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PASSOS, M.J. Efeito da Proteína Morfogenética Óssea 15 (BMP-15) e do Hormônio Folículo Estimulante (FSH) sobre o desenvolvimento de folículos ovarianos bovinos. 2012. 239 f. Dissertação (MESTRADO EM BIOTECNOLOGIA) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2012. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2017-09-01T12:13:07Z No. of bitstreams: 1 2012_dis_mjpassos.PDF: 1449315 bytes, checksum: 7fe646701a8e49c19398e06093742c0d (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2017-09-27T15:07:44Z (GMT) No. of bitstreams: 1 2012_dis_mjpassos.PDF: 1449315 bytes, checksum: 7fe646701a8e49c19398e06093742c0d (MD5) / Made available in DSpace on 2017-09-27T15:07:44Z (GMT). No. of bitstreams: 1 2012_dis_mjpassos.PDF: 1449315 bytes, checksum: 7fe646701a8e49c19398e06093742c0d (MD5) Previous issue date: 2012 / This study aims to 1) evaluate the effect of BMP-15 and FSH, alone or in different combinations on the in vitro development, survival and antrum formation of bovine secondary follicles and 2) quantify the levels of mRNA expression for BMP-15 receptors (BMPR-IB and BMPR-II), FSH receptor and Kit Ligand in follicles grown in vivo and in vitro. With regard to culture, secondary follicles were microdissected and cultured for twelve days in α-MEM+ alone, or supplemented with BMP-15 and/or FSH, alone or in various associations. After culture, follicular diameter were evaluated, viability and rate of antrum formation. For the analyzes gene expression, by real time PCR, were used follicles cultured in the different treatments for comparisons of the levels of mRNA. After the culture period it was found that the association of BMP+FSH (0-12) stimulated a significant increase in follicular diameter in relation to other treatments (p<0.05), as well as stimulated the formation of antral cavity more effectively the control (MEM) and BMP-15 alone (p<0.05). However, for survival, after 12 days treatment BMP+FSH (0-12) significantly reduced follicular viability when compared to other treatments (p<0.05). The results showed that the levels of mRNA for BMP receptor type I (BMPR-IB) were significantly higher in follicles cultured with MEM when compared to other treatments, while levels of mRNA for the receptor type II (BMPR-II) did not differ between treatments. The highest levels of mRNA for the FSH receptor were detected in follicles cultured with BMP-15 alone. In conclusion, this study demonstrated that combination of BMP-15+FSH (D0-D12) accelerates the growth of bovine preantral follicles cultured for 12 days, but this effect reduces the follicular viability, affecting the ultrastructural integrity of the follicles. Furthermore, the addition of BMP alone increased expression levels of RNAm for FSHR after 12 days of culture , showing that BMP-15 and FSH may contribute significantly to the in vitro development of preantral follicles cultured for 12 days in this specie. / Este trabalho tem como objetivo 1) avaliar o efeito da BMP-15 e do FSH, sozinhos ou em diferentes associações sobre o crescimento, sobrevivência e formação de antro em folículos secundários bovinos e 2) avaliar os níveis de expressão do RNAm para os receptores de BMP-15 (BMPR-IB e BMPR-II) em folículos crescidos in vitro. Com relação ao cultivo, folículos secundários foram microdissecados e cultivados por doze dias em α-MEM+ sozinho, ou suplementado com BMP-15 e/ou FSH, sozinhos ou em diferentes associações. Após o cultivo, foram avaliados o diâmetro folicular, a viabilidade e a taxa de formação de antro. A análise de expressão gênica foi realizada por PCR em tempo real, utilizando folículos cultivados nos diferentes tratamentos, para comparações dos níveis de RNAm. Após o período de cultivo verificou-se que a utilização da BMP+FSH (0-12) estimulou um aumento significativo no diâmetro folicular em relação aos demais tratamentos (p<0,05), bem como estimulou a formação da cavidade antral de forma mais efetiva que o controle (MEM) e a BMP-15 sozinha (p<0,05). No entanto, em relação à sobrevivência, após12 dias o tratamento BMP+FSH (0-12) reduziu significativamente a viabilidade folicular em relação aos demais tratamentos (p<0,05). Os resultados mostraram que os níveis de RNAm para o receptor de BMP-15 do tipo I (BMPR-IB) foram significativamente maiores nos folículos cultivados com MEM quando comparado aos demais tratamentos, enquanto que os níveis de RNAm para o receptor do tipo II (BMPR-II) não diferiram entre os tratamentos. Os maiores níveis de RNAm para o receptor de FSH foram detectados em folículos cultivados com BMP-15 sozinha. Em conclusão, este estudo demonstrou que a combinação de BMP-15 + FSH durante todo o período de cultivo (D0-D12) acelera o crescimento de folículos pré-antrais bovinos cultivados por 12 dias, mas este efeito reduz a viabilidade folicular, afetando a integridade ultra-estrutural dos folículos. Além disso, a adição de BMP isoladamente aumentou os níveis de expressão de RNAm para FSHR após 12 dias de cultivo,mostrando que BMP-15 e FSH podem contribuir significativamente para o desenvolvimento in vitro de folículos pré-antrais cultivados por 12 dias nesta espécie.

