Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2016-09-22T19:02:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2016-09-29T19:20:07Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Made available in DSpace on 2016-09-29T19:20:08Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) Previous issue date: 2016-09 / O câncer é apontado como a segunda maior causa de morte em todo o mundo, com previsão de em breve ser tornar a primeira. O câncer de próstata está entre os 5 tipos de câncer mais diagnosticados em homens, sendo que o câncer hepático está em segundo lugar em taxa de mortalidade entre homens e mulheres. Indícios apontam para uma ativação das vias da inflamação associados a uma inibição das vias de morte celular no processo de carcinogênese. A regulação destas vias torna-se alvo importante e complementar no controle do câncer, sendo estimulada a busca de biomoléculas com este potencial. As plantas são importante fonte de descoberta de novas biomoléculas com ampla utilização para o tratamento de diversas patologias. A família Myrtaceae possui diversas espécies que são apontadas como fortes candidatos em potencial nesta busca, incluindo as do gênero Myrciaria. A espécie Myrciaria plinioides não possui estudos referentes suas propriedades terapêuticas ou a atuação em vias de sinalização envolvidas na inflamação ou na carcinogênese. Neste contexto, este estudo teve por objetivo avaliar a atividade do extrato etanólico de M. plinioides em células de carcinoma hepatocelular (HepG2) e próstata (LNCaP) , através da análise de expressão dos marcadores p38-α, pp38-α, NF-κB e caspase-3, envolvidos na carcinogênese, e o efeito sobre a viabilidade celular através do método de MTT. A viabilidade das células foi alterada significativamente, em ambas as linhagens celulares quando tratadas com o extrato etanólico. A análise da expressão proteica demonstra significativa inibição da expressão de p38-α e caspase-3 nas células LNCaP, quando tratadas com extrato etanólico de M. plinioides seguido de LPS. Em células HepG2, somente houve alteração na expressão da caspase-3 na concentração de 200 μg/mL, com ou sem adição de LPS após tratamento com extrato. Os resultados deste estudo demonstraram redução da viabilidade celular nas duas linhagens tumorais, expressão diferenciada de proteínas envolvidas em apoptose, o que leva a indícios da ativação de mecanismos distintos pelo extrato em cada tipo celular. Estudos futuros para averiguar o mecanismo celular e a indução de morte em células tumorais de câncer de próstata e de fígado podem contribuir para a identificação e elucidação de novas biomoléculas com potencial antitumoral. / Cancer is touted as the second leading cause of death worldwide, forecast to soon be making the first. Prostate cancer is among the five most cancers diagnosed in men, and liver cancer is second in mortality between men and women. Evidence points to the activation of pathways of inflammation associated with an inhibition of cell death pathways in carcinogenesis. The regulation of these pathways becomes important and complementary target in cancer control, and stimulated the search for biomolecules with this potential. The plants are important source of discovery of new biomolecules with wide use for the treatment of various diseases. The Myrtaceae family has many species that are identified as potential candidates strong in this search, including the Myrciaria genre. The species Myrciaria plinioides not have studies on its therapeutic properties or performance in signaling pathways involved in inflammation or carcinogenesis. In this context, this study aimed to evaluate the activity of the ethanol extract of M. plinioides in hepatocellular carcinoma cells (HepG2) and prostate (LNCaP) by expression analysis of p38-α markers, PP38-α, NF-kB and caspase-3, involved in carcinogenesis, and the effect on cell viability by the MTT method. The viability of cells was significantly altered in both cell lines when treated with ethanolic extract. Protein expression analysis demonstrates significant inhibition of p38-α expression and caspase-3 in LNCaP cells, when treated with ethanolic extract of M. plinioides followed by LPS. In HepG2 cells there was only a change in the expression of caspase-3 at a concentration of 200 / ml, with or without addition of LPS after treatment with extract. The results showed reduction of cell viability in both tumor lines, differential expression of proteins involved in apoptosis, leading to evidence of activation by distinct mechanisms in each extract cell type. Further studies to investigate the cellular mechanism, and induction of death in tumor cells of prostate and liver cancer may contribute to the identification and elucidation of new biomolecules with antitumor potential.

