O transplante intrauterino de células-tronco (TIUCT) é um método de tratamento de doenças genéticas, congênitas, hematológicas e imunológicas em um feto durante a gestação. Em pesquisa básica este modelo permite o estudo da dinâmica de migração, enxertia e estado funcional de diferentes tipos de células-tronco (CT). Estas células podem ser transplantadas em diferentes momentos do período gestacional, que pode ser dividido em três momentos do desenvolvimento fetal, sendo estes, diferentes funcionalmente. A escolha deste momento para o transplante influenciará tanto no comportamento celular quanto no resultado. Para o TIUCT são utilizadas as CT mesenquimais derivadas da medula óssea ou fetais ou hematopoiéticas. Para esta pesquisa utilizamos células-tronco derivadas da polpa dentária imatura humana (CTIPDh) as quais apresentam potencial pluripotente e propriedades imunomodulatórias. Nosso principal objetivo foi avaliar a capacidade migratória, bem como de proliferação e endereçamento (homing) das CTIPDh durante o terceiro período gestacional do desenvolvimento fetal no modelo canino. Todos os procedimentos experimentais foram elaborados sob protocolo anestésico apropriado e aprovados pelo comitê de ética da FMVZ da USP. Foram transplantadas via intraperitoneal (IP) 1x106 CTIPDh GFP+ em cada feto, durante procedimento cirúrgico de laparotomia exploratória com ultrassonografia guiada intraoperatóriamente em quatro fetos com idade gestacional aproximada de 45 dias, e outros dois fetos os quais não receberam o transplante, utilizados como controle. Avaliamos os fetos pré e pós-transplante através do ultrasson. Após sete dias, realizamos a ovário-salpingo-histerectomia (OSH) para a colheita dos fetos. Em seguida coletamos seus órgãos e tecidos os quais foram fixados em paraformoldeído a 4% e criopreservados a temperatura de -80oC. Analisamos a biodistribuição das CTIPDh dentro dos órgãos e tecidos em criocortes de 5µm sob microscopia Confocal. Constatamos o homing das CTIPDh nos órgãos derivados das linhas germinativas endodermais, ectodermais e mesodermais. No estômago e intestinos as CTIPD/GFP+ foram identificadas tanto no espaço intraglandular, como na camada muscular da mucosa; no fígado no parenquima hepático; no coração especialmente no tecido muscular do miocárdio; no cérebro nos vasos da substância branca, e cerebelo entre células de Purkinje. Na placenta estas células foram encontradas especialmente junto aos vasos. Quantificamos as CTIPD GFP+ utilizando a citometria de fluxo. Comparativamente dentre os órgãos analisados, obtivemos resultados expressivos do homing celular no miocárdio (~50%), no baço e fígado. Nossos resultados foram confirmados através das análises de imunohistoquímica e imunofluorescência utilizando os anticorpos Anti-núcleo (HuNu), Anti-CTIPD e Anti-GFP humanos. Concluímos que as CTIPDh apresentam grande potencial migratório e proliferativo após o TIUCT em fetos caninos. Estas células indiferenciadas demonstraram homing, especialmente nos tecidos: hematopoiéticos fetais (placenta, fígado e baço), tecido epitelial e glandular de órgãos, bem como de nichos perivasculares de CT. Estes dados sugerem que as CTIPD através do TIU, é uma alternativa viável, segura e promissora para o tratamento de doenças genéticas, congênitas, hematológicas e imunológicas. / Intra-uterine stem cells transplantation (IUSCT) is a method for the treatment of genetic, congenital, hematological, and immunological diseases. In basic research it provides a model for studying the dynamics of migration, graft and functional status of different types of stem cells. The cells can be transplanted in different moments of gestational period, which can be divided into quarters that are not functionally equivalent. The choice of the cells and quarter where the stem cells will be applied can influence cells behavior and results of transplantation. Fetal and adult hematopoietic or bone marrow derived mesenchymal stem cells (MSCs) were mainly used for IUSCT. We previously obtained human immature dental pulp stem cell (IDPSCs), which showed pluripotent potential and immune-compatible properties. The goal of our study was to evaluate migration capacity, proliferation and homing of IDPSCs after IUSCT during the third fetal period in dogs. All experimental procedures were approved by the Ethical Committee of the School of Veterinary Medicine and Animal Science of São Paulo University and were performed under appropriate anesthesia. 1x106 of undifferentiated GFP-positive human IDPSCs were transplanted following laparotomy and intraperitoneal injection under intra-operative ultrasound control into 5 fetuses at the 45 days of gestation. Five fetuses, which did not receive IDPSCs, were used as a control. Ultrasound analyses were performed daily before collection of the fetuses. After 7 days ovarian hysterectomy was performed, fetuses were collected; organs and tissues were isolated and fixed in 4% paraformaldehyde or cryopreserved. Biodistribution of IDPSCs within the organs and tissues were analyzed on cryosections (5µm) under Confocal Microscopy. Homing of IDPSCs was observed in organs derived from three germ lines, endoderm, ectoderm and mesoderm. In stomach and in intestine GFP IDPSCs were found in intraglandular space as well as in muscularis mucosae. In liver they appeared in hepatic parenchyma; in heart in myocardium and in brain in bold vessels, in cerebellum within Purkinje cells. Using Flow cytometry assay GFP IDPSCs graft was quantified. Among the different organs an expressive homing was observed in myocardium of heart (~50%), in spleen and liver. The IDPSCs were also found in canine placenta, especially in blood vessels. These data were confirmed using anti-human nucleus (HuNu), anti-GFP and anti-IDPSCs anti-bodies. Human IDPSCs showed high migration and proliferation potential after IUSCT in dog fetuses. Undifferentiated IDPSCs demonstrated homing in fetal hematopoietic (placenta), epithelial (gastric glands) and perivascular stem cells niches. Our data suggest that IDPSCs is a new promising source for genetic, congenital, hematological, and immunological treatment for those diseases through IUSCT.

