Ovarian cancer is a deadly disease that kills an estimated 15,000 women annually in the United States. It is estimated that approximately 10% of ovarian cancers are due to familial inheritance. The most commonly mutated genes in familial ovarian cancer are BRCA1 and BRCA2. It has been reported that cells carrying the BRCA1 185delAG mutation undergo an enhanced caspase-3 mediated apoptotic response. Here, we report on the transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer cells. Cells transfected with the BRCA1 185delAG truncation protein (BRAt) showed increased levels of active caspase 3, increased cleavage of caspase 3 substrates, PARP and DFF45, and decreased XIAP and cIAP1 following staurosporine (STS) treatment. BRAt also reduced Akt phosphorylation and over expression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild type cells. Further, BRAt expression increased chemosensitivity in platinum resistant ovarian cancer cells. Similarly, maspin protein has been shown to sensitize breast carcinoma cells to STS-induced apoptosis. We provide the first evidence that BRAt is sufficient to induce maspin protein in IOSE cells. IOSE cell lines carrying the BRCA1 185delAG mutation showed higher maspin levels than wild-type BRCA1 IOSE cell lines. BRCA1 wild-type IOSE cells were transfected with BRAt protein and showed increased maspin mRNA levels and increased nuclear maspin protein levels as compared to control cells. Additionally, both heterozygous carriers of the BRCA1 185delAG mutation and cells transfected with BRAt protein show an increased ability to activate the maspin promoter as compared to control cells. The transcription factor AP1 is at least partially required for full activation of the maspin promoter in BRAt cells, as siRNA directed towards c-jun decreased activation of the full-length maspin promoter. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis by increasing nuclear maspin expression, suggesting a possible mechanism by which ovarian cancer patients with germline BRCA1 mutations may respond better to chemotherapy.

This thesis tests the hypothesis that increased neuronal cell death in SIDS infants is related to the ability of risk factors, such as prone sleeping, to expose infants to intermittent hypercapnic hypoxia (IHH). Based on the hypothesis that the NMDA system is linked to neuronal death, by way of excitotoxicity, correlations were also sought between cell death and changes in NMDA receptor (NR1) expression in brainstem nuclei controlling cardiorespiratory function. The first aim of this study was to verify that increased neuronal cell death occurs in SIDS infants. To verify a piglet model of SIDS risk factors, brainstem changes were examined in piglets exposed to IHH, and comparisons were made to changes seen in SIDS infants. The NMDA receptor was characterised in controls for both the human infant and the piglet groups. Comparisons of neuronal changes were made with SIDS infants, and piglets exposed to IHH. Non-radioactive in-situ hybridisation and immunohistochemistry were performed on formalin fixed and paraffin embedded brainstem tissue to identify markers of cell death (caspase-3, active caspase-3, and TUNEL), and to examine NR1 mRNA and protein expressions. Staining was quantified using computerised image analysis software. Eight nuclei from the brainstem medulla (caudal in piglets, and mid in infants), and two nuclei from the rostral pons (infants) were studied. The first dataset included human infants aged 1-6 months with a diagnosis of SIDS (n=15) or non-SIDS (n=10). The second dataset comprised developing piglets aged 13-14 days, with controls (n=6), against those exposed to IHH for 2 (n=6) or 4 (n=5) days. Increased neuronal cell death was not verified in the SIDS infants, but abnormalities in NR1 expression were present in selected nuclei of the medulla. Piglets exposed to IHH had increased neuronal cell death and changes in NR1 in selected nuclei of the medulla. There was also a positive correlation between increased cell death and high NR1 levels. Preliminary data showed that SIDS infants who usually slept prone had some differences in NR1 compared to those who did not usually sleep prone. From these findings, it was concluded that IHH may underlie the abnormalities in NMDA receptor expression that are present in the brainstem of SIDS infants. Although IHH can induce an increase in neuronal cell death, its significance in the aetiology of SIDS is not known. In piglets, IHH induced cell death correlated with high NMDA expression in some brainstem nuclei, supporting the hypothesis that excitotoxicity may be involved in the mechanism for cell death. Moreover, this thesis presents for the first time, �preliminary pathological proof� of an association between prone sleeping and abnormal NMDA receptor expression in SIDS infants.

