Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T14:23:59Z No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T14:24:19Z (GMT) No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) / Made available in DSpace on 2016-02-04T14:24:19Z (GMT). No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10 sobre a de TNF. Macrófagos derivados de medula óssea de camundongos deficientes no gene HO-1 têm menor carga parasitária, quando infectados por L. chagasi em comparação aos macrófagos de camundongos selvagens. Além disso, pacientes com LV apresentam maiores níveis de heme-oxigenase 1 e de heme no soro. Nossas observações indicam que heme é capaz de induzir necroptose em macrófagos humanos, e de que moléculas da via da necroptose estão associadas com a resistência na infecção por Leishmania. A molécula RIPK1 controla a replicação de Leishmania por um mecanismo independente da produção de IL-1β, enquanto que a molécula PGAM5 depende de IL-1βpara controlar o crescimento do parasita. Por fim, encontramos que essas proteínas participam do controle da replicação por Leishmania em um modelo experimental de Leishmaniose cutânea. Esses achados indicam um potencial deletério para a HO-1 na infecção por L. chagasi, e um papel protetor da necroptose na infecção porLeishmania. / Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to cell death pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania infantum chagasi infection, the main etiologic agent of human VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are involved in resistance to Leishmania infection. Our observations indicate that HO-1 is induced in macrophages infected with L. infantum chagasi and pharmacological induction for HO-1 by CoPP increases parasite load of infected macrophages and reduces production on inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that favors L. chagasi replication through a higher IL-10/TNF-α ratio in macrophages. We also observed that bone marrow derived macrophages knockout to HO-1 gene have a significant lower parasite load when infected by L. infantum chagasi than their wild type counterparts. Beyond this, we found that patients with VL presented higher systemic concentrations of HO- 1 and heme than healthy individuals. We found that heme is able to induce programmed necrosis “necroptosis” in human cells and that molecular players from necroptosis pathway contribute to resistance to Leishmania infection. RIPK1 controls Leishmania replication through a mechanism independent of IL-1β production, while PGAM5 requires IL-1β to control Leishmania replication. Finally, we found that RIPK1 and PGAM5 play an important role in controlling Leishmania replication in a cultaneous leishmaniasis experimental model. Our findings argue that HO-1 has a critical role in L. chagasi replication and necroptosis pathway is involved in resistance against Leishmania infection.

abstract: Since the molecular biology revolution in the 1980s, ease of gene editing had led to the resurgence of Oncolytic Virotherapy. Countless viruses have been engineered yet only three are approved for clinical use worldwide, with only one being approved by the U.S Food and Drug Administration (FDA). Vaccinia virus (VACV) has a large genome, contains many immune evasion genes and has been thoroughly studied, making it a popular candidate for an oncolytic platform. VACV mutants with deletions in the E3 immune evasion protein have been shown to have oncolytic efficacy but the mechanism of tumor selectivity has not been fully elucidated. These mutants have been shown to be regulated by the necroptosis pathway, a pathway that has been shown to be deficient in certain cancers. Using a pan-cancer screening method that combines dye exclusion assays, western blot analysis, and viral growth curve, the role of necroptosis in regulating VACV replication and oncolytic efficacy in cancer was further characterized. Results demonstrate a preliminary correlation between necroptosis, viral replication, and oncolytic efficacy. This correlation is clearest in breast cancer and melanomas yet may apply to other cancer subgroups. This data was also used to guide the development of a receptor-interacting protein kinase 3 (RIP3) matched pair mouse model in the E0771 mouse breast cancer line which can be used to further study the role of necroptosis and oncolytic efficacy in vivo. Understanding the contribution necroptosis plays in oncolytic efficacy can guide to design enhance the design of clinical trials to test VACV E3L mutants and may lead to better efficacy in humans and an improvement in clinical oncology. / Dissertation/Thesis / Masters Thesis Biology 2020

Oxidative cell death contributes to neuronal cell death in many neurological diseases such as stroke, brain trauma, and Alzheimer's disease. In this study, we explored the involvement of poly(ADP-ribose)-polymerase (PARP) in oxidative stress-induced necroptosis. We showed that PJ34, a potent and specific inhibitor of PARP, can completely inhibit glutamate-induced necroptosis in HT-22 cells. This protective effect was still observed 8 h after glutamate exposure followed by PJ34 treatment. These results suggest that PARP activation plays a critical role in glutamate-induced necroptosis. We also examined the interaction between PARP and a necroptosis inhibitor called necrostatin-1 (Nec-1). Previously, we showed that Nec-1 protects against glutamate-induced oxytosis by inhibiting the translocation of cellular apoptosis-inducing factor (AIF), a downstream target of PARP-1 activation. In this study, Nec-1 reduced PARP activity but had no effect on the expression of PARP-1 in cells treated with glutamate. Nec-1 also did not protect against cell death mediated by the PARP activator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), although PJ34 did protect against MNNG-mediated cell death. These findings suggest that Nec-1 is not a direct PARP inhibitor and that its signaling target is located upstream of PARP.

