Krüppel-like factors (KLFs) are a family of transcription factors with 3 Cys2/His2 zinc fingers that regulate cell differentiation and developmental processes. EKLF is involved in primitive and definitive erythropoiesis; KLF2 is implicated in the development of primitive erythroid and endothelial cells of the vasculature. Using light and electron microscopy, the yolk sacs and dorsal aortae from EKLF/KLF2 double knockout (KO) E9.5 (embryonic day 9.5) were examined to determine whether these KLFs have compensatory functions in morphology of blood cells and vessels. EKLF/KLF2 double KO E9.5 erythroid, endothelial, and mesothelial cells had more severely abnormal morphology than WT and KLF2-/-. Flow cytometry and cytospins were used to determine maturational effects of single and EKLF/KLF2 double KO primitive erythroid cells double-labeled with anti-TER119 and anti-CD71. EKLF KO and EKLF/KLF2 double KO erythroid cells display defective erythroid maturation. EKLF and KLF2 have overlapping roles in the development of embryonic erythroid and endothelial cells.

The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)

During pregnancy, 4 - 7% of women suffer from major depressive disorder. When antidepressive treatment is needed, selective serotonin reuptake inhibitors (SSRIs) are the most commonly used. Although severe complications from SSRI treatment are rare, association with a number of adverse pregnancy and fetal outcomes has been found. Also, antenatal depression per se has been shown to affect pregnancy outcomes. The overall aim of this thesis was to examine the effects of SSRIs on human placenta and embryo. In the first study, gene expression was investigated in placenta from depressed, SSRI-treated and healthy pregnant women, using microarray analysis. Antenatal depression and SSRI treatment induced alterations in gene expression, but only 20 genes in common were noted. Validation with qRT-PCR showed that six out of seven selected genes were altered in SSRI-treated women compared with controls, and two genes were altered between depressed women and controls. In study two, the protein levels in placenta from depressed, SSRI-treated and healthy pregnant women were investigated, focusing on the NGF signaling pathway. NGF, phosphorylated Raf-1, ROCK2 and phosphorylated ROCK2, were altered in both SSRI-treated and depressed women, although the proteins were regulated differently in the two groups. In the third study, human embryos were treated with fluoxetine. Embryo development and protein expression were studied. Fluoxetine had some effect on the timing of embryo developmental stages. Also, several proteins were uniquely found in fluoxetine-treated embryos compared with untreated embryos. Fluoxetine also altered the levels of proteins secreted from the embryo. In the fourth study, the human neuroblastoma cell line SH-SY5Y/TrkA was treated with TPA and NGF. The activation of Raf-1 was investigated and the involvement of Ras and PKC was studied. Both NGF and TPA activated Raf-1, but to a different extent and via different pathways. The NGF-induced activation of Raf-1 was mediated via Ras, while TPA induced signaling via PKC. In conclusion, SSRI treatment and antenatal depression influence placental gene and protein expression. These findings may affect placental development and function, which in turn could affect fetal development. Also, direct exposure of embryos to fluoxetine has some effects on embryo development and protein expression, which may affect the development of the fetus.