Cytosolic double stranded DNA (dsDNA) is sensed as a “danger signal” by host cells. Detection of viral and bacterial nucleic acid is emerging as a major route for cells to identify an infection by a pathogen. Recognition of cytoplasmic DNA causes death of some cells and interferon (IFN) and cytokine induction, which are appropriate anti-viral responses. Responses to cytoplasmic DNA may not only be relevant to certain retrovirus, DNA virus and bacterial infections, but could also be generated by reverse transcription of endogenous retro-elements. Introduction of DNA into the cytoplasm of bone marrow derived macrophages (BMM) causes upregulation of MHC Class I, induction of IFNβ and other cytokines and cell death. Both cytokine induction and cell death were independent of recognition of “CpG motifs” through TLR9. In order to determine whether a single receptor was likely to mediate these responses, the types of DNA eliciting these responses was compared. Both cellular activation to produce cytokines and IFNβ, as well as cell death were seen only with dsDNA but not single stranded DNA (ssDNA). Both responses increased with increasing DNA length, with little detectable effect of a double stranded 22bp oligonucleotide (ODN). The sequences of DNA leading to optimal induction of IFNβ and death were different. Although all dsDNA induced death of primary macrophages, poly(dA):(dT) was a particularly potent and rapid pro-death stimulus. In contrast, poly(dA):(dT) was a relatively poor stimulus for IFNβ, even at doses which were minimally toxic, or in cells which are resistant to DNA induced cell death. The alternating co-polymer poly(dA-dT) was the most potent inducer of IFNβ. This data suggests that separate DNA receptors mediate cell death and IFNβ induction in response to dsDNA Transfected dsDNA also rapidly activated caspase 3, a classical pro-apoptotic caspase, in BMM as early as 2½ minutes post-transfection with DNA. Caspase 3 is an effector caspase which is activated by an upstream initiator caspase. Although the apical caspase in the DNA detection system has not been defined, use of Bcl2 overexpressing BMM and caspase 2-/- BMM showed that DNA-dependent caspase 3 activation did not occur via the mitochondrial damage or the caspase 2 activation pathways. The inflammatory caspase, caspase 1 was also activated in response to DNA transfection, although whether caspase 1 is responsible for cleavage of caspase 3 has not been established. Caspase 1 activation suggests the involvement of the inflammasome, which is important for processing pro-inflammatory cytokines such as IL-1β into their biologically active forms. Furthermore, there is recent evidence suggesting that DNA-transfected cells die by a caspase 1-dependent cell death called pyroptosis. Other work in our lab identified the HIN-200 family member and candidate lupus susceptibility factor p202 as a candidate receptor for cytoplasmic dsDNA; p202 bound stably and rapidly to transfected DNA. Here, knockdown studies revealed p202 to be a regulatory protein limiting DNA-induced caspase 1 and 3 activation. Conversely, the related pyrin domain-containing HIN-200 factor AIM2 (p210), a candidate tumour suppressor, was required for caspase 1 and 3 activation by cytoplasmic dsDNA. Recently published work suggests that AIM2 multimerises along the length of the DNA leading to the formation of an inflammasome complex. The pyrin domain of AIM2 recruits the adaptor protein ASC through homotypic pyrin domain interactions. ASC subsequently recruits caspase 1, which results in its auto-activation. The inhibitory effect of p202 on caspase activation is likely to be due to its lack of a pyrin signalling domain. p202 rapidly binds to cytoplasmic DNA, and may reduce the clustering of AIM2 pyrin domains which results in caspase activation. Consistent with this proposal, DNA-dependent caspase activation correlated inversely with p202 expresssion in 3 mouse strains. This work defines HIN-200 proteins as a new class of pattern recognition receptors mediating responses to dsDNA. Work in this thesis aimed to understand the biological role and mechanism of responses to cytoplasmic DNA. Responses to cytoplasmic DNA are likely to be relevant not only to infectious disease but also to autoimmune diseases such as systemic lupus erythmatosus (SLE), where DNA appears to act as an adjuvant, and even tumour progression where there is evidence for a role for active endogenous retro-elements. In addition, responses to DNA may limit transfection efficiency and the efficacy of non-viral gene therapy.

Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.

Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

Depression is a physiological disorder that may be treated by increasing the body¡¦s amount of one or a few of the following neurotransmitters: serotonin, dopamine and norepinephrine. Although there are seven distinct classes of antidepressants, selective serotonin reuptake inhibitors (SSRIs) and tetracyclic antidepressants are widely prescribed and generally regarded as the first-line drugs in the treatment of depression. However, many physiological roles of some SSRIs appear to be dissociated with the inhibition of serotonin reuptake. For instance, paroxetine, a member of SSRIs and maprotiline, a member of tetracyclic antidepressant, have been shown to induce apoptosis or to prevent other agents from inducing apoptosis in several cell lines. Thus the effects of these two drugs on the apoptosis are still controversial. The aim of this study is to investigate the molecular mechanisms of paroxetine and maprotiline in induction of cell death in human osteosarcoma and murine neuroblastoma cells. First, WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of paroxetine and maprotiline. Then immunoblotting was used to measure the activity of apoptotic markers caspase-3 and mitogen-activated protein kinases (MAPKs) to survey the apoptotic pathways induced by these two antidepressants. The experimental results may be helpful to understand the pharmacological and toxicological effects of these two antidepressants in cells from important organs. Results showed that paroxetine caused apoptosis via the activation of caspase-3 in cultured human osteosarcoma cells (MG63). Although paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine was also found to induce [Ca2+]i increases but pretreatment with BAPTA/AM, a Ca2+ chelator, prevented paroxetine-induced [Ca2+]i increases, and thus did not protect cells from death. These results suggest that paroxetine caused Ca2+-independent apoptosis via the activation of p38 MAPK-associated caspase-3 in MG63 cells. Maprotiline was also found to induce apoptosis through increased caspase-3 activation in murine neuroblastoma Neuro-2a cells. Induction of JNK phosphorylation contributed to the activation of caspase-3 resulting in maprotiline-induced Neuro-2a cell apoptosis. Thus, it appears that maprotiline induced apoptosis via JNK/caspase-3-dependent signaling pathways. Blockage of activation of ERK was found to increase the activation of caspase-3 leading to an enhancement of maprotiline-induced apoptosis. These data suggest that ERK was a survival signal to oppose maprotiline-caused apoptotic effect in Neuro-2a cells. Thus the activation of caspase-3 by maprotiline appears to depend on the activation of JNK and the inactivation of ERK. [Ca2+]i measurement in the presence of maprotiline showed that the antidepressant induced [Ca2+]i increases. Interestingly, pretreatment with BAPTA/AM could suppress maprotiline-induced ERK phosphorylation, enhance caspase-3 activation and increase maprotiline-induced apoptosis. In conclusion, this study demonstrates that maprotiline induced apoptosis in murine neuroblastoma cells through activation of JNK-associated caspase-3 pathways. Maprotiline also evoked an anti-apoptotic response that was both Ca2+- and ERK-dependent. This thesis contains some published data in the journal of Toxicology and Applied Pharmacology and some data were submitted in the journal of Toxicology Letters.

This thesis tests the hypothesis that increased neuronal cell death in SIDS infants is related to the ability of risk factors, such as prone sleeping, to expose infants to intermittent hypercapnic hypoxia (IHH). Based on the hypothesis that the NMDA system is linked to neuronal death, by way of excitotoxicity, correlations were also sought between cell death and changes in NMDA receptor (NR1) expression in brainstem nuclei controlling cardiorespiratory function. The first aim of this study was to verify that increased neuronal cell death occurs in SIDS infants. To verify a piglet model of SIDS risk factors, brainstem changes were examined in piglets exposed to IHH, and comparisons were made to changes seen in SIDS infants. The NMDA receptor was characterised in controls for both the human infant and the piglet groups. Comparisons of neuronal changes were made with SIDS infants, and piglets exposed to IHH. Non-radioactive in-situ hybridisation and immunohistochemistry were performed on formalin fixed and paraffin embedded brainstem tissue to identify markers of cell death (caspase-3, active caspase-3, and TUNEL), and to examine NR1 mRNA and protein expressions. Staining was quantified using computerised image analysis software. Eight nuclei from the brainstem medulla (caudal in piglets, and mid in infants), and two nuclei from the rostral pons (infants) were studied. The first dataset included human infants aged 1-6 months with a diagnosis of SIDS (n=15) or non-SIDS (n=10). The second dataset comprised developing piglets aged 13-14 days, with controls (n=6), against those exposed to IHH for 2 (n=6) or 4 (n=5) days. Increased neuronal cell death was not verified in the SIDS infants, but abnormalities in NR1 expression were present in selected nuclei of the medulla. Piglets exposed to IHH had increased neuronal cell death and changes in NR1 in selected nuclei of the medulla. There was also a positive correlation between increased cell death and high NR1 levels. Preliminary data showed that SIDS infants who usually slept prone had some differences in NR1 compared to those who did not usually sleep prone. From these findings, it was concluded that IHH may underlie the abnormalities in NMDA receptor expression that are present in the brainstem of SIDS infants. Although IHH can induce an increase in neuronal cell death, its significance in the aetiology of SIDS is not known. In piglets, IHH induced cell death correlated with high NMDA expression in some brainstem nuclei, supporting the hypothesis that excitotoxicity may be involved in the mechanism for cell death. Moreover, this thesis presents for the first time, �preliminary pathological proof� of an association between prone sleeping and abnormal NMDA receptor expression in SIDS infants.

