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Thesis (S.T.M.)--Dallas Theological Seminary, 1996. / Includes bibliographical references (leaves 47-49).

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-07-24T13:25:47Z No. of bitstreams: 1 julianapolisseni.pdf: 208270479 bytes, checksum: 5c1499cab892f30dc1d6d2d17268e467 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-09T13:28:51Z (GMT) No. of bitstreams: 1 julianapolisseni.pdf: 208270479 bytes, checksum: 5c1499cab892f30dc1d6d2d17268e467 (MD5) / Made available in DSpace on 2017-08-09T13:28:51Z (GMT). No. of bitstreams: 1 julianapolisseni.pdf: 208270479 bytes, checksum: 5c1499cab892f30dc1d6d2d17268e467 (MD5) Previous issue date: 2013-05-14 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Objetivou-se avaliar sistemas de cultivo propostos para utilização de embriões humanos: sistema de cultivo único e sistema de cultivo sequencial, comparando com sistema de cultivo utilizado nos modelos experimentais bovino e ratas Wistar. Especificamente avaliou-se taxa de clivagem e blastocisto, cinética do desenvolvimento embrionário, quantificação da expressão dos genes em blastocistos bovinos e avaliação da viabilidade e qualidade por morfologia e Tunel de embriões oriundos de ratas Wistar. Para bovinos, complexos cumulus-oócitos foram maturados, fecundados in vitro. No experimento 1 os zigotos foram distribuídos nos sistemas de cultivo: CR2aa, SOFaa, sistema de cultivo seqüencial ECM®/Multiblast® e sistema de cultivo único Global®. Avaliou-se a taxa de clivagem e de blastocisto e expressão dos genes PRDX, HSP70.1, GLUT1 e GLUT5, nos blastocistos. No experimento 2, o cultivo embrionário foi avaliado sobre o desenvolvimento de embriões bovinos biopsiados. Os zigotos foram distribuídos nos sistemas de cultivo: CR2aa, sistema de cultivo seqüencial G1®/G2® e ECM®/Multiblast®, sistema de cultivo único Global®. Para todos os grupos, os embriões 8-16 células foram biopsiados e retornados para cultivo. Avaliou-se taxa de clivagem e de blastocisto. Para ratas Wistar, utilizou-se animais, do Centro de Biologia da Reprodução (CBR/UFJF), com doze semanas de vida, superovuladas com 150 Ul/kg de PMSG e 75 Ul/kg de hCG, intraperitonealmente. No experimento 1 a coleta embrionária procedeu-se 24 horas após o hCG e avaliou-se a viabilidade embrionária No experimento 2 ratas foram distribuídas entre os grupos controle e superovulado e os embriões foram coletados 48 e 72 h após a administração de hCG. Peso dos ovários, número total de estruturas embrionárias e grau de qualidade de embriões foram analisados. No experimento 3 a coleta embrionária procedeu-se 72 h após a administração de hCG. Avaliou-se a qualidade de embriões cultivados in vitro oriundos de ratas superovuladas através da taxa de blastocisto e do índice apoptótico. Já no experimento 4 a coleta dos embriões foi realizada 48h após a administração do hCG. Embriões recuperados foram distribuídos aleatoriamente segundo os grupos: sistema de cultivo sequencial ECM°/Multiblast®, sistema de cultivo único Global®, KSOM. Avaliou-se taxa de blastocisto. A análise estatística foi realizada pelo teste do qui-quadrado, teste de Student, ANOVA e a expressão gênica foi analisada com REST®. No experimento 1 bovino as taxas de clivagem foram semelhantes entre os grupos, mas a taxa de blastocisto total foi menor no sistema sequencial e a taxa de blastocisto expandido foi maior no sistema de cultivo único. Além disso, o cultivo de embriões no sistema único resultou em maior expressão de GLUT1 e GLUT5 (P <0,01) e níveis de expressão semelhantes de HSP70.1 e PRDX1 em comparação com os embriões cultivados em meios sequenciais. No experimento 2 bovino tanto a taxa de clivagem quanto a taxa de blastocisto foi semelhante entre os grupos. Para ratos, no experimento 1 98,2% dos embriões coletados foram considerados viáveis. No experimentos 2 o grupo superovulado apresentou ovários com maior peso e maior número de embriões, comparado com o grupo controle. Entretanto, os embriões do grupo superovulado apresentaram menor número de células 72 horas após a