The inflammatory and anti-inflammatory macrophages have been implicated in many diseases including rheumatoid arthritis, inflammatory bowel disease and chronic rhinosinusitis. Recent studies suggest targeting macrophage function and activation may represent a potential target to treat these diseases. Herein, I investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of normal human pro- and anti-inflammatory macrophage subsets. It has been shown that human monocytes are highly susceptible to the cytotoxic effects of SMs, however, differentiated macrophages (M0) develop resistance to the cytocidal abilities of SMs. Whether human macrophage subsets are also resistant to the cytotoxic effects of SM remains unknown. My results show that differentiation of M0 macrophages towards M1 state rendered them highly susceptible to SM-induced cell death, whereas M2a, M2b and M2c differentiated subsets were resistant, with M2c being the most resistant. SM-induced cell death in M1 macrophages was mediated by apoptosis as well as necroptosis and activated both extrinsic and intrinsic pathways of apoptosis. The susceptibility of M1 macrophages to SM-induced cell death was attributed to the IFN-𝛾-mediated polarization as JAK inhibitor reversed their susceptibility. In contrast, M2c and M0 macrophages experienced cell death through necroptosis pathway following simultaneous blockage of the IAPs pathways by SM-LCL161 and the caspase pathways by the pan-caspase inhibitors (zVAD.fmk). I investigated the molecular mechanism governing SM-induced cell death in M1 macrophages. My results show that in contrast to the cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-⍺, the canonical and non- canonical NF-𝜅B pathways. The susceptibility of M1 macrophages to SM-induced cell death was found to be dependent on IFN-𝛾-mediated differentiation through the JAK-STAT pathway and subsequent activation of IRF-1. In addition, the selective cell death in SM-treated M1 macrophages is mediated by simultaneous degradation of cellular IAP-2 (cIAP-2) and RIPK-1/3 through the activation of mTORC signaling pathway. Overall, the results suggest that survival of human macrophages is critically linked to the activation of the IAPs pathways. Moreover, agents blocking cIAP-1/2, mTORC and IRF-1 can be exploited therapeutically to address inflammation-related diseases. These observations hold a promising therapeutic strategy to limit the activation of proinflammatory M1 macrophages and eventually controlling the M1-associated diseases.

Stroke is the third leading cause of death and disability in the United States. At present, intravenous administration of tissue plasminogen activator (t-PA) is the only thrombolytic therapy approved by the FDA for the treatment of acute ischemic stroke. There are no other effective treatments available so far. The discovery of new drugs and new treatments for stroke to reduce mortality and disability is an urgent medical research priority. In this study, the protective effects and mechanisms of two novel agents Gly14 humanin (HNG) and necrostatin-1 (Nec-1) were examined. HNG, a highly potent neuropeptide against amyloid toxicity, exhibited anti-apoptotic properties on cerebral ischemia injury. HNG reduced infarct volume after ischemia/reperfusion injury with pre-treatment or post-treatment (i.c.v. and i.p.) in a middle cerebral artery occlusion model in mice and decreased neurological deficits induced by ischemia. The protection of HNG was mediated by inhibiting ERK activation and activating PI3K/Akt pathway. Inhibition of the PI3K/Akt pathway blocked the protective effects of HNG. Nec-1 is a specific inhibitor of necroptosis, a newly identified cell death, and was reported to reduce infarct volume even when it was administered at 6 h post-ischemia in a mouse stroke model. Interestingly, this small molecule protected against glutamate-induced oxidative toxicity in a hippocampal HT-22 cell line. It inhibited the translocation of apoptosis-inducing factor from the mitochondria to the nucleus, increased the cellular glutathione level, and decreased free radical formation after glutamate treatment. More importantly, Nec-1 inhibited BNIP3-mediated caspase-independent cell death. Cerebral ischemia/reperfusion injury involves the activation of different pathways that lead to neuronal cell death. Given this multifactorial pathnogenesis, it is possible that a cocktail of neuroprotective agents would be superior to monotherapy. In this study, a cocktail of HNG and Nec-1 was examined in vitro and in vivo. HNG and Nec-1 exerted synergistic neuroprotection on oxygen-glucose deprivation-induced cell death and cerebral ischemia/reperfusion injury. This study provided a new therapeutic strategy for the treatment of stroke by the combination of anti-apoptosis and anti-necroptosis therapy.