Submitted by Romulus Lima (romulus.lima@ufpe.br) on 2015-03-13T11:52:35Z No. of bitstreams: 2 DISSERTAÇÃO Matheus de Oliveira Pessoa.pdf: 1655237 bytes, checksum: cb17d863af7e79cde501c39393d25566 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T11:52:35Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Matheus de Oliveira Pessoa.pdf: 1655237 bytes, checksum: cb17d863af7e79cde501c39393d25566 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-07-30 / Diante da crise energética mundial, o Brasil vem tentando seguir os passos de países como Alemanha, Holanda e Noruega, investindo em tecnologias que busquem minimizar os efeitos deletérios ao meio ambiente. Nesse sentido, várias pesquisas vêm se destacando no âmbito do aproveitamento do biogás, produto do tratamento de esgoto que faz uso da tecnologia anaeróbia, que tem como produto final o gás metano, 21 vezes mais poluente que o clássico monóxido de carbono. Desta forma, este trabalho teve por objetivo geral estudar e avaliar a produção de biogás a partir de efluentes domésticos, urbanos e lixiviados. Para tanto utilizou o efluente e biomassa das Estações de Tratamento de Esgoto Domético (ETE) Mangueira e Cabanga, da Estação de Tratamento de Esgoto Industrial Brasil Kirin e da Estação de Tratamento de Lixiviado Muribeca. A pesquisa foi desenvolvida em 4 fases distintas, todas objetivando avaliar a produção de biogás, usando substratos e biomassas diferentes. Na fase1realizou um Pré-Tratamento com Peróxido de Hidrogênio, utilizando o substrato – Esgoto bruto da ETE Mangueira e a biomassa – Lodo da mesma ETE. Na fase 2 foi monitorada a produção de biogás em um Reator UASB em escala Piloto, utilizando os mesmos substrato e biomassa da fase anterior. Na fase 3 foi avaliada a produção de biogás através dos Ensaios de Atividade Metanogênica Específica (AME), utilizando como substrato os Esgotos brutos da ETE Mangueira, da ETE Cabanga, da ETEI Brasil Kirin e da ETL da Muribeca, e biomassa os Lodos da ETE Mangueira e da ETEI Brasil Kirin. E por ultimo a fase 4, avaliação da produção de biogás através dos ensaios BMP - Biochemical Methane Production, com os mesmos substratos e biomassa da fase 3. Os resultados mostraram que os modelos existentes na literatura não expressam de forma coerente a produção de biogás. Foi constatado que a DBO5 é um parâmetro de extrema importância para esta previsão e que os ensaios AME e BMP são ferramentas mais precisas na determinação do biogás gerado a partir de efluentes.