The lens is responsible for focusing light into the retina. It accomplishes this through its maturation from an epithelial cell into a fiber cell. A large amount of research has been done on cellular differentiation. Nevertheless, we still lack knowledge on many different aspects of differentiation, including a complete theory on the mechanism behind differentiation. Due to the lens’ unique structure and cell types, this is an ideal model for studying differentiation. Our research has shown that αB crystallin, a small heat shock protein, is able to modulate cytochrome C levels and protect the mitochondria under oxidative stress. Also, cytochrome C release is often followed by caspase 3 activation. In addition, research has shown that low levels of caspase 3 activation is essential in driving differentiation. My work examined if αB crystallin could modulate cytochrome C to lower caspase 3 levels to allow for differentiation rather than apoptosis. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

La production de globules rouges dépend du taux apoptose des précurseurs érythroïdes et est principalement régulé par l'érythropoïétine (Epo). La privation en Epo aboutit à l'activation de la caspase-3 (casp-3) qui clive GATA-1 ce qui entraîne l'apoptose des érythroblastes immatures. L'activation de la casp-3 est également indispensable aux modifications morphologiques caractéristiques observées au cours de la différenciation érythroïde terminale humaine, sans qu'il n'y ait ni d'apoptose ni de clivage de GATA-1. L'objectif de cette thèse était de mettre en évidence si Hsp70 inductible, dont un des rôles principaux est la régulation de l'apoptose, est impliquée dans la protection sélective des substrats de la casp-3 activée au cours de la différenciation érythroïde terminale humaine. Nous avons mis en évidence que lors de la différentiation érythroïde terminale pendant la phase d'activation des caspases, Hsp70 a une expression nucléo-cytoplasmique constitutive et co-localise avec GATA-1 dans le noyau. La localisation nucléaire d'Hsp70 est régulée par l'Epo : après privation des cellules en Epo, il y a une importante diminution de la localisation nucléaire d'Hsp70 et GATA-1 est clivée. L'inhibition de l'expression d'Hsp70 par une approche siRNA a comme conséquence le clivage de GATA-1 lors de l'activation de la casp-3 avec un arrêt de différenciation et une augmentation de la mort cellulaire. Hsp70 est une nouvelle protéine anti-apoptotique de la différenciation érythroïde terminale. Nous proposons un modèle dans lequel l'Epo détermine le destin des érythroblastes (apoptose vs différenciation) en aval de la casp-3 en régulant la localisation nucléaire d'Hsp70.

The effect of the natural essential oil carvacrol or the anesthetic propofol on cell viability, cell cycle distribution, reactive oxygen species (ROS), intracellular free Ca2+ concentrations ([Ca2+]i) and caspase-3-associated apoptosis in human normal cells or non-normal cells is unclear. Human gingival fibroblasts (HGF), human oral cancer cell line (OC2) and human glioblastoma cell line (DRTBG-05MG, HGB) were used in this study. Cell viability was measured by detecting reagent water soluble tetrazolium salt-1 (WST-1). Apoptosis was detected by Annexin V/propidium iodide (PI) staining, cell cycle distribution was detected by PI staining, and ROS was detected by membrane-permeable probe dichlorofluorescein diacetate (DCFH-DA) or hydroethidine (HE) staining. Apoptosis, cell cycle distribution and ROS were analyzed by flow cytometry. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Caspase-3 expression was detected by western blotting. Carvacrol at 200-800 £gM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 1,000 £gM carvacrol almost killed all OC2 or HGB cells, but in HGF cells, 200-800 £gM carvacrol did not significantly kill cells and 1,000 £gM carvacrol decreased only about 63% of cell viability. Similarly, propofol at concentrations between 300 and 600 £gM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 700 £gM propofol almost killed all OC2 or HGB cells, but in HGF cells, 300-600 £gM propofol did not significantly kill cells and 700 £gM propofol decreased about 62% of cell viability. In OC2 or HGB cells, carvacrol (200 £gM, 400 £gM or 600 £gM) or propofol (300 £gM, 400 £gM or 500 £gM) induced apoptosis, increased ROS production, evoked cell cycle arrest and activated caspase-3. The caspase-3 inhibitor (DEVD-CHO) partially decreased the apoptotic effect induced by carvacrol or propofol. On the other hand, in OC2 or HGB cells, carvacrol at concentrations between 400 £gM and 1,000 £gM induced a [Ca2+]i rise in a concentration-dependent manner and the signal was reduced by removal of extracellular Ca2+. In HGF cells, carvacrol at 1000 £gM did not induce immediate [Ca2+]i rises in Ca2+-containing or Ca2+-free medium. Similarly, propofol at concentrations between 400 £gM and 1,000 £gM induced a [Ca2+]i rise in a concentration-dependent manner in OC2 or HGB cells, but not in HGF cells. This cytotoxic effect was not reversed in carvacrol-treated groups, but was partially reversed in propofol-treated groups when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA-AM) in OC2 or HGB cells. The apoptotic effect of propofol was also partially decreased by BAPTA-AM treatment in OC2 and HGB cells. In OC2 and HGB cells, carvacrol- or propofol-induced Ca2+ signal was not altered by L-type voltage-gated Ca2+ channel blocker nifedipine, store-operated Ca2+ channel blocker econazole or SK&F96365) and protein kinase C (PKC) activator phorbol myristate acetate (PMA), but was inhibited by PKC inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol- or propofol-induced [Ca2+]i rises. Incubation with carvacrol or propofol also abolished TG or BHQ-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol- or propofol-induced [Ca2+]i rises. Together, first, in HGF cells, carvacrol (200-800 £gM) or propofol (300-600 £gM) did not induce [Ca2+]i rises and cell death. Second, in OC2 or HGB cells, carvacrol induced [Ca2+]i rises and cell death that might involve ROS- and caspase-3-associated apoptosis. Third, in OC2 or HGB cells, propofol induced [Ca2+]i rises and cell death that might involve ROS-, Ca2+- and caspase-3-associated apoptosis. Lastly, in OC2 or HGB cells, carvacrol or propofol induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive, non store-operated Ca2+ channels.