administração de hCG. No experimento 3 a taxa de blastocisto foi de 83,17%, com taxa apoptótica de 10.32 ± 8.91%. No experimento 4 a taxa de blastocisto total foi menor em sistema sequencial (13,6%) quando comparado com KSOM (24,0%) e sistema de cultivo único (32,9%)(p<0.05). Concluiu-se que o cultivo de embriões em meio único é mais favorável em comparação com a de meios sequenciais, a superovulação foi bem sucedida no modelo rato e a técnica de TUNEL se mostrou viável na avaliação da qualidade embrionária de embriões de ratas Wistar. / The objective was to evaluate culture systems proposed to use in human embryos: single culture system and sequential culture system, compared with culture used in experimental model bovine and Wistar rats. Specifically we evaluated the cleavage and blastocyst rate, embryo kinetics, quantification of gene expression in bovine blastocysts and evaluated the viability and quality by morphology and Tunel of embryos derived from female rats. For bovine cumulus-oocyte complexes were matured, fertilized in vitro. In experiment 1 zygotes were distributed in culture systems: CR2aa, SOFaa, sequential culture system ECM®/Multiblast® and single culture system Global®. We evaluated cleavage and blastocyst rate and gene expression PRDX, HSP70.1, GLUT1 and GLUT5 in blastocysts. In experiment 2, the embryo development was evaluated on the development of bovine embryos biopsied. The zygotes were distributed in culture systems: CR2aa, sequential culture system G1e/GZ) and ECM®/Multiblast®, single culture system Global®. For all groups, the 8-16 cells embryos were biopsied and returned to culture. We evaluated cleavage and blastocyst rate. For Wistar rats, we used animals of the Center for Biology of Reproduction (CBR/UFJF), with twelve weeks of life and superovulated with 150IU/kg of PMSG and 75 IU/kg hCG intraperitoneally. In experiment 1 the embryo collection proceeded 24 hours after hCG and we evaluated embryo viability In experiment 2 rats were distributed between the control and superovulated and embryos were collected at 48 and 72 h after hCG administration. Weight of ovaries, the total number of embryonic structures and quality of embryos were analyzed. In experiment 3 embryo collection proceeded to 72 h after hCG administration. We evaluated the quality of embryos cultured in vitro from superovulated rat through the blastocyst rate and apoptotic index. Already in experiment 4 embryo collection was performed 48 h after hCG administration. Embryos recovered were randomized according to the groups: sequential culture system ECM®/Multiblast®, single culture system Global®, KSOM. We evaluated the rate of blastocyst. Statistical analysis was performed using the chi-square test, Student's test, ANOVA and gene expression was analyzed with REST®. In experiment 1 bovine cleavage rate was similar between groups, but the total blastocyst rate was lower in the sequential system and expanded blastocyst rate was higher in single culture system. Furthermore, the embryo cultured in single culture resulted in higher expression of GLUT1 and GLUT5 (P <0.01) and similar expression levels of HSP70.1 and PRDX1 compared to embryos cultured in sequential media. In experiment 2 both cleavage andblastocyst rate was similar between bovine groups. For rats in experiment 1, 98.2% collected embryos were were considered viable. In experiments 2 superovulated group showed ovaries with greater weight and greater number of embryos, compared with control group. However, embryos from superovulated group showed smaller number of cells 72 hours after hCG administration. In experiment 3 the blastocyst rate was 83.17%, with 8.91% ± 10:32 apoptotic rate of. In experiment 4 the total blastocyst rate was lower in sequential system (13.6%) compared to KSOM (24.0%) and single culture system (32.9%) (p <0.05). It was concluded that the culture of embryos in single medium is more favorable than sequential media, superovulation was successful in a rat model and the TUNEL technique proved feasible in evaluating the quality of embryos from embryonic Wistar rats.

Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.

<p dir="ltr">Cancer biological mechanisms are a vastly researched area in the field, yet they are not well understood in the various contexts in which cancer is found. Cancerous tumors often exist in harsh, stressful environments for normal cells, but cancer cells can thrive in these conditions. The tumor microenvironment (TME) typically has low oxygen levels (hypoxia), high acidity, and low nutrition. Exposure to the TME leads to many metabolic changes in the cells, enabling cancer to continue proliferating and migrating. However, these metabolic changes are not well understood, especially at the single-cell level. The ability to monitor cells in real time to determine the physical characteristics they undergo is critical to understanding the impact of these metabolic changes. Conventional methods focus on determining the genomic and proteomic changes in large numbers of cells, which may be overlooked if the changes are homogeneous across samples. In this work, we demonstrate the power of using multiple imaging techniques in combination with biochemical methods to visualize metabolic changes and determine the causes in various cancer cells under extreme hypoxia conditions.</p><p dir="ltr">The changes in the microtubule network that occur under hypoxia at the single-cell level are not widely researched. The use of confocal fluorescence microscopy can determine microtubule polymerization in conjunction with eGFP-transfected EB3, a protein that assists in microtubule polymerization. We have determined that hypoxic HeLa cells produce finger-like protrusions when exposed to hypoxia that help with cell migration and, ultimately, cancer cell metastasis. The formation of these protrusions is facilitated by localized mitochondria activities in the protrusions.</p><p dir="ltr">The metabolic changes in lipid droplets (LDs) under hypoxia at the single-cell level remain an elusive topic. The use of stimulated Raman spectroscopy (SRS) and coherent anti-Stokes Raman scattering (CARS) can determine the quantity and spatial-temporal distribution of LDs in cancer cells. We have found that LDs redistribute to the endoplasmic reticulum (ER) and increase in intensity in hypoxic MIA PaCa-2 and A549 cells. Time-lapse CARS microscopy revealed a release-accumulate process of these LDs on ER in hypoxia. We also studied the impact of carbon sources on LD formation and found that MIA PaCa2 cells prefer direct lipid uptake while glucose is also essential to reduce lipotoxicity. The use of hyperspectral stimulated Raman scattering (hSRS) also reveals that the content of the LDs changes to include less cholesteryl ester and a decrease in lipid saturation level.</p><p dir="ltr">Collectively, these findings shed new light on the understanding of cytoskeleton dynamics and lipid metabolism in hypoxic conditions. The discoveries made within this research would lead to better treatment strategies for effective treatment of hypoxia-resistant cancer cells.</p>

早期獨立音樂因非歸屬在主流唱片公司旗下，因為自身資源稀少，缺乏行銷管道和預算，音樂人除了要努力以外，亦需要有運氣，才能抓到機會曝光其作品，至於要成為當代明星更是難上加難。然而，21世紀的數位時代改變整個音樂產業結構，不管是MP3技術或是網路的無遠弗屆，都直接影響到傳統音樂產業營銷模式；尤其是音樂檔案的共享讓消費者開始於線上聆聽和下載音樂，改變了傳統的音樂消費習慣，數位音樂收益也隨之上升。當音樂消費從線下轉為線上，能讓過去資源稀少的音樂人有更多的行銷管道和曝光機會；而網路也讓音樂作品更快速被全世界聽到，像是Adele、One Republic等目前當紅明星都是從過去獨立音樂創作開始，透過線上平台而曝光並崛起。反觀臺灣的音樂市場現況，卻未因應數位時代的轉變創造出更多音樂明星，而臺灣目前音樂市場上有影響力的主流明星多半仍為2000年左右出道的歌手，呈現出明星斷層問題。 為了探究以上問題，本次聚焦探討目前臺灣獨立音樂產業結構，過去臺灣獨立音樂產業相關文獻較多以單向探討為主，例如針對文化政策、大眾媒體、獨立廠牌、Live House等問題作線性研究，較少以整體產業作多向分析。而本次研究希望透過多面的交錯關係分析獨立音樂產業結構，並以Mollard （2002）所提出的文化體系來解釋決策者、創作者、中介者及群眾四大組群做各面向關係，試圖分析四大群組在獨立音樂產業的缺口，以了解臺灣目前獨立音樂與主流音樂的斷層缺口，並建構出臺灣獨立音樂文化體系運作之規則。 本研究發現，數位時代下的網路平台力量不足以帶動整個音樂市場需求，甚至有減緩創造主流的現象，包含缺乏守門機制、網路分眾、樂評需求降低等因素。另外，四大群組中的中介者則在獨立音樂文化體系中扮演最重要的角色，擔任創作者與群眾之間的橋梁，因此，如何去培養群眾對於獨立音樂的包容性和多元的聆聽習慣為必須思考的方向之一。此外，透過培養音樂市場需求刺激創作供給，讓環境能夠利於創作者成長，再加政府落實文化補助、擴增硬體建設及法規制定相關的文化政策才能真正建立穩固及永續的獨立音樂文化體系。然而，目前四大群組仍存在需填補的缺口，透過檢視四大群組的關係才能更順利鏈結人才、創作品質、傳遞文化等產業關鍵能力，進而拉近獨立音樂與主流音樂之距離，以創造下一波音樂流行的可能。