A encefalopatia hipóxico-isquêmica neonatal, ou simplesmente hipóxia-isquemia (HI) neonatal, é uma das principais causas de morbidade e mortalidade em neonatos humanos. De 20% a 50% dos recém-nascidos com HI severa morrem no período perinatal. Quando sobrevivem, 25% apresentam deficiências neuropsicológicas, como dificuldade de aprendizado, epilepsia e paralisia cerebral. Devido a isso, a eficácia de possíveis agentes neuroprotetores tem sido testada em modelos animais. Há razão para se pensar que a progesterona tem um forte potencial para o tratamento da HI neonatal, já que a sua utilização tem se mostrado benéfica em pesquisas relacionadas com lesão cerebral traumática, lesão cerebral isquêmica e outros modelos de lesão do sistema nervoso central (SNC) em adultos. Inúmeros estudos têm mostrado que o modelo animal de HI de Rice e Vannucci (1981) em animais neonatos, utilizado no presente trabalho, pode produzir lesões no sistema nervoso central relativamente previsíveis, e que estas lesões encefálicas parecem semelhantes às observadas clinicamente em humanos (SALMASO et al., 2014). Para a realização do modelo de HI foram utilizados ratos Wistar com idade de 7 dias (P7). Após a oclusão da carótida esquerda, os animais foram colocados em câmaras para exposição à atmosfera hipóxica com 8% O2/92% N2 por 90 minutos. Os animais foram divididos em cinco grupos experimentais: SHAM, HI, HI+PROG-PRÉ (PRÉ), HI+PROG-PÓS (PÓS), HI+PROG-PRÉ/PÓS (PP). Os termos PRÉ e PÓS referem-se à administração de progesterona (na dose de 5 mg/kg) antes ou após o procedimento de HI neonatal . Dependendo do grupo experimental, os animais foram tratados com progesterona imediatamente antes da isquemia e/ou 6 e 24 horas após o início da hipóxia. Foram analisados o peso corporal dos animais (imediatamente antes da isquemia e 6, 24 e 48 horas após o início da hipóxia), o volume de lesão cerebral, além da expressão das proteínas p-Akt e caspase-3 pela técnica de Western blotting. / Neonatal hypoxic-ischemic encephalopathy or simply neonatal hypoxia-ischemia (HI) is a main cause of morbidity and mortality in human neonates. Moreover, 25% of survivors show neuropsychological dysfunctions such as learning difficulties, epilepsy and cerebral palsy. Because of this, the effectiveness of potential neuroprotective agents has been tested in animal models. There is a reason to suppose that progesterone has a strong potential for the treatment of neonatal HI since its use has been shown to be beneficial in researches related to traumatic brain injury, ischemic brain injury and other central nervous system injury models (CNS) in adults. Several studies have shown that the newborn animal model of HI developed by Rice and Vannucci (1981), and used in the present study, can produce lesions in the central nervous system which are predictable and similar to those observed clinically in humans. In order to perform the HI model we used 7 days old (P7) Wistar rats. After occlusion of the left carotid, the animals were placed in hypoxic chambers and exposed to the hypoxic atmosphere (8% O2/92% N2 for 90 minutes). The animals were divided into five groups: SHAM, HI, HI+PROG-PRÉ (PRÉ), HI+PROG-PÓS (PÓS), HI+PROG-PRÉ/PÓS (PP).The PRÉ and PÓS terms refer to the administration of progesterone (5 mg/kg) before and/or after the HI procedure. Progesterone was administered immediately before ischemia, 6 and 24 hours after the beginning of hypoxia, depending on the experimental group. Body weight was evaluated immediately before ischemia and/or 6 and 24 hours after the start of hypoxia. The volume of brain damage, in addition to the expression of p-Akt and caspase-3 were also evaluated.