Orientador: Paula Rahal / Coorientador: / Banca: / Resumo: 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... / Abstract: 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 ... / Doutor

Macrophage apoptosis is an important process in the pathophysiology of atherosclerosis. Oxidized low-density lipoproteins (OxLDL) are a major component of lesions and potently induce macrophage apoptosis. Cannabinoid receptor 2 (CB2), the predominant macrophage cannabinoid receptor, modulates several macrophage processes associated with ongoing atherosclerosis; however, the role of CB2 in macrophage apoptosis is unknown. To determine if CB2 influences a macrophage apoptotic pathway relevant to atherosclerosis, we examined the effect of CB2 deficiency on OxLDL-induced macrophage apoptosis. In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) analysis of resident peritoneal macrophages detected significantly fewer apoptotic CB2-/- macrophages than CB2+/+ macrophages after incubation with OxLDL (27.9 ± 4.7% vs. 61.9 ± 8.5%, P < 0.001) or 7-ketocholesterol (7KC) (18.9 ± 10.5% vs. 54.1 ± 6.9%, P < 0.001), an oxysterol component of OxLDL. Caspase-3 activity; proteolytic conversion of procaspase-3; and cleavage of a caspase-3 substrate, PARP, were also diminished in 7KC-treated CB2-/-macrophages. Furthermore, the deactivation of the prosurvival kinase, Akt, in response to 7KC was impaired in CB2-/-macrophages. These results suggest that CB2 expression increases the susceptibility of macrophages to OxLDL-induced apoptosis, in part, by modulating the effect of oxysterols on the Akt survival pathway and that CB2 may influence atherosclerosis by modulating lesional macrophage apoptosis.

Connexin43 (Cx43) is one of the most abundant gap junction proteins in the central nervous system. Abnormal opening of Cx43 hemichannels after ischemic insults causes apoptotic cell death. In this study, we found persistently increased expression of Cx43 8 h to 7 d after hypoxia/ischemia (HI) injury in neonatal rats. Pre-treatment with Gap26 and Gap27, two Cx43 mimetic peptides, significantly reduced cerebral infarct volume. Gap26 treatment at 24 h after ischemia improved functional recovery on muscle strength, motor coordination, and spatial memory abilities. Further, Gap26 inhibited Cx43 expression and reduced active astrogliosis. Gap26 interacted and co-localized with Cx43 together in brain tissues and cultured astrocytes. After oxygen glucose deprivation, Gap26 treatment reduced the total Cx43 level in cultured astrocytes; but Cx43 level in the plasma membrane was increased. Degradation of Cx43 in the cytoplasm was mainly via the ubiquitin proteasome pathway. Concurrently, phosphorylated Akt, which phosphorylates Cx43 on Serine373 and facilitates the forward transport of Cx43 to the plasma membrane, was increased by Gap26 treatment. Microdialysis showed that increased membranous Cx43 causes glutamate release by opening Cx43 hemichannels. Extracellular glutamate concentration was significantly decreased by Gap26 treatment in vivo. Finally, we found that cleaved caspase-3, an apoptosis marker, was attenuated after HI injury by Gap26 treatment. Effects of Gap27 were analogous to those of Gap26. In summary, our findings demonstrate that modulation of Cx43 expression and astroglial function is a potential therapeutic strategy for ischemic brain injury.

Muscle loss from lack of activity is a serious issue for immobilized patients on Earth and in human spaceflight, where the low gravity environment prevents normal muscle activity. Simulating muscle loss in cultured cells is an important step in understanding how this condition occurs. This work evaluates different means of simulating muscle loss and selects the one that most closely mimics the cellular responses seen in animals and humans. To simulate the microgravity environment of spaceflight, mouse skeletal muscle cells were grown in a rotary cell culture system (RCCS). Growing the cells within a natural gelled substrate was compared against growing them on the surface of small plastic beads. Changes after culture under simulated microgravity were characterized by assessing proteins and genes known to change during muscle loss. The structure of the cells was also evaluated by microscopy. The mouse skeletal muscle cells grown on plastic beads in the RCCS had significant changes in multiple key genes associated with muscle loss and demonstrated physical characteristics expected of mature tissue in live animals. This model is a valuable platform for exploring muscle loss mechanisms and testing new drugs.