The aortic valve functions in a complex mechanical environment which leads to force dependent cellular and tissue responses. Characterization of these responses provides a fundamental understanding of valve pathogenesis. The aim of this work was to develop an ex vivo organ culture system capable of simulating physiological aortic pressures and flow rates, and study the biological characteristics of native porcine aortic valves cultured in the system. Collagen, sGAG and elastin content of the valve leaflets were measured and cusp morphology, cell phenotype, cell proliferation and apoptosis were examined. Presence of endothelial cells (ECs) on the leaflet surface was also evaluated. The differences in collagen, sGAG and elastin contents were not significant (p greater than0.05) between the cultured and fresh valve leaflets. The cultured valves maintained the structural integrity of the leaflets while preserving the native morphology and cell phenotype. Cell phenotype in leaflets incubated statically under atmospheric conditions decreased compared to fresh and cultured valve leaflets, indicating the importance of mechanical forces in maintaining the natural biology of the valve leaflets. ECs were retained on the surfaces of cultured leaflets with no remodeling of the leaflets. The number of apoptotic cells in the cultured leaflets was significantly (p less than 0.05) less than in the statically incubated leaflets and comparable to fresh leaflets. The sterile ex vivo organ culture system thus maintained the viability and native biological characteristics of the aortic valves that were cultured under dynamic conditions for a period of 48 hours.

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.

Made available in DSpace on 2015-03-26T13:40:07Z (GMT). No. of bitstreams: 1 texto completo.pdf: 553604 bytes, checksum: a791b2e9f9e28a7c00d1599297d486ed (MD5) Previous issue date: 2007-07-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cow urine can be considered an agriculturist input that makes it possible for agriculturists to reduce property dependence on external products, especially in production of vegetables in organic system. The objective of this work was to evaluate the production of lettuce submitted to cow urine concentrations via soil and leaves. The experiment was conducted over the period from 1/13/2006 to 3/22/2006, at the Universidade Federal de Viçosa (UFV) campus, in a protected environment and organic culture system. It consisted of 12 treatments, conducted in subdivided parcel scheme, in randomized block design, with four repetitions; the application ways of cow urine (soil and leaf) were allocated in the parcels and the concentrations of cow urine solutions (0.00, 0.25, 0.50, 0.75, 1.00, and 1.25%) were allocated in the sub parcels. Weekly, starting after seven days of seedling transplant, 5, 5, 10, 20 and 20 mL of solution per plant were applied. Cultivar utilized was Regina 2000. Parcel consisted of 4 rows, and the space between them was 0.25 x 0.25 m, a total of 28 plants; the six central plants of the two central rows were considered as useful. Throughout the cycle the state of nitrogen was evaluated and in harvest, number of leaves, leaf area, leaf, stem and root fresh and dry mass, stem length, head volume, head fresh and dry mass, commercial productivity and leaf, stem and root levels of N, P, K, Ca, Mg, S, Na, Zn, Fe, Mn, Cu and B. Data were submitted to variance analysis, Tukey test at 5% probability, and regression analysis. When applied via soil, urine concentrations provided significant response in almost all phytotechnical characteristics, with the exception of leaf area; when applied via leaves, there was no effect on the number of leaves, leaf area, stem dry mass, root fresh mass and head volume. The application of cow urine either via leaves or via soil