BACKGROUND: Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is the most severe form of cutaneous adverse drug reaction and is characterized by extensive epidermal destruction of the skin and mucosal surfaces. Controversy remains regarding the immunopathogenesis of the disease. It has long been assumed that CD8 cytotoxic T cells mediate cell death by releasing cytotoxic granules and soluble granulysin that trigger keratinocyte apoptosis. However, this does not explain the massive cell death or inflammation that is observed clinically. We have preliminary evidence from transcriptional profiling of patient skin samples suggesting that the cell death pathways necroptosis and pyroptosis may mediate SJS/TEN. Herein we utilize retrospectively and prospectively collected patient samples to investigate these cell death pathways. OBJECTIVE: The goals of this study are two-fold: (i) to investigate cell death pathways in retrospectively-collected (SJS/TEN) patient skin samples and (ii) to directly test the cell death mediators and pathways mediating SJS/TEN using a novel in vitro model. METHODS: Clinically and histopathologically confirmed SJS/TEN skin specimens and control skin specimens from non-blistering T cell mediated drug reactions and healthy skin were obtained following retrospective analysis from a multi-centered patient database. Gene expression profiling is being performed using the NanoString nCounter® System on these samples as a second patient cohort to confirm and expand on preliminary study findings. In parallel, we have optimized the use of a novel human skin platform for an in vitro model of SJS/TEN. We also collected human serum from a prospective study of SJS/TEN and control patients and have optimized and are actively collecting blister fluid from SJS/TEN and control patients in an ongoing prospective study for use in this model. RESULTS: Through an extensive pathology database and medical record search of potential cases at Brigham and Women's Hospital, we identified a second patient cohort of SJS/TEN, non-blistering delayed-type drug hypersensitivity reactions and healthy controls. We identified and are collecting thorough demographic, clinical and laboratory data on 61 potential candidates for SJS/TEN, 4 for Drug Reaction with Eosinophilia Syndrome (DRESS), and 200 for Morbilliform Drug Eruptions (MDE). This second cohort is in the final step of analysis with review by an expert clinician to confirm cases. In parallel, we have designed an expansive gene panel to confirm cell death mediator and marker transcription in our bank of skin samples. This 815 gene panel uses the pre-designed panel from Nanostring®, spiked with an additional 30 genes specific to apoptosis, pyroptosis, and necroptosis. We reviewed multiple potential in vitro skin models and identified GenoSkin® as the most suitable human skin platform for our in vitro model. We collected serum from 6 SJS/TEN patients and 6 non-blistering drug reaction patients and 3 healthy controls, and are actively collecting blister fluid from SJS/TEN and thermal burn control patients for analysis in this model. CONCLUSIONS: Our preliminary data suggest necroptosis and pyroptosis induced by soluble death mediators tumor necrosis factor (TNF) alpha and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as the main cell death pathways responsible for SJS/TEN. We have successfully identified a large number of potential patient samples of both cases and controls to perform transcriptional profiling using a self-designed gene panel to confirm and expand upon our preliminary data. We have successfully collected prospectively patient serum and are actively collecting patient blister fluid for analysis in an optimized in vitro model using GenoSkin®. SJS/TEN is severely understudied and lacks a standard protocol for care. This stems from uncertainty surrounding disease pathobiology. It is critical that we use innovative approaches to interrogate the mechanism mediating disease to advance the field, and, most importantly, to improve the quality of care for these patients.