Os tecidos ósseos alógenos são utilizados nas cirurgias de reconstrução ortopédicas e odontológicas. O número crescente de transplantes de ossos na última década beneficia diversos pacientes. Os profissionais dos Bancos de Tecidos utilizam protocolos que reduzem os riscos de transmissão de doenças infectocontagiosas, entretanto, não eliminam esta possibilidade. Portanto, é importante que tecidos sejam esterilizados por um método eficaz, sendo usualmente aplicada a radiação ionizante. Porém, a radiação ionizante pode acarretar alterações estruturais e biológicas nos ossos em relação à dose. O objetivo deste trabalho foi estudar os efeitos da aplicação da radiação gama e de feixe de elétrons, nas doses de 15 kGy, 25 kGy e 50 kGy nos tecidos ósseos desmineralizados preservados liofilizados ou congelados e avaliar as possíveis alterações no colágeno, na concentração de proteínas totais, BMP-2 e BMP-7. Para tanto foram utilizadas as técnicas de espectroscopia Raman, Bradford e ELISA. Foram utilizadas 5 diáfises de femur humano e parte das amostras foram desmineralizadas. As proteínas ósseas foram extraídas e quantificadas. Com os resultados da espectroscopia Raman, observamos que a eficiência da desmineralização foi de 85 a 90% porém com alterações na estrutura do colágeno. Alterações também foram observadas nos ossos congelados e irradiados nas doses de 25 kGy e 50 kGy. Nos resultados da quantificação de proteínas totais e específicas, ocorreram diminuições gradativas das concentrações médias das proteínas em relação à dose de radiação nos grupos estudados. Nas doses de radiação usualmente aplicadas aos tecidos ósseos, 15 kGy ou 25 kGy, as reduções nas concentrações das BMP-2 e BMP-7, foram menores que 20%. As reduções na dose de 50 kGy foram entre 27% a 53%, sendo influenciadas pelo tipo de radiação e pelo tipo de preservação dos ossos. / Allogenic bone tissues are used in orthopedic and dental reconstructive surgery. The growing number of bone transplants in the last decade benefits many patients. The professionals of Tissue Banks use protocols that reduce the risk of transmission of infectious diseases, however do not eliminate this possibility. Therefore, it is important that tissues are sterilized by an effective method, usually being applied ionizing radiation. However, ionizing radiation can cause structural and biological changes in the bones relative to the dose. The objective of this work was to study the effects of the application of gamma radiation and electron beam at doses of 15 kGy, 25 kGy and 50 kGy in the bone tissues demineralized preserved freeze-dried or frozen and to evaluate possible changes in collagen, the protein concentration total, BMP-2 and BMP-7. For both Raman spectroscopy techniques, Bradford and ELISA were used. They used 5 diaphyses human femur and part of the samples was demineralized. Bone proteins were extracted and quantified. With the results of Raman spectroscopy, we found that the efficiency of demineralization was 85-90% but with changes in the structure of collagen. Changes were also observed in frozen and irradiated bone at doses of 25 kGy and 50 kGy. The results of quantification of total and specific proteins, there were gradual decreases in average concentrations of proteins in relation to radiation dose in both groups. In the radiation doses usually applied to the bone tissue (15 kGy and 25 kGy) reductions in concentrations of BMP- 2 and BMP-7, were lower than 20%. The reductions at a dose of 50 kGy were between 27% to 53%, being influenced by the type of radiation and the kind of preservation of the bones.

Installation of low-grade weirs in agricultural drainage ditches is being evaluated as an innovative, and cost effective, management practice that decreases nutrient concentrations and loads by increasing water volume and hydraulic residence time of the ditch. Results revealed that weirs significantly increased (P = 0.029) hydraulic residence time (HRT) and ditch water volumes, leading to considerable reductions in outflow water volumes (61%). Furthermore, ditches with weirs achieved greater (P = 0.09) cumulative outflow load reductions (96%) and greater (P = 0.029) concentration reductions during the biogeochemical reduction phase of the experiment. Similarly, field research from Terrace Ditch in Yazoo County, MS yielded significant percentage concentration reductions for baseflow (53%), stormflow (63%), and load (65%). Results from the experimental approach and field scale research offer promising insight into the future of low-grade weir’s establishment as an additional best management practice in agricultural landscapes.