Aims: This study was aimed to investigate the role of GSDME-mediated pyroptosis in cardiac injury induced by Doxorubicin (DOX), and to evaluate the role of BH3-only protein Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) in regulation of DOX-induced pyroptosis. Main methods: HL-1 cardiomyocytes and C57BL/6J mice were treated by DOX to establish DOX-induced cardiotoxicity in vitro and in vivo models, respectively. Cell transfection was applied to regulate the expression of caspase-3, GSDME and Bnip3. Western blot was used for measuring expression of protein level. LDH-cytotoxicity assay was used to detect the LDH release. The Flow cytometry analysis was used to detect the cell death. Echocardiography was used to determine the cardiac function. HE staining was used for observing pathological feature of heart tissues. Key findings: Our results showed that GSDME-mediated pyroptosis was involved in DOX-induced cardiotoxicity in vivo. We showed that HL-1 cardiomyocytes exposed to DOX exhibited morphological features of pyroptosis in vitro. We also showed that DOX induced activation of caspase-3 and eventually triggered GSDME-dependent pyroptosis, which was reduced by the silence or inhibitor of caspase-3. We further showed that knockdown of GSDME inhibited DOX-induced cardiomyocyte pyroptosis in vitro. Finally, DOX increased the expression of Bnip3, whereas silencing of Bnip3 blunted cardiomyocyte pyroptosis induced by DOX, which was regulated through caspase-3 activation and GSDME cleavage. Significance: Our findings revealed a novel pathway that cardiomyocyte pyroptosis is regulated through Bnip3-caspase-3-GSDME pathway following DOX treatment, suggesting that Bnip3-dependent pyroptosis may offer a novel therapeutic strategy to reduce cardiotoxicity induced by DOX.

Os campos elétricos alternados de frequência intermediária (10 KHz a 1MHz) e baixa intensidade têm efeitos inibitórios sobre o crescimento de várias linhagens celulares de tumores humanos e murinos. O objetivo geral do presente trabalho é conhecer os efeitos biológicos da aplicação externa de Campos Elétricos Alternados de intensidades de 10 V e 5 V nas frequências de 200kHz, 1Mhz e 2Mhz; em culturas de células de melanoma murino (B16F10) e fibroblastos humanos normais (FN1), durante 24 horas. Foram estudados os efeitos biológicos antiproliferativos e pró-apoptóticos através das seguintes análises: Citometria de Fluxo para identificar a distribuição das populações celulares nas fases do ciclo celular, Colorimétrico MTT para avaliar a viabilidade celular e Peroxidação Lipídica para avaliar a produção de radicais livres lipídicos poliinsaturados. Os resultados mostraram que em fibroblastos humanos normais o campo elétrico alternado induziu efeito antiproliferativo, indutor de necrose e apoptose, porém em menor efeito tóxico quando comparado às células de melanoma (B16F10), principalmente na frequência de 200 kHz. Por outro lado, na frequência de 2MHz e intensidade 10V, as células de melanoma presentes no sobrenadante e aderentes expressaram diferencialmente número de células mortas por apoptose (Anexina-V) e caspase-3 fosforilada. Os diferentes efeitos produzidos entre as duas linhagens estudadas atentam para o fato de que a compreensão dos mecanismos biofísicos da regulação do reparo e da proliferação celular envolve fenômenos celulares e vias de sinalização altamente complexas. / Electric fields of alternating intermediate frequency and low intensity have inhibitory effects on the growth of various tumor cell lines. The objective of this study was to evaluate the biological effects of external application of external alternating electric field (CEAE) intensities of 10 V and 5 V in the frequency 200kHz, 1MHz and 2MHz; in cultured murine melanoma cells (B16F10) and human fibroblasts normal (FN1). We studied the antiproliferative and pro-apoptotic by flow cytometry to identify the distribution of cell populations in the phases of the cell cycle, Colorimetric MTT to evaluate cell viability and lipid peroxidation to evaluate the production of free radicals lipid acids. The results showed that in fibroblasts (FN 1) the normal CEAE induced antiproliferative effect, inducing apoptosis and necrosis, but at a lower cytotoxic potential when applied to melanoma cells (B16F10), mainly in the frequency of 200 kHz. Moreover, the frequency of 2MHz and intensity 10V, the melanoma cells in supernatant and the members expressed differentially number of dead cells by apoptosis and caspase-3 phosphorylated. The different effects between the two strains studied to undermine the fact that understanding the biophysical mechanisms of regulation of repair and cell proliferation involves cellular phenomena and signaling pathways highly specific and complex.