Submitted by Marivaldo Rodrigues Figueiro (marivaldo.figueiro@embrapa.br) on 2018-05-02T19:49:49Z No. of bitstreams: 1 Tese_Marivaldo_Rodrigues_Figueiro.pdf: 1104812 bytes, checksum: 07b57b57c50c0ce48e486b2965bd2e74 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-04T17:05:00Z (GMT) No. of bitstreams: 1 figueiró_mr_dr_jabo.pdf: 1104812 bytes, checksum: 07b57b57c50c0ce48e486b2965bd2e74 (MD5) / Made available in DSpace on 2018-05-04T17:05:00Z (GMT). No. of bitstreams: 1 figueiró_mr_dr_jabo.pdf: 1104812 bytes, checksum: 07b57b57c50c0ce48e486b2965bd2e74 (MD5) Previous issue date: 2018-03-19 / O maior rebanho bubalino da América Latina encontra-se no Brasil, fato que pode fazer do país um grande exportador de material genético da espécie. Nesse contexto, o desenvolvimento e o uso das biotécnicas da reprodução animal surgem como eixo central para aumentar a capacidade de multiplicação de material genético superior e promover o melhoramento animal. Desta forma, este estudo trem como objetivo validar protocolos que proporcionem a obtenção de embriões com maior qualidade e menor acúmulo lipídico, os quais estão apresentados em dois capítulos. O capitulo I, é composto de revisão bibliográfica abordando os estudos relacionados aos principais aspectos na maturação in vitro de oócitos bubalinos, importância dos lipídeos nos oócitos e embriões produzidos in vitro, efeitos da redução lipídica no desenvolvimento oocitário e embrionário e o emprego da L-carnitina na produção embrionária. No capítulo II, está apresentado um artigo composto de experimento I, o qual foi a determinado a menor concentração de SFB no meio MIV que promovesse manutenção das taxas de desenvolvimento embrionário, No experimento II, foi avaliado a adição de 5 mM de L-carnitina nos meios de maturação e consequentemente forma submetidos à avaliação lipídica, por meio de técnicas de coloração em microscopia óptica e confocal. Onde concluímos que não houve diferenças em relação ao acúmulo lipídico embrionário e que possível reduzir a concentração de SFB até 5% nos meios de maturação in vitro para produção de embriões em bubalinos e a suplementação do meio com L-carnitina não proporciona aumento na produção embrionária. / The largest buffaloes herd in Latin America is in Brazil, a fact that can make the country a great exporter of genetic material of the species. In this context, the development and use of biotechnics of animal reproduction arise as a central axis to increase the multiplication capacity of higher genetic material and promote animal improvement. In this way, this study train as objective validate protocols that provide the obtaining of embryos with higher quality and lower lipid accumulation, which are presented in two chapters. Chapter I, is composed of a bibliographical revision addressing the studies related to the main aspects of the in vitro maturation of oocytes buffaloes, importance of the lipids in the oocytes and in vitro embryos produced, effects of the reduction lipid in oocitário and embryonic development and the use of L-carnitine in embryonic production. In chapter II, an article composed of experiment I, which was determined the smallest concentration of fetal bovine serum (FBS) in the IVM environment that promoted the maintenance of the embryo development rates. In the experiment II, was evaluated the addition of 5 mM of L-carnitine in the means of maturation and consequently form subjected to the lipid evaluation, by means of coloring techniques in optical and confocal microscopy. Where we concluded that there were no differences in relation to embryonic lipid accumulation and that it could reduce the concentration of FBS up to 5% in the IVM methods for the production of embryos in buffaloes and the supplementation of the medium with L-carnitine does not provide increase in embryonic production.

Made available in DSpace on 2015-05-14T13:00:18Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 4056353 bytes, checksum: ec317eee623ca7f537cb2773d854de24 (MD5) Previous issue date: 2011-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ca2+ is an intracellular messenger that controls a wide range of physiological functions through changes in its cytosolic concentration ([Ca2+]c). The increase in [Ca2+]c is derived of mobilization from intracellular stores or influx through channels, especially voltage-gated (Cav), present on cell surface. According to scientific literature, sea urchins embryogenesis is a process induced exclusively by the release of Ca2+ from the endoplasmic reticulum, and Ca2+ influx is not necessary for this process. However, there are studies in several species of the animal kingdom where Ca2+ influx is crucial for embryogenesis. Therefore, the aim of this study was to evaluate the involvement of Ca2+ influx at fertilization and embryonic development of Echinometra lucunter, a species of sea urchins with wide distribution on Brazilian coast. Thereby, eggs and embryos of E. lucunter were treated with various pharmacological tools and fertilization and embryonic development were monitored. Incubation of gametes in Ca2+ free medium inhibited fertilization and embryo treatment with Ca2+ chelators blocked embryonic development, suggesting that