O transplante intrauterino de células-tronco (TIUCT) é um método de tratamento de doenças genéticas, congênitas, hematológicas e imunológicas em um feto durante a gestação. Em pesquisa básica este modelo permite o estudo da dinâmica de migração, enxertia e estado funcional de diferentes tipos de células-tronco (CT). Estas células podem ser transplantadas em diferentes momentos do período gestacional, que pode ser dividido em três momentos do desenvolvimento fetal, sendo estes, diferentes funcionalmente. A escolha deste momento para o transplante influenciará tanto no comportamento celular quanto no resultado. Para o TIUCT são utilizadas as CT mesenquimais derivadas da medula óssea ou fetais ou hematopoiéticas. Para esta pesquisa utilizamos células-tronco derivadas da polpa dentária imatura humana (CTIPDh) as quais apresentam potencial pluripotente e propriedades imunomodulatórias. Nosso principal objetivo foi avaliar a capacidade migratória, bem como de proliferação e endereçamento (homing) das CTIPDh durante o terceiro período gestacional do desenvolvimento fetal no modelo canino. Todos os procedimentos experimentais foram elaborados sob protocolo anestésico apropriado e aprovados pelo comitê de ética da FMVZ da USP. Foram transplantadas via intraperitoneal (IP) 1x106 CTIPDh GFP+ em cada feto, durante procedimento cirúrgico de laparotomia exploratória com ultrassonografia guiada intraoperatóriamente em quatro fetos com idade gestacional aproximada de 45 dias, e outros dois fetos os quais não receberam o transplante, utilizados como controle. Avaliamos os fetos pré e pós-transplante através do ultrasson. Após sete dias, realizamos a ovário-salpingo-histerectomia (OSH) para a colheita dos fetos. Em seguida coletamos seus órgãos e tecidos os quais foram fixados em paraformoldeído a 4% e criopreservados a temperatura de -80oC. Analisamos a biodistribuição das CTIPDh dentro dos órgãos e tecidos em criocortes de 5µm sob microscopia Confocal. Constatamos o homing das CTIPDh nos órgãos derivados das linhas germinativas endodermais, ectodermais e mesodermais. No estômago e intestinos as CTIPD/GFP+ foram identificadas tanto no espaço intraglandular, como na camada muscular da mucosa; no fígado no parenquima hepático; no coração especialmente no tecido muscular do miocárdio; no cérebro nos vasos da substância branca, e cerebelo entre células de Purkinje. Na placenta estas células foram encontradas especialmente junto aos vasos. Quantificamos as CTIPD GFP+ utilizando a citometria de fluxo. Comparativamente dentre os órgãos analisados, obtivemos resultados expressivos do homing celular no miocárdio (~50%), no baço e fígado. Nossos resultados foram confirmados através das análises de imunohistoquímica e imunofluorescência utilizando os anticorpos Anti-núcleo (HuNu), Anti-CTIPD e Anti-GFP humanos. Concluímos que as CTIPDh apresentam grande potencial migratório e proliferativo após o TIUCT em fetos caninos. Estas células indiferenciadas demonstraram homing, especialmente nos tecidos: hematopoiéticos fetais (placenta, fígado e baço), tecido epitelial e glandular de órgãos, bem como de nichos perivasculares de CT. Estes dados sugerem que as CTIPD através do TIU, é uma alternativa viável, segura e promissora para o tratamento de doenças genéticas, congênitas, hematológicas e imunológicas. / Intra-uterine stem cells transplantation (IUSCT) is a method for the treatment of genetic, congenital, hematological, and immunological diseases. In basic research it provides a model for studying the dynamics of migration, graft and functional status of different types of stem cells. The cells can be transplanted in different moments of gestational period, which can be divided into quarters that are not functionally equivalent. The choice of the cells and quarter where the stem cells will be applied can influence cells behavior and results of transplantation. Fetal and adult hematopoietic or bone marrow derived mesenchymal stem cells (MSCs) were mainly used for IUSCT. We previously obtained human immature dental pulp stem cell (IDPSCs), which showed pluripotent potential and immune-compatible properties. The goal of our study was to evaluate migration capacity, proliferation and homing of IDPSCs after IUSCT during the third fetal period in dogs. All experimental procedures were approved by the Ethical Committee of the School of Veterinary Medicine and Animal Science of São Paulo University and were performed under appropriate anesthesia. 