The local release of complexed siRNA from biomaterials enables targeted therapy of specific cells and tissues. This thesis focused on gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA) as a drug delivery system for siRNA. The siRNA-loaded cGM were aggregated with SaOS-2 cells or human mesenchymal stem cells (hMSC) to microtissues and stimulated with osteogenic supplements. Cell survival and tissue formation in microtissues could be improved by incorporating cGM in spheroid cultures. We observed hydroxyapatite deposition in the particles in dependence of medium and cell type. Osteogenic stimulation with BMP-2 and simultaneous silencing of BMP-2 antagonist chordin accelerated matrix mineralization of the microtissues. Higher cross-linking degree of cGM positively influenced chordin silencing and alkaline phosphatase (ALP) activity as a marker for osteogenic differentiation. These higher cross-linked cGM mineralized in an osteogenic medium within 8–9 days, in presence and absence of cells. The effects of pre-differentiated and chordin-silenced microtissues were investigated by simulation of in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). Increased ALP activity and osteoprotegerin (OPG) secretion were observed after 14 days compared to co-cultures with siRNA-free controls. These results indicate that the pre-differentiated and silenced microtissues can induce osteogenic differentiation of surrounding unstimulated cells. Using the microtissue approach with siRNA complexed with tyrosine-modified low molecular weight polyethyleneimine (P10Y/P5Y) as transfection reagent was not successful. The results of this thesis indicate that the pre-differentiation of microtissues with BMP-2 in combination with chordin silencing stimulates and enhances osteogenic differentiation of other stem cells. As a combination of biomaterial, RNAi, and autologous cells, microtissues could be a promising approach to regenerating bone defects.:Chapter I: Controlled release of siRNA for bone tissue engineering Chapter II: Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration Chapter III: Mineralizing gelatin microparticles as cell carrier and drug delivery system for siRNA for bone tissue engineering Chapter IV: Tyrosine-modified polyethylenimines for siRNA transfection in microtissues Chapter IV: Final discussion

For an agricultural watershed, best management practice (BMP) is a conservational way to prevent non-point source pollution, soil and water loss and mitigate groundwater declination. In this dissertation, several BMPs of tail water recovery system, conservation tillage system and crop rotation were selected and evaluated in order to demonstrate the impacts of those activities on stream water quality and quantity. Besides, a land use change scenario was also evaluated. In order to evaluate the scenarios comprehensively, Soil and Water Assessment Tool (SWAT) and Annualized Agricultural Non-point Source Pollution (AnnAGNPS) were applied to simulate surface hydrology scenarios, and Modular flow (MODFLOW) models was used to simulate groundwater level change. This dissertation contains several novel methods regarding to model simulation including (i) using satellite imagery data to detect possible tail water recovery ponds, (ii) simulating surface and groundwater connected, (iii) selecting land use change area based on local trend and spatial relationship, (iv) comparing scenarios between two models. The outcomes from this dissertation included scenarios comparison on surface water quantity and quality, groundwater level change for long term simulation, and comparison between surface water models.

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El conocimiento de los mecanismos moleculares implicados en la modulación de las funciones biológicas de los osteoblastos, células responsables de la formación de hueso, es indispensable como punto de partida para el diseño de nuevas estrategias terapéuticas aplicables en las patologías óseas. La acción del trifosfato de adenosina (ATP) sobre los receptores purinérgicos de la membrana plasmática ha mostrado regular importantes funciones celulares de los osteoblastos. Los resultados expuestos en esta Tesis describen por primera vez, el efecto del ATP sobre la expresión de proteínas inductoras de formación de hueso en osteoblastos de calvaria de rata (OBCs), como así también la modulación de dicho suceso por la infección con los virus de Sarampión (MEV) y Junín (JUNV). Mediante diferentes técnicas, como ensayos de proliferación, determinación de deposiciones de calcio, citometría de flujo y reacción en cadena de la polimerasa en tiempo real, se demostró que la activación por ATP de los receptores P2Y2 estimula la proliferación y diferenciación de los OBCs. Además, se observó que estos receptores modulan positivamente la expresión de proteínas asociadas a la diferenciación osteoblástica como las proteínas morfogenéticas