extracellular Ca2+ is essential for both processes. Cav blockers nifedipine, diltiazem and verapamil were also effective in blocking fertilization and embryo development, showing the importance of these channels to embryogenesis of E. lucunter. Inhibitory effect on embryo development is not associated with modulation of ABC superfamily proteins, since embryonic development was not affected, even under inhibition of these proteins. Verapamil inhibitory effect was reversed by prior addition of valinomycin which may be related to a probable increase of [Ca2+]c induced by K+ ionophore. ouabain, a Na+/K+-ATPase blocker that activates the reverse mode of Na+ /Ca2+, also reversed inhibition of development induced by verapamil. The reversal was not observed when compounds were added to embryos after verapamil, suggesting a temporal profile in inhibitory effect of these blockers. Inhibitory effects of verapamil and Ca2+ chelator EGTA were time-dependent, being absent 50 minutes after fertilization, suggesting that Ca2+ influx is seminal only in the first minutes of embryonic development. However, intracellular Ca2+ is essential for embryogenesis, since treatments with BAPTA-AM (chelator of intracellular Ca2+) and chlorpromazine or Trifluoperazine (Ca2+-calmodulin complex blockers) inhibited E. lucunter embryogenesis. Additionally, it was found that the rotundifolone, a plant-derived compound with vasorelaxing activity, attributed to the blockade of Cav, inhibited E. lucunter embryonic development showing an inhibitory profile similar to observed in vascular smooth muscle. Therefore, these results suggest that Ca2+ influx is essential for Echinometra lucunter embryogenesis and certify the embryonic development of this animal as appropriated pharmacological model for the investigation of natural and synthetic products that interferes in Ca2+ cellular dynamics. / O Ca2+ é um mensageiro intracelular que controla uma ampla variedade de funções fisiológicas por meio de alterações na sua concentração citosólica ([Ca2+]c). O aumento na [Ca2+]c é derivado da liberação a partir de estoques intracelulares ou do influxo através dos canais, principalmente os sensíveis à voltagem (Cav), presentes na superfície celular. De acordo com a literatura científica, a embriogênese de ouriços-do-mar é um processo regulado exclusivamente pela liberação de Ca2+ a partir do retículo endoplasmático, sendo o influxo dispensável para esse processo. Entretanto, há relatos em diversas espécies do reino animal onde o influxo de Ca2+ é crucial para a embriogênese. Portanto, o objetivo deste trabalho foi avaliar a participação do influxo de Ca2+ na fertilização e no desenvolvimento embrionário de Echinometra lucunter, uma espécie de ouriço-do-mar com ampla distribuição na costa Brasileira. Dessa forma, óvulos e embriões de E. lucunter foram tratados com diversas ferramentas farmacológicas e a fertilização e o desenvolvimento embrionário monitorados. A incubação dos gametas em meio isento de Ca2+ inibiu a fertilização e o tratamento dos embriões com quelantes de Ca2+ bloqueou o desenvolvimento embrionário, sugerindo que o Ca2+ extracelular é fundamental para ambos os processos. Os bloqueadores de Cav nifedipina, diltiazem e verapamil também foram eficazes no bloqueio da fertilização e do desenvolvimento embrionário, indicando a importância desses canais para a embriogênese de E. lucunter. O efeito inibitório sobre o desenvolvimento embrionário não está associado à modulação de proteínas da superfamília ABC, uma vez que o desenvolvimento embrionário ocorreu de forma normal, mesmo com a inibição dessas proteínas. O efeito inibitório do verapamil foi revertido pela adição prévia de valinomicina e tal fato pode estar relacionado a um provável aumento da [Ca2+]c induzido por este ionóforo de K+. A ouabaína, um bloqueador da Na+/K+-ATPase, capaz de ativar o modo reverso do trocador Na+/Ca2+, também reverteu a inibição do desenvolvimento induzida pelo verapamil. Essa reversão não foi observada quando os compostos foram adicionados aos embriões após o verapamil, sugerindo uma relação temporal no efeito inibitório desses bloqueadores. O efeito inibitório do verapamil e do quelante de Ca2+ EGTA foi dependente de tempo, sendo ausente 50 minutos após a fertilização, sugerindo que o influxo de Ca2+ é um fator determinante apenas nos primeiros minutos do desenvolvimento embrionário. Contudo, o Ca2+ intracelular é indispensável para a embriogênese, uma vez que os tratamentos com o BAPTA-AM, um quelante intracelular de Ca2+, e com trifluoperazina ou clorpromazina, bloqueadores do complexo Ca2+-calmodulina, inibiram a embriogênese de E. lucunter. Adicionalmente, foi verificado que a rotundifolona, um composto de origem vegetal com atividade vaso-relaxante atribuída ao bloqueio dos Cav, inibiu o desenvolvimento embrionário de E. lucunter, obtendo um perfil inibitório similar ao observado em musculatura lisa vascular. Portanto, esses resultados sugerem que o influxo de Ca2+ é essencial para a embriogênese de Echinometra lucunter e legitima o desenvolvimento embrionário desse animal como um excelente modelo farmacológico para a prospecção de produtos naturais e sintéticos bioativos que interferem na dinâmica celular do Ca2+.