1x106 of undifferentiated GFP-positive human IDPSCs were transplanted following laparotomy and intraperitoneal injection under intra-operative ultrasound control into 5 fetuses at the 45 days of gestation. Five fetuses, which did not receive IDPSCs, were used as a control. Ultrasound analyses were performed daily before collection of the fetuses. After 7 days ovarian hysterectomy was performed, fetuses were collected; organs and tissues were isolated and fixed in 4% paraformaldehyde or cryopreserved. Biodistribution of IDPSCs within the organs and tissues were analyzed on cryosections (5µm) under Confocal Microscopy. Homing of IDPSCs was observed in organs derived from three germ lines, endoderm, ectoderm and mesoderm. In stomach and in intestine GFP IDPSCs were found in intraglandular space as well as in muscularis mucosae. In liver they appeared in hepatic parenchyma; in heart in myocardium and in brain in bold vessels, in cerebellum within Purkinje cells. Using Flow cytometry assay GFP IDPSCs graft was quantified. Among the different organs an expressive homing was observed in myocardium of heart (~50%), in spleen and liver. The IDPSCs were also found in canine placenta, especially in blood vessels. These data were confirmed using anti-human nucleus (HuNu), anti-GFP and anti-IDPSCs anti-bodies. Human IDPSCs showed high migration and proliferation potential after IUSCT in dog fetuses. Undifferentiated IDPSCs demonstrated homing in fetal hematopoietic (placenta), epithelial (gastric glands) and perivascular stem cells niches. Our data suggest that IDPSCs is a new promising source for genetic, congenital, hematological, and immunological treatment for those diseases through IUSCT.

No description available.

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.

This dissertation investigates the origins of tunable and efficient photochemistry in three different photoactive proteins, bacteriorhodopsin (BR), rhodopsin (RHO) and green fluorescent protein (GFP). In all cases, significant differences exist between the photoreactivity of model chromophores in solution and in the protein environment, in terms of excited state lifetime and efficiency of the primary photochemical process (opsin proteins) or the type of reaction (excited state proton transfer versus C=C double bond photoisomerisation for GFP). The work presented here investigates for each case to what extent the protein environment is necessary to alter the photochemistry of model chromophores in solution. For GFP and BR steric and electrostatic interactions between the protein pocket and the chromophore are shown to be likely responsible for shaping the excited state surface along which the photoreactions take place. For RHO it is suggested, contrary to current belief, that selection of a reactive ground state conformer might be the main effect generating the observed differences between solution and protein environment. The solution photochemistry of structurally modified retinal protonated Schiff bases, taken as model chromophores for the opsin proteins, is studied with continuous wave irradiation experiments and ultrafast transient spectroscopies. Surprisingly large differences are observed for the isomerisation reaction depending on the starting configuration (trans or cis) of the photoactive double bond. The current model for BR based on the tuning of the excited state barrier encountered along the isomerisation coordinate is expanded to include the changes in selectivity, speed and efficiency observed for a series of all-trans derivatives. For 11-cis, the photoisomerisation in solution is proposed to take place along a barrierless isomerisation coordinate, in contrast with the models currently available in literature. It is suggested that the protein might be discriminating between ground state conformers rather than significantly changing the topography of the reaction coordinate. For GFP, excited state Raman spectra are recorded for the wild-type protein, two mutants and a model chromophore in solution. It is suggested that the high frequency vibrational modes observed in the excited state spectra of the proteins but not of the model chromophore in DMSO are a sign of a tighter chromophore environment that inhibits the photoisomerisation reaction occurring in solution.