No description available.

La nécroptose est une mort cellulaire régulée impliquée dans les pathologies inflammatoires, ischémiques et dégénératives. RIPK1 serait une cible thérapeutique intéressante car son activité kinase serait à l’origine de leur initiation ou aggravation. Nous avons caractérisé les effets biologiques de deux nouveaux inhibiteurs de RIPK1. La sibiriline protège les souris d’une hépatite autoimmune et 6E11 protège les cellules endothéliales de l’aorte de la mort par ischémie froide/réoxygénation, suggérant des propriétés intéressantes pour ces inhibiteurs. Ces composés diffèrent par leur mode d’action, d’interaction et de sélectivité, et restent à optimiser en vue d’une utilisation thérapeutique. / Necroptosis is a regulated cell death pathway involved in inflammatory, ischemic or degenerative diseases. RIPK1 would be an interesting therapeutic target since its kinase activity is probably responsible of their initiation or aggravation. We have characterized the biological effects of two new RIPK1 inhibitors. Sibirilin protects mice from autoimmune hepatitis and 6E11 protects endothelial aortic cells from death due to cold ischemia/reoxygenation, suggesting interesting properties for these inhibitors. These compounds differ in their mode of action, interaction and selectivity, and still need to be optimized for therapeutic use.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-17T20:53:38Z No. of bitstreams: 1 DayaneFreitas.pdf: 3248256 bytes, checksum: ea021e1cfc7eeadb7ac1ec7bc8cfd5a3 (MD5) / Made available in DSpace on 2017-05-17T20:53:38Z (GMT). No. of bitstreams: 1 DayaneFreitas.pdf: 3248256 bytes, checksum: ea021e1cfc7eeadb7ac1ec7bc8cfd5a3 (MD5) Previous issue date: 2016-03-14 / The Euterpe oleracea Mart seed. (Acai) has demonstrated different biological activities, such as antioxidant, anti-inflammatory, antihypertensive, antinociceptive and antineoplastic. In this new study, the aim was to analyze the antineoplastic activity of fractions derived from hydroalcoholic extract of Euterpe oleracea Mart. seed in cell line MCF-7, and to identify the compounds responsible for the antineoplastic action by mass spectrometry using electrospray ionization source, and a ciclotrônico analyzer coupled to a Fourier transform (ESI-FT-ICR MS). The MCF-7 cell line was treated with 10, 20, 40 and 60 μg L-1 with fractions hexane (FH), chloroform (FC) and ethyl acetate (FAE) of the hydroalcoholic extract acai seed for 24, 48 and 72 hours. After treatment the cell viability was measured using the assay with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and the cell type of death was assessed using annexin - Iodide propidium (PI) assay. Data were analyzed statistically by analysis of variance (ANOVA) or Student's t test, us appropriate. It was observed that all the fractions caused significant reduction of cell viability of MCF-7, but the FAE was the most cytotoxic (p <0.001). In the test Annexin-PI, there was no significant labeling annexin but increased PI staining was significant (p <0.001). This study showed that the FAE was more effective in reducing cell viability, following necroptose mechanism in MCF-7 cell. The FAE is composed of epicatechin, proanthocyanidin A2 and trimeric and tetrameric procyanidins. / A semente de Euterpe oleracea Mart. (açaí) tem demonstrado diferentes atividades biológicas, tais como: antioxidante, anti-inflamatória, anti-hipertensiva, antinociceptiva e antineoplásica. Neste estudo, o objetivo foi analisar a atividade antineoplásica de frações do extrato hidroalcoólico da semente de Euterpe oleracea Mart. na linhagem celular MCF-7, assim como identificar os compostos responsáveis pela ação antineoplásica por espectrometria de massas utilizando fonte de ionização por eletrospray e um analisador ciclotrônico acoplado a uma transformada de Fourier (ESI- FT-ICR MS). A linhagem MCF-7 foi tratada com 10, 20, 40 e 60 μg L-1 com as frações hexânica (FH), clorofórmica (FC) e de acetato de etila (FAE) do extrato hidroalcoólico da semente do açaí por 24, 48 E 72 horas. Após o tratamento a viabilidade celular foi mensurada usando o ensaio com 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) e o tipo de morte celular foi avaliado com o ensaio Anexina - Iodeto de propídeo (PI). Os dados foram analizados estatisticamente por análise de variância (ANOVA) ou por teste T student, quando apropriado. Foi observado que todas as frações causaram redução significativa da viabilidade celular da MCF-7, porém a FAE foi a mais citotóxica (p < 0,001). No ensaio de anexina-pi, não foi observado marcação significativa para anexina porém o aumento da marcação de PI foi significativo (p<0,001). O presente estudo demonstrou que a FAE foi a mais eficaz na redução da viabilidade celular, seguindo mecanismo de necroptose na célula MCF-7. A FAE é composta por epicatequina, proantocianidina A2 e procianidinas trimérica e tetramérica.