<p>Infertility is the disease of the reproductive system and is estimated to affect more than 10% of the people of reproductive age. Assisted reproductive technologies (ART) are methods designed to alleviate infertility problems. <i>In vitro </i>embryo production is part of most infertility treatments and the efficiency of ART is low due to the lack of reliable methods to measure embryo viability. In order to improve the success rate of ART procedures, the current study was designed to investigate the use of an optical analyzer technology known as the Biodynamic Imaging (BDI) system for viability assessment. BDI is a novel approach that is able to measure intracellular dynamic processes that are directly related to functional events. During a series of experiments, 13 different biomarkers of oocytes and embryos were monitored by the BDI microscope and used for machine learning and evaluation of BDI sensitivity. We monitored cellular mechanisms essential for proper embryo development such as (1) extrusion of first and second polar body (2) energy status and mitochondrial activity, and (3) viability of embryos with different cellular composition. We were able to identify several biomarkers that have the potential to indicate viability: (1) slope, (2) NSD, (3) Knee (4) Floor, and (5) R² could consistently differentiate between oocytes and embryos of different viability. In addition, the BDI microscope could successfully predict the energy status of embryos by identifying 4 biomarkers (Slope, Knee, Floor, and Dy). Finally, a lipidomic analysis was done to evaluate the lipid composition of oocytes with different cytoplasm integrities. This analysis demonstrated that there is a difference in lipid subclasses among oocytes with dark vs. light cytoplasm. The results indicate that the BDI is useful for viability assessment of oocytes and embryos and may be helpful for the improvement of the efficiency of assisted reproductive technologies.</p>

A poluição do ar é associada a diferentes patologias inclusive as que afetam o sistema nervoso central. O objetivo deste estudo foi avaliar o efeito do material particulado fino (PM2,5) da cidade de São Paulo nas células de Purkinje de embriões de galinha. Dose única de PM2,5 em suspensão (1,5 ou 20,0 Pg.100Pl-1) foi injetada em ovos fertilizados de galinha em E0 foram artificialmente incubados por 18 dias (E18). Análises morfométricas no cerebelo foram realizadas em material submetido à reação imuno-histoquímica com anticorpos dirigidos à calbindina (CB), ao fator neurotrófico derivado do encéfalo (BDNF) e à caspase 3 (CAS) para avaliação da densidade de células de Purkinje, de seus dendritos e da taxa de apoptose nestas células. A expressão do BDNF no cerebelo foi determinada pelo método de immunoblotting. A ocorrência de elementos traço e de peroxidação lipídica no cerebelo foi avaliada, assim como a atividade da superóxido dismutase (SOD) e da catalase. Não foram observadas diferenças no desenvolvimento geral e na taxa de mortalidade dos embriões submetidos ao PM2,5 em comparação aos controles. Embora o PM2.5 em suspensão tenha apresentado elevada quantidade de elementos traço, depleção na concentrações de elementos como Cu, Mg, Mn, Se e Zn foi observada no cerebelo dos animais. Comparados ao controle salina, animais submetidos às concentrações de PM2,5 apresentaram diminuição da densidade de células de Purkinje CB+ nos grupos PM2,5 (18% no PM2,51,5 Pg e 23% no grupo PM2,5 20,0 Pg). Aumento de 45% na densidade de dendritos das células de Purkinje no grupo exposto a PM2,5 20,0 Pg foi observado. Redução significativa na expressão de BDNF no cerebelo e também na densidade de células de Purkinje BDNF+ no grupo PM2,5 20,0 Pg foi observada em relação aos controles e ao grupo PM2,51,5 Pg. A ocorrência de células de Purkinje apoptóticas não variou entre os grupos deste estudo da mesma forma em que a peroxidação lipídica e a atividade de SOD também não variaram. Contudo, foi observada maior atividade da catalase no cerebelo dos animais expostos ao material particulado. Conclusão: O material particulado fino da cidade de São Paulo afetou o desenvolvimento embrionário das células de Purkinje no modelo empregado