Submitted by MILENE ROCHA RIBEIRO (mrocharibeiro@yahoo.com.br) on 2018-07-12T17:13:42Z No. of bitstreams: 1 tese milene Final 2018.pdf: 2347108 bytes, checksum: e7698208bc381aa0ba18d041fd938d1b (MD5) / Approved for entry into archive by Paula Torres Monteiro da Torres (paulatms@sjrp.unesp.br) on 2018-07-13T18:00:11Z (GMT) No. of bitstreams: 1 ribeiro_mr_do_sjrp.pdf: 2347108 bytes, checksum: e7698208bc381aa0ba18d041fd938d1b (MD5) / Made available in DSpace on 2018-07-13T18:00:11Z (GMT). No. of bitstreams: 1 ribeiro_mr_do_sjrp.pdf: 2347108 bytes, checksum: e7698208bc381aa0ba18d041fd938d1b (MD5) Previous issue date: 2018-06-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O ZIKV é um vírus de RNA, não segmentado, de fita simples e sentido positivo membro da família Flaviviridae. O genoma viral possui uma arquitetura típica de flavivírus, com cerca de 11kb de comprimento, que codifica três proteínas estruturais (Capsídeo, precursor-Membrana, Envelope) e sete proteínas não-estruturais (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). Nas Américas, emergiu rapidamente após surto na ilha da Páscoa, Chile. No Brasil, o surto iniciou em 2015, aumentando consideravelmente casos de microcefalia em recém-nascidos. Aliada a esses casos, também foi observada a ocorrência de síndrome neurológica de Guillain-Barré. Essas associações transformaram o impacto da transmissão e infecções por ZIKV em uma preocupação de saúde pública global. O vírus é transmitido principalmente pelos mosquitos do gênero Aedes, que possuem ampla distribuição e apresentam grandes adaptações a ambientes urbanos. Além de transmissão vetorial, pode ser transmitido via sexual e materno-fetal. O objetivo deste trabalho foi comparar as infecções por uma cepa contemporânea de ZIKV com DENV2 em modelo de explantes de placenta humana a termo, bem como quantificar expressão de citocinas, interferons do tipo I, II e III e marcadores de apoptose induzida via infecção viral. Os resultados demonstram que os explantes da placenta a termo são permissivos e apoiam a infecção por ZIKV. A quantificação da carga viral entre infecções ZIKV e DENV2 foram similares. No entanto, DENV2 apresentou decréscimo na liberação de carga viral em 24 horas pós-infecção. A cinética da replicação viral coincidiu com a expressão de citocinas pró-inflamatórias e o aumento de apoptose no tecido infectado. Clivagem de caspase 3 foi parcialmente dependente de TNF- α e o tratamento com Anti-TNF-α diminuiu significativamente essa ativação mediada por infecção viral. Cumulativamente, este modelo demonstra que os tecidos placentários humanos são alvo de infecção por ZIKV e que a infecção é patogênica para o tecido placentário. Palavras-chave: placenta humana, Flavivírus, explantes, apoptose, interferon, caspase 3. / ZIKV is a non-segmented, single-stranded, positive-sense RNA virus and a member of the Flaviviridae family. Its genome has a typical 11 kB-long flavivirus architecture that encodes three structural proteins (Capsid, PrecursorMembrane, Envelop) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). In the Americas, the viruses emerged rapidly after outbreak on Pascoa Island, Chile. The outbreak reached Brazil in 2015, substantially increasing cases of microcephaly in newborns. In addition to microcephaly, cases associated with neurological diseases such as GuillainBarré syndrome have made ZIKV a global public health concern. The virus is mainly transmitted by mosquitoes of the genus Aedes, which are widely distributed and which have adapted well great to urban environments. In addition to vector transmission, ZIKV can be transmitted via sexual and maternal-fetal routes, the virus has been isolated is from sperm, amniotic fluid and central nervous systems of stillborn fetuses. The goal of this report was compare ZIKV-infected to DENV2 in full-term human placenta explant model. Quantify expression of cytokines, type I, II and III interferons and markers of induced-apoptosis by viral infection. The results demonstrated that full-term placenta explants are permissive and support ZIKV infection. Viral loads in ZIKV and DENV2 infections were similar. However, DENV2 presented a decrease in viral load release at 24 hours post infection (h.p.i). The kinetics of viral replication coincided with the expression of proinflammatory cytokines and the increase of apoptosis in the infected tissue. Apoptosis was partially dependent on TNF-α. Anti-TNF-α treatment significantly decreased the activated-caspase 3 mediated viral infection. Cumulatively, this model demonstrates that human placental tissues are targets of ZIKV-infection and that the infection is pathogenic to placental tissue.