Made available in DSpace on 2016-08-19T12:59:16Z (GMT). No. of bitstreams: 1 Dissertacao-DayanneSilvaFreitas.pdf: 3246572 bytes, checksum: 53f34629ab61070b06e48c3c8284366d (MD5) Previous issue date: 2016-03-14 / The Euterpe oleracea Mart seed. (Acai) has demonstrated different biological activities, such as antioxidant, anti-inflammatory, antihypertensive, antinociceptive and antineoplastic. In this new study, the aim was to analyze the antineoplastic activity of fractions derived from hydroalcoholic extract of Euterpe oleracea Mart. seed in cell line MCF-7, and to identify the compounds responsible for the antineoplastic action by mass spectrometry using electrospray ionization source, and a ciclotrônico analyzer coupled to a Fourier transform (ESI-FT-ICR MS). The MCF-7 cell line was treated with 10, 20, 40 and 60 μg L-1 with fractions hexane (FH), chloroform (FC) and ethyl acetate (FAE) of the hydroalcoholic extract acai seed for 24, 48 and 72 hours. After treatment the cell viability was measured using the assay with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and the cell type of death was assessed using annexin - Iodide propidium (PI) assay. Data were analyzed statistically by analysis of variance (ANOVA) or Student's t test, us appropriate. It was observed that all the fractions caused significant reduction of cell viability of MCF-7, but the FAE was the most cytotoxic (p <0.001). In the test Annexin-PI, there was no significant labeling annexin but increased PI staining was significant (p <0.001). This study showed that the FAE was more effective in reducing cell viability, following necroptose mechanism in MCF-7 cell. The FAE is composed of epicatechin, proanthocyanidin A2 and trimeric and tetrameric procyanidins. / A semente de Euterpe oleracea Mart. (açaí) tem demonstrado diferentes atividades biológicas, tais como: antioxidante, anti-inflamatória, anti-hipertensiva, antinociceptiva e antineoplásica. Neste estudo, o objetivo foi analisar a atividade antineoplásica de frações do extrato hidroalcoólico da semente de Euterpe oleracea Mart. na linhagem celular MCF-7, assim como identificar os compostos responsáveis pela ação antineoplásica por espectrometria de massas utilizando fonte de ionização por eletrospray e um analisador ciclotrônico acoplado a uma transformada de Fourier (ESI- FT-ICR MS). A linhagem MCF-7 foi tratada com 10, 20, 40 e 60 μg L-1 com as frações hexânica (FH), clorofórmica (FC) e de acetato de etila (FAE) do extrato hidroalcoólico da semente do açaí por 24, 48 E 72 horas. Após o tratamento a viabilidade celular foi mensurada usando o ensaio com 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) e o tipo de morte celular foi avaliado com o ensaio Anexina - Iodeto de propídeo (PI). Os dados foram analizados estatisticamente por análise de variância (ANOVA) ou por teste T student, quando apropriado. Foi observado que todas as frações causaram redução significativa da viabilidade celular da MCF-7, porém a FAE foi a mais citotóxica (p < 0,001). No ensaio de anexina-pi, não foi observado marcação significativa para anexina porém o aumento da marcação de PI foi significativo (p<0,001). O presente estudo demonstrou que a FAE foi a mais eficaz na redução da viabilidade celular, seguindo mecanismo de necroptose na célula MCF-7. A FAE é composta por epicatequina, proantocianidina A2 e procianidinas trimérica e tetramérica.