e promoveu a ativação de mecanismos antioxidantes. A composição elementar do material particulado da cidade de São Paulo pode estar associada à perturbação da poda dendrítica nas células de Purkinje / Air pollutants are associated to several diseases including those related to central nervous system. The aim of this work was to evaluate the effect of urban fine particles (PM2.5) in chicken embryo Purkinje cells. A single dose of PM2.5 suspension in two different concentrations (1.5 or 20.0 Pg.100Pl-1) was injected in fresh laid fertilized eggs on E0. Control groups were performed (intact and saline groups). After 18 days of embryo incubation (E18), no differences in general abnormal development and mortality ratio were found between groups. Cerebellar morphometrical analysis for neuronal density, dendritic outgrowth and cellular apoptosis were scored in anti-CB, anti-BDNF and anti-CAS immunelabeled Purkinje cells. Measurements of trace elements, lipid peroxidation, superoxide dismutase and catalase were performed as well. PM2.5 suspensions presented high amounts of metals but the cerebellar tissue presented metal depletion. Compared to control animals (saline group) both PM2.5 doses exposed embryos showed a decreased number of Purkinje cell CB+ (ca. 18% for PM2.5 1.5 Pg and 23% for PM2.5 20.0 Pg) and increased Purkinje cell dendritic branches density in the PM2.5 20.0Pg exposed embryos (ca. 45% for PM2.5 20.0Pg). Interestingly, a significant reduction of Purkinje cell BDNF expression was observed in the PM2.5 20 Pg group embryos when compared to those of the control and PM2.5 1.5Pg groups. No alterations in the number of Purkinje cells CAS+ were observed. The lipid peroxidation and SOD activity showed similar levels in the cerebellar tissue of all experimental groups, although significant higher levels of CAT were detected in PM2.5 20 Pg group cerebella. In conclusion, urban fine particles impair the embryonic development of Purkinje cells in chick embryo model as well promote the antioxidant defense activation. PM2.5 elemental compounds may disrupt the physiological dendritic pruning of Purkinje cells

La maladie de Parkinson (PD) est considérée comme la deuxième maladie neurodégénérative la plus fréquente. L'examen post-mortem de patients parkinsoniens et des modèles physiologiques d’études de la maladie de Parkinson suggèrent la participation de la mort cellulaire programmée, l'inflammation et l'autophagie dues au stress oxydatif, à des mutations ou l’agrégation de protéines au sein des neurones DA. Les aptamères peptidiques sont de petites protéines combinatoires, consistitués d’une plateforme (dans notre cas, la thiorédoxine humaine, hTRX) et une boucle variable insérée dans le domaine actif de hTRX. Deux aptamères peptidiques ont été identifiés par la sélection fonctionnelle. L’aptamère peptide 32 (Apta-32) ,est spécifique liant deux paralogues T32 impliqués dans le processus d'endocytose. L’aptamère peptidique 34(Apta-34) lie à une cible "T34", une protéine pro-apoptotique ayant un rôle dans la voie apoptotique provenant du noyau. Le travail de cette thèse visait à étudier la fonction anti-apoptotique de nos deux aptamères peptidiques dans deux modèles d’étude de la maladie de Parkinson: un modèle cellulaire (in vitro) et un modèle transgénique D. melanogaster (in vivo). Deux toxines majeures ont été appliquées dans ce travail, 6-hydroxindopamine (6-OHDA) et le paraquat, un pesticide couramment utilisé. Nos observations montrent que la drosophile exprimant Apta-32 dans tous les neurones ont montré une meilleure résistance après 48h de traitement avec le paraquat comparé à deux autre aptamères peptidiques, Apta-34 et Apta-TRX (sans boucle de contrôle variable). Une autre étude a révélé un défaut dans la phagocytose des corps apoptotiques au cours du développement embryonnaire de la drosophile exprimant Apta-32 dans les macrophages, ce qui suggère qu’Apta-32 pourrait participer à et peut-être interférer avec le processus de l’autophygie, et que Apta-32 pourrait protéger contre l'autophagie induite par paraquat dans les neurones. / Parkinson’s disease is considered as the second most common neurodegenerative disease. Although the