didn t alter the level of N, P, K, Ca, Mg, S, Na, Zn, Fe, Mn, Cu and B on plant or stem; as to the roots, there was significant response only to P, K, Fe and Mn when the urine was applied via soil and to Na when applied via leaves. The linear increment of SPAD index was observed with applied urine concentration, as well as quadratic response along time. The highest head productivity (17.00 Mg ha-1) was registered in plants that received urine solution via leaves in a concentration of 1.25%, corresponding to the increase of 28.1% in productivity, compared to control; when applied via soil, at the optimal concentration of 1.01%, productivity was of 14.92 Mg ha-1, corresponding to the increase of 47.3% in productivity, compared to control. The effects observed on lettuce growth and productivity, because of the applied urine solution are due to factors other then only quantity of nutrients used in the solutions. / A urina de vaca pode ser considerada um insumo agrícola que possibilita aos agricultores reduzir a dependência de produtos externos à propriedade, sobretudo na produção de hortaliças no sistema orgânico. O objetivo deste trabalho foi avaliar a produção de alface submetida a concentrações de urina de vaca aplicadas via solo e folhas. O experimento foi conduzido no período de 13/1/2006 a 22/3/2006, na Universidade Federal de Viçosa (UFV), em ambiente protegido e sistema orgânico de cultivo. Foi constituído de 12 tratamentos, conduzidos no esquema de parcelas subdivididas, em delineamento de blocos ao acaso, com quatro repetições; nas parcelas foram alocadas as via de aplicação da urina de vaca (solo e foliar) e nas subparcelas, as concentrações das soluções de urina de vaca (0,00, 0,25, 0,50, 0,75, 1,00 e 1,25%). Semanalmente, iniciando após sete dias do transplante das mudas, foram aplicados 5, 5, 10, 20 e 20 mL de solução por planta. A cultivar utilizada foi Regina 2000. A parcela foi constituída por quatro fileiras, em espaçamento de 0,25 x 0,25 m, totalizando 28 plantas; foram consideradas como úteis as seis plantas centrais das duas fileiras centrais. Ao longo do ciclo avaliou-se o estado de nitrogênio e na colheita, número de folhas, área foliar, massa fresca e seca de folhas, caule e raízes, comprimento de caule, volume de cabeça, massa seca e fresca da cabeça, produtividade comercial e teores dos elementos N, P, K, Ca, Mg, S, Na, Zn, Fe, Mn, Cu e B na massa seca da folha, do caule e da raiz. Os dados foram submetidos à análise de variância, teste de Tukey, a 5% de probabilidade e análise de regressão. Quando aplicadas via solo, as concentrações de urina proporcionaram resposta significativa em quase todas as características fitotécnicas avaliadas, com exceção da área foliar; quando aplicadas via folhas, não houve efeito sobre número de folhas, área foliar, massa seca de caule, massa fresca de raiz e volume de cabeça. A aplicação da urina de vaca tanto via folhas quanto via solo não alterou os teores de N, P, K, Ca, Mg, S, Na, Zn, Fe, Mn, Cu e B na folha e no caule; quanto às raízes, somente houve resposta significativa para o P, K, Fe e Mn quando a urina foi aplicada via solo e de Na, quando aplicada via folhas. O incremento linear do índice SPAD foi observado com o aumento da concentração de urina aplicada, assim como resposta quadrática ao longo do tempo. A maior produtividade de cabeça (17,00 Mg ha-1) foi registrada em plantas que receberam solução de urina via folhas à concentração de 1,25%, correspondendo ao aumento de 28,1% em produtividade, comparada à da testemunha; quanto aplicada via solo, na concentração ótima de 1,01%, a produtividade foi de 14,92 Mg ha-1, correspondendo ao aumento de 47,3% em produtividade, comparada à da testemunha. Os efeitos observados sobre crescimento e produção da alface, em razão das soluções de urina aplicada, são devido a fatores outros que não somente quantidade de nutrientes veiculados nas soluções.

