal influencia sobre las concentraciones séricas de IGF-I y P-III-P. / Direct detection of recombinant peptidic hormones erythropoietin (EPO) and growth hormone (GH), very similar to endogen molecules and with a short half life in blood, is difficult in antidoping control. The main objective of this work is to determine indirect biomarkers' values of EPO, soluble transferrin receptor, insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P), in selected populations of athletes, and the effect of exercise and different types of training on their concentration in serum. The comparison of results obtained by the different assays showed the need of extensive validation of the analytical techniques before their use in the antidoping field. Excepting P-III-P, proposed biomarkers for the detection of rhEPO and rhGH abuse are not directly influenced by the athletic level, exercise or different training workload along the sport season. Age is the main factor affecting IGF-I and P-III-P concentrations in serum.

No description available.