cause of the progressive cell loss of PD remains unclear to date, programmed cell death, inflammation and autophagy due to oxidative stress, gene mutations or protein aggregations within DA neuron have been suggested as potential causes. Peptide aptamers are small combinatorial proteins, with a variable loop inserted into a scaffold protein, human thioredoxin, hTRX. They are used to facilitate dissection of signaling networks by modulating specific protein interactions and functions. Two peptide aptamers were identified by functional selection which inhibit Bax-dependent cell death in mammalian models. One peptide aptamer (Apta-32) is binding two paralogues involved in endocytotic trafficking T32. The second peptide aptamer (Apta-34) is binding to a target "T34", a pro-apoptotic protein mediating apoptosis emanating from the nucleus. The work of my PhD thesis aimed to investigate the anti-apoptotic function of our two peptide aptamers in different PD models including cell model (in vitro), brain tissue slice and D. melanogaster (in vivo) ; in particular their impact on neuron survival after exposure to specific toxins. Two major toxins were applied in this work, 6-hydroxindopamine (6-OHDA) and Paraquat, a commonly used pesticide. Our observations indicated that Drosophila expressing Apta-32 in all neurons showed more resistance 48h after treatment with Paraquat, compared to drosophila expressing Apta-34 or TRX. Another study revealed a defect in phagocytosis of apoptotic bodies in drosophila embryo’s expressing Apta-32 in macrophage, suggesting Apta-32 could be involved in, and perhaps interfere with, the process of autophagy. This suggests that Apta-32 could protect against paraquat induced autophagy in neurons.

A poluição do ar é associada a diferentes patologias inclusive as que afetam o sistema nervoso central. O objetivo deste estudo foi avaliar o efeito do material particulado fino (PM2,5) da cidade de São Paulo nas células de Purkinje de embriões de galinha. Dose única de PM2,5 em suspensão (1,5 ou 20,0 Pg.100Pl-1) foi injetada em ovos fertilizados de galinha em E0 foram artificialmente incubados por 18 dias (E18). Análises morfométricas no cerebelo foram realizadas em material submetido à reação imuno-histoquímica com anticorpos dirigidos à calbindina (CB), ao fator neurotrófico derivado do encéfalo (BDNF) e à caspase 3 (CAS) para avaliação da densidade de células de Purkinje, de seus dendritos e da taxa de apoptose nestas células. A expressão do BDNF no cerebelo foi determinada pelo método de immunoblotting. A ocorrência de elementos traço e de peroxidação lipídica no cerebelo foi avaliada, assim como a atividade da superóxido dismutase (SOD) e da catalase. Não foram observadas diferenças no desenvolvimento geral e na taxa de mortalidade dos embriões submetidos ao PM2,5 em comparação aos controles. Embora o PM2.5 em suspensão tenha apresentado elevada quantidade de elementos traço, depleção na concentrações de elementos como Cu, Mg, Mn, Se e Zn foi observada no cerebelo dos animais. Comparados ao controle salina, animais submetidos às concentrações de PM2,5 apresentaram diminuição da densidade de células de Purkinje CB+ nos grupos PM2,5 (18% no PM2,51,5 Pg e 23% no grupo PM2,5 20,0 Pg). Aumento de 45% na densidade de dendritos das células de Purkinje no grupo exposto a PM2,5 20,0 Pg foi observado. Redução significativa na expressão de BDNF no cerebelo e também na densidade de células de Purkinje BDNF+ no grupo PM2,5 20,0 Pg foi observada em relação aos controles e ao grupo PM2,51,5 Pg. A ocorrência de células de Purkinje apoptóticas não variou entre os grupos deste estudo da mesma forma em que a peroxidação lipídica e a atividade de SOD também não variaram. Contudo, foi observada maior atividade da catalase no cerebelo dos animais expostos ao material particulado. Conclusão: O material particulado fino da cidade de São Paulo afetou o desenvolvimento embrionário das células de Purkinje no modelo empregado e promoveu a ativação de mecanismos antioxidantes. A composição elementar do material particulado da cidade de São Paulo pode estar associada à perturbação da poda dendrítica nas células de Purkinje / Air pollutants are associated