Orientador: Ibsen Bellini Coimbra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T06:48:06Z (GMT). No. of bitstreams: 1 Bombini_MarianaFreschi_M.pdf: 1816531 bytes, checksum: 7a3edba503588d6825d034f24c884991 (MD5) Previous issue date: 2012 / Resumo: A utilização de células-tronco mesenquimais (CTM) para a reconstrução da cartilagem articular é uma promissora alternativa terapêutica, devido à vulnerabilidade do tecido a lesões e processo degenerativo irreversível. O objetivo deste estudo foi investigar o potencial condrogênico de CTM de líquido amniótico humano (CTM-LA) em sistema de cultura de micromass (alta densidade celular) com TGF-?3 por 21 dias. Métodos: 53 líquido amnióticos (LA) foram obtidos de mulheres submetidas à amniocentese durante o segundo trimestre de gravidez. A indicação da amniocentese foi feita pela obstetrícia, conforme protocolo específico do serviço de medicina fetal da UNICAMP. Foram selecionadas células-tronco mesenquimais, caracterizadas por citometria de fluxo. Estas células foram expandidas para obter um número populacional para o desenvolvimento do micromass. O micromass foi realizado em placa de cultura de 96 poços com fundo em "v", em cada poço foram pipetados 10?l contendo 5x105 CTM-LA e meio para diferenciação condrogênica contendo TGF-?3. Esta condição se manteve por 21 dias, e então, o potencial condrogênico foi avaliado pela presença da proteína do colágeno II pela técnica de western blotting, também foi avaliada a expressão gênica do Sox-9, colágeno II e agrecano pela técnica da PCR em tempo real (qRT-PCR). Comparamos CTM-LA em monocamada a CTM-LA submetidas ao sistema de cultura de micromass e como controle positivo utilizamos a cartilagem adulta humana. Resultados: Confirmamos o potencial condrogênico pela diferenciação das CTM-LA em condrócitos através da expressão dos genes SOX-9, colágeno tipo II e agrecano, bem como a proteína do colágeno II. A expressão de SOX-9 em micromass foi significativamente maior do que na cartilagem adulta. Conclusão: A condrogênese foi desenvolvida a partir da combinação de uma fonte de célula tronco recém descrita proveniente do líquido amniótico humano com o sistema de cultura de micromass. Esta fonte apresentou alto potencial condrogênico e dessa forma, fortes evidências para aplicações clínicas. Os resultados são promissores e sugerem a possibilidade de investimentos em bancos de líquido amniótico / Abstract: Introduction: The use of mesenchymal stem cells (MSC) for reconstruction of articular cartilage, leads to a promising therapeutic alternative, due to the tissue vulnerability to injuries and irreversible degenerative process. The aim of this study was to investigate the chondrogenic potential of MSC from human amniotic fluid in Micromass system (high-density cell culture) with TGF-?3 for 21 days. Methodology: The amniotic fluid was obtained from 53 pregnant women. The micromass was performed using MSC that was cultured in monolayer and chondrocytes from adult human normal cartilage as control groups. After 21 days, the chondrogenic potential was determined by metabolic products released from the cell, such as SOX-9, type II collagen and aggrecan. This study was approved by the ethics committee. Results: The proteic production of type II collagen was observed by Western Blotting. The genetic expression of SOX-9 was analyzed by PCR in real time, and this was found to be significantly higher than in adult cartilage. The same procedure was used to determinate the genetic expression of aggrecan and type II collagen, verifying positive result for both. Conclusion: Chondrogenesis was developed from the unique combination of the newly discovered source of mesenchymal stem cells from human amniotic fluid with micromass, and it demonstrated a satisfactory expression. Thus, this source is extremely viable for clinical applications, and the results suggest the possibility of investments in human amniotic fluid banks / Mestrado / Clinica Medica / Mestra em Clínica Médica