óseas (BMP-2, 4, 5 y 7), sialo-proteína ósea, colágeno y fosfatasa alcalina. Se demostró la participación de la vía de la fosfatidil inositol 3 quinasa / proteína quinasa B (PI3K/AKT) en la estimulación inducida por el ATP sobre la proliferación y diferenciación de los OBCs. También, el empleo de diferentes nucleótidos sugiere que la expresión génica de BMP-1, 3, y 6 estaría regulada por los subtipos P2Y6, P2Y4,1,12,13 y P2Y6,1,12,13 respectivamente. Además, por técnicas de microscopía convencional y de fluorescencia, western blot y ensayos de infectividad, se demostró que MEV o JUNV pueden infectar a los OBCs y modular la expresión de varias proteínas relacionadas con la diferenciación de los osteoblastos. La infección con MEV mostró analogía con datos reportados en la literatura, de biopsias de pacientes con otosclerosis, enfermedad de los huesecillos del oído que causa sordera. Sin embargo, a diferencia de lo observado para el tratamiento con ATP, esta modulación no ocurre a través de la vía PI3K/AKT. La activación de los receptores P2Y1, 12, 13 inhibe la multiplicación o liberación de MEV. En cambio en el caso de los OBCs infectados con JUNV, los tratamientos con nucleótidos no modificaron los títulos virales, sin embargo, generaron péptidos truncados derivados de la nucleoproteína viral característicos de la infección persistente de cultivos celulares por JUNV. Asimismo, se comprobó la capacidad de JUNV de infectar persistentemente a los OBCs. Estos resultados permiten especular acerca de las probables alteraciones inducidas por el virus en la respuesta de los OBCs a estímulos externos y ubica a estos sistemas virus-célula como modelos de estudio de enfermedades óseas de probable etiología viral como la otosclerosis. Los resultados aquí presentados contribuyen a la comprensión de los mecanismos involucrados en la modulación de la proliferación y maduración de osteoblastos por receptores purinérgicos y también como respuesta a la infección viral. / The knowledge of molecular mechanisms involved in the modulation of osteoblasts biological functions, cells responsible for bone formation, is essential as a starting point to design new therapeutic strategies applicable in bone diseases. The adenosine triphosphate (ATP) action on purinergic receptors of plasma membrane has shown to regulate important cellular functions in osteoblasts. The results presented in this thesis describe, for the first time, the ATP effect on the expression of proteins related to bone formation in rat calvarial osteoblasts (OBCs), as well as the modulation of this event when infected with Measles (MEV) and Junin (JUNV) virus. Using different techniques, such as proliferation trials, calcium deposition determination, flow cytometry and realtime quantitative polymerase chain reaction, it was demonstrated that the P2Y2 receptor activation by ATP, stimulates OBCs proliferation as well as differentiation. Moreover, it was observed that these receptors positively modulate those proteins associated with osteoblast differentiation such as bone morphogenetic proteins (BMP-2, 4, 5 and 7), bone sialoprotein, collagen and alkaline phosphatase. It has been demonstrated that the phosphatidyl inositol 3 kinase / protein kinase B (PI3K/AKT) pathway is involved in the proliferation and differentiation of OBCs. The use of different nucleotides also suggests that BMP-1, 3, and 6 gene expression would be regulated by P2Y6, P2Y4, 1, 12, 13 and P2Y6, 1, 12 and 13 subtypes receptors respectively. In addition, by conventional microscopy and fluorescence techniques, western blot and infectivity assays, it was demonstrated that both viruses are able to infect OBCs and modulate several proteins related to these cells differentiation. These results are in accordance with reports dealing with biopsies from patients suffering otosclerosis, a human disease of the ear ossicles which causes hearing impairment. However, by contrast to that observed for the treatment with ATP, viral modulation does not occur via PI3K/AKT. The P2Y1, 12, 13 receptors activation inhibit the MEV release or multiplicity; however in the case of OBCs infected with JUNV, the treatments with different nucleotides did not modify the viral titer but it induced the appearance of truncated peptides derived from the viral nucleoprotein, a salient feature of persistent infection of JUNV in vitro. In addition and accordance to the results shown here, JUNV was able to infect OBCs, persistently. On the other hand, results allow speculation about the viral modulation of OBCs response to external stimuli and the perspectives of viruscell systems as useful models in the study of otosclerosis, a bone disease associated to viral infection. The results presented here contribute to understand the mechanisms involved in the modulation of proliferation and maturation in osteoblasts by purinergic receptors as well as in response to viral infection.