to several diseases including those related to central nervous system. The aim of this work was to evaluate the effect of urban fine particles (PM2.5) in chicken embryo Purkinje cells. A single dose of PM2.5 suspension in two different concentrations (1.5 or 20.0 Pg.100Pl-1) was injected in fresh laid fertilized eggs on E0. Control groups were performed (intact and saline groups). After 18 days of embryo incubation (E18), no differences in general abnormal development and mortality ratio were found between groups. Cerebellar morphometrical analysis for neuronal density, dendritic outgrowth and cellular apoptosis were scored in anti-CB, anti-BDNF and anti-CAS immunelabeled Purkinje cells. Measurements of trace elements, lipid peroxidation, superoxide dismutase and catalase were performed as well. PM2.5 suspensions presented high amounts of metals but the cerebellar tissue presented metal depletion. Compared to control animals (saline group) both PM2.5 doses exposed embryos showed a decreased number of Purkinje cell CB+ (ca. 18% for PM2.5 1.5 Pg and 23% for PM2.5 20.0 Pg) and increased Purkinje cell dendritic branches density in the PM2.5 20.0Pg exposed embryos (ca. 45% for PM2.5 20.0Pg). Interestingly, a significant reduction of Purkinje cell BDNF expression was observed in the PM2.5 20 Pg group embryos when compared to those of the control and PM2.5 1.5Pg groups. No alterations in the number of Purkinje cells CAS+ were observed. The lipid peroxidation and SOD activity showed similar levels in the cerebellar tissue of all experimental groups, although significant higher levels of CAT were detected in PM2.5 20 Pg group cerebella. In conclusion, urban fine particles impair the embryonic development of Purkinje cells in chick embryo model as well promote the antioxidant defense activation. PM2.5 elemental compounds may disrupt the physiological dendritic pruning of Purkinje cells

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No. of bitstreams: 2 Tese - Poliana Carneiro Martins - 2017.pdf: 2303821 bytes, checksum: 3e74a5fc40c60b212e0c79f9270d5b43 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-26 / Two experiments were carried on to evaluate the effects of the inclusion of conjugated linoleic acid (CLA) on the diets of breeders and broilers, and the alterations caused by post-hatch fasting. In the first experiment, two commercial flocks of breeders aged 58 weeks old were used, and one of them was supplemented for 26 days with 0.025% CLA. At the end of the supplementation, a completely randomized design was applied, composed by two treatments (0 and 0.025% CLA), to evaluate the physical quality and composition of the eggs. Subsequently, 270 eggs per treatment were distributed in two hatcheries, in a randomized block design, determined according to the hatchery used. The incubation parameters were evaluated and the fatty acid profile of the feed, yolk and yolk sac were determined. After hatching, post-hatching diets (0 or 0.025% CLA) were provided for 12 hours, and 320 chicks were housed in a completely randomized design, in a 2x2 scheme (maternal CLA x post-hatching CLA), totalizing four treatments and eight replicates of 10 birds each. The organs relative weight, intestinal development and humoral and cellular immunity were evaluated in embryos, newly hatched chicks, 12 hours after hatching, and at seven days old, as the pre-starter performance. The inclusion of CLA in the diet was able to alter the physical and bromatological characteristics of the eggs and yolk sacs of the progeny from supplemented breeders, also changing the fatty acid profile, but without causing embryonic mortality. The intestine was also influenced by CLA, both macroscopically and histologically. The CLA offered to the breeders influenced the intestinal development of the chicks from the embryonic phase and continued to exert effect after hatching, associated with the progeny supplementation. Breeder supplementation led to a better pre-starter performance of the progeny. In the second experiment, 320 male and female chicks were distributed in a completely randomized design, in a 2x2 factorial scheme, considering post-hatch fasting (access to water for 