A encefalopatia hipóxico-isquêmica neonatal, ou simplesmente hipóxia-isquemia (HI) neonatal, é uma das principais causas de morbidade e mortalidade em neonatos humanos. De 20% a 50% dos recém-nascidos com HI severa morrem no período perinatal. Quando sobrevivem, 25% apresentam deficiências neuropsicológicas, como dificuldade de aprendizado, epilepsia e paralisia cerebral. Devido a isso, a eficácia de possíveis agentes neuroprotetores tem sido testada em modelos animais. Há razão para se pensar que a progesterona tem um forte potencial para o tratamento da HI neonatal, já que a sua utilização tem se mostrado benéfica em pesquisas relacionadas com lesão cerebral traumática, lesão cerebral isquêmica e outros modelos de lesão do sistema nervoso central (SNC) em adultos. Inúmeros estudos têm mostrado que o modelo animal de HI de Rice e Vannucci (1981) em animais neonatos, utilizado no presente trabalho, pode produzir lesões no sistema nervoso central relativamente previsíveis, e que estas lesões encefálicas parecem semelhantes às observadas clinicamente em humanos (SALMASO et al., 2014). Para a realização do modelo de HI foram utilizados ratos Wistar com idade de 7 dias (P7). Após a oclusão da carótida esquerda, os animais foram colocados em câmaras para exposição à atmosfera hipóxica com 8% O2/92% N2 por 90 minutos. Os animais foram divididos em cinco grupos experimentais: SHAM, HI, HI+PROG-PRÉ (PRÉ), HI+PROG-PÓS (PÓS), HI+PROG-PRÉ/PÓS (PP). Os termos PRÉ e PÓS referem-se à administração de progesterona (na dose de 5 mg/kg) antes ou após o procedimento de HI neonatal . Dependendo do grupo experimental, os animais foram tratados com progesterona imediatamente antes da isquemia e/ou 6 e 24 horas após o início da hipóxia. Foram analisados o peso corporal dos animais (imediatamente antes da isquemia e 6, 24 e 48 horas após o início da hipóxia), o volume de lesão cerebral, além da expressão das proteínas p-Akt e caspase-3 pela técnica de Western blotting. / Neonatal hypoxic-ischemic encephalopathy or simply neonatal hypoxia-ischemia (HI) is a main cause of morbidity and mortality in human neonates. Moreover, 25% of survivors show neuropsychological dysfunctions such as learning difficulties, epilepsy and cerebral palsy. Because of this, the effectiveness of potential neuroprotective agents has been tested in animal models. There is a reason to suppose that progesterone has a strong potential for the treatment of neonatal HI since its use has been shown to be beneficial in researches related to traumatic brain injury, ischemic brain injury and other central nervous system injury models (CNS) in adults. Several studies have shown that the newborn animal model of HI developed by Rice and Vannucci (1981), and used in the present study, can produce lesions in the central nervous system which are predictable and similar to those observed clinically in humans. In order to perform the HI model we used 7 days old (P7) Wistar rats. After occlusion of the left carotid, the animals were placed in hypoxic chambers and exposed to the hypoxic atmosphere (8% O2/92% N2 for 90 minutes). The animals were divided into five groups: SHAM, HI, HI+PROG-PRÉ (PRÉ), HI+PROG-PÓS (PÓS), HI+PROG-PRÉ/PÓS (PP).The PRÉ and PÓS terms refer to the administration of progesterone (5 mg/kg) before and/or after the HI procedure. Progesterone was administered immediately before ischemia, 6 and 24 hours after the beginning of hypoxia, depending on the experimental group. Body weight was evaluated immediately before ischemia and/or 6 and 24 hours after the start of hypoxia. The volume of brain damage, in addition to the expression of p-Akt and caspase-3 were also evaluated.