12 hours; access to water and food for 12 hours) and post-hatch diet (0; 0.025% CLA). The birds were vaccinated at 15 days old against Newcastle disease virus. The metabolizability of diets from four to seven days old, intestinal and organs development, and cellular and humoral immunity were evaluated weekly up to 35 days old. Post-hatch fasting affected performance, and CLA was not able to minimize the negative effects of fasting. However, birds were able to recover up to 35 days old. Post-hatch fasting decreased the immune response of birds. In both assays, CLA generally positively influenced humoral and cellular immunity. Among the evaluated organs, the liver was the main one to have its relative weight altered due to the use of CLA. Supplementing only progeny did not provide as much benefit as compared to supplementation of the breeder or both. The obtained results demonstrate the inclusion level of 0.025% of CLA is safe for use in breeders and may bring benefits to their progeny. / Foram conduzidos dois experimentos para avaliar os efeitos da inclusão de ácido linoleico conjugado (CLA) na dieta de matrizes e frangos de corte, e as alterações causadas pelo jejum pós-eclosão. No primeiro experimento, foram acompanhados dois lotes de matrizes com 58 semanas de idade, sendo um suplementado 26 dias com 0,025% de CLA. Ao final da suplementação, adotou-se o delineamento inteiramente casualizado, composto por dois tratamentos (0 e 0,025% CLA), para avaliação da qualidade física e da composição dos ovos. Posteriormente, 270 ovos por tratamento foram distribuídos em duas incubadoras, aplicando-se o delineamento em blocos casualizados, determinados pela incubadora utilizada. Avaliaram-se os parâmetros de incubação e determinou-se o perfil de ácidos graxos da ração, gema e saco vitelínico. Após a eclosão, dietas pós-eclosão (0 ou 0,025% de CLA) foram fornecidas por 12 horas, e 320 pintos foram alojados em delineamento inteiramente casualizado e esquema 2x2 (CLA materno x CLA pós-eclosão), totalizando quatro tratamentos e oito repetições de 10 aves cada. Avaliou-se o peso relativo de órgãos, o desenvolvimento intestinal e a imunidade humoral e celular em embriões, neonatos, após 12 horas, e aos sete dias de idade, e o desempenho pré-inicial. A inclusão de CLA na dieta foi capaz de alterar características físicas e bromatológicas dos ovos e sacos vitelínicos da progênie de matrizes suplementadas, causando também alteração do perfil de ácidos graxos, porém sem provocar a morte dos embriões. O intestino também foi influenciado pelo CLA, tanto macroscopicamente, quanto a nível histológico. O CLA oferecido às matrizes influenciou o desenvolvimento intestinal dos pintos desde a fase embrionária e continuou a exercer efeito após a eclosão, aliado à suplementação conjunta da progênie. A suplementação da matriz garantiu melhor desempenho pré-inicial da progênie. No segundo experimento, 320 pintos machos e fêmeas foram distribuídos em delineamento inteiramente casualizado, em esquema fatorial 2x2, considerando os fatores jejum alimentar pós-eclosão (acesso à água por 12 horas; acesso à água e alimento por 12 horas) e a dieta pré-inicial (0; 0,025% de CLA). As aves foram vacinadas aos 15 dias de idade contra o vírus da doença de Newcastle. Avaliou-se a metabolizabilidade das dietas de quatro a sete dias de idade, e o desenvolvimento intestinal e dos órgãos, e a imunidade celular e humoral, semanalmente até os 35 dias de idade. O jejum pós-eclosão afetou o desempenho, e o CLA não foi capaz de minimizar os efeitos negativos do jejum. No entanto, as aves conseguiram se recuperar até os 35 dias de idade. O jejum pós-eclosão prejudicou a resposta imune das aves. Nos dois ensaios, de modo geral, o CLA influenciou positivamente a imunidade humoral e celular. Entre os órgãos avaliados, o fígado foi o principal a sofrer alterações em seu peso relativo em decorrência da utilização de CLA. A prática de suplementar apenas a progênie não trouxe tantos benefícios quando comparada à suplementação da matriz ou de ambos. Os resultados aqui obtidos demonstraram que o nível de inclusão de 0,025% de CLA é seguro para utilização em matrizes e pode trazer benefícios à progênie.