El aumento de la actividad minera en el Perú hace necesario el desarrollo de tecnologías rápidas y económicas de detección de contaminantes para su monitoreo y control. Implementando conocimientos de biología molecular y de la regulación génica podemos construir un circuito genético sintético que posibilite el monitoreo de sustancia toxicas que generen estrés oxidativo como son los compuestos cianurados. El objetivo de esta investigación es desarrollar un circuito genético sintético conformado por el promotor de la proteína del shock por fagos (psp) de Escherichia coli y las secuencia codificante del gen de la proteína verde fluorescente (GFP). La construcción de dicho circuito se logró usando estrategias de clonamiento por topoisomerasas y clonamiento clásico con enzimas de restricción, se usó la reacción en cadena de la polimerasa (PCR) para confirmar que todos los segmentos del circuito estén presentes en el vector. Los estudios preliminares de la actividad del nuevo circuito se realizaron transformando genéticamente células competentes de E. coli. La observación de dichas bacterias muestra una expresión de GFP continua, lo que indica que el circuito sintético está siendo activado sin estar en presencia de agentes de estrés oxidativo, lo que suponía una posible interacción con otros sistemas de regulación de estrés en la célula. Due to the increase of mining activity in Peru new technologies that can detect and monitor hazardous pollutants in a faster and cheaper way must be developed. Implementing molecular biology knowledge about genetic regulation we are able to construct a synthetic genetic circuit that can allow the monitoring of toxic substances that generate oxidative stress such us cyanide compounds. The objective of this research is to develop a synthetic genetic circuit from the promoter of the phage shock protein operon from E. coli and the complementary DNA of the green fluorescent gene (GFP). The construction of the circuit was achieved using classic cloning strategies with restriction enzymes and also more advanced strategies such us topoisomerase cloning, the polymerase chain reaction (PCR) was used to confirm the presence of all the desire segments in the vector. Preliminary studies of the circuit activity were carried out by genetically transforming competent E. coli cells. The observation of the bacteria shows a continuous expression of GFP without any inducer, this indicates that the synthetic circuit is being activated through a possible interaction with other stress response pathway.

Em 1978, o trabalho realizado por Stephenson e Zamecnik demonstrou a capacidade de um oligonucleotídeo de impedir a expressão de uma proteína específica. Atualmente, duas tecnologias são mais utilizadas para este propósito: os oligodeoxiribonucleotídeos antisense e o RNA de interferência (siRNA), que se aproveitam da capacidade de anelação entre as fitas complementares. A maior diferença entre as duas técnicas é a maquinaria proteica recrutada, isso é, o complexo RISC atua no funcionamento do siRNA, e a protease RNase H atua na clivagem da fita de RNA quando hibridizada com DNA. Apesar da grande aplicabilidade destas tecnologias, tanto para doenças metabólicas quanto para canceres, o veículo de entrega e proteção dessas sequências é de fundamental importância, visto que a aplicação desses oligonucleotídeos livres está sujeita à rápida degradação e ineficiência. A modificação das bases é uma das estratégias para conferir maior estabilidade às sequências, porém estas tem sido relacionadas a um aumento da toxicidade. Nessa dissertação, a quitosana, um polissacarídeo catiônico é utilizado para síntese de nanopartículas e encapsulamento dos oligodeoxiribonucleotídeos antisense (ASO). Para isso, foram realizadas modificações na quitosana comercial como despolimerização, trimetilação ou conjugação com PEG, seguida da síntese das nanopartículas com a adição de tripolifosfato de sódio (TPP) pelo método de gelatinização ionotrópica. A estabilidade das nanopartículas foi medida em função do tempo, da variação de temperatura e da diferença de pH. Além disso, a toxicidade dessas nanopartículas foi analisada através da viabilidade celular em diferentes linhagens, NB-4, HepaRG, HTC e BHK-570. A expressão da proteína verde fluorescente (GFP) na célula NB-4 foi utilizada para avaliar a entrega do ASO desenhado, sendo sua fluorescência monitorada por microscopia confocal. Os resultados demonstram que as nanopartículas se mantiveram estáveis durante o período de tempo analisado, assim como com a temperatura variando de 22 a 45&deg;C e em pH ácido. Cada linhagem celular respondeu de forma diferente ao tratamento com as nanopartículas sem ASO, sendo a linhagem saudável BHK-570 com a maior resistência. Ademais, todas as células apresentaram viabilidade reduzida quando tratadas com concentrações na ordem de 1011 nanopartículas/mL a base de quitosana trimetilada. A fluorescência das células NB-4 quando tratada com as nanopartículas com ASO diminuiu consideravelmente nas 18 primeiras horas, seguida de um aumento após 42 horas. Dessa forma, pode-se concluir que as nanopartículas de quitosana propostas nessa dissertação apresentaram uma excelente alternativa para a entrega de material genético, principalmente para o trato gastro-intestinal, devido à sua estabilidade em pH ácido. / The property of an oligonucleotide to interfere in the expression of a protein was observed in 1978 by Stephenson and Zamecnik. To perform such interference, there are today, two main techniques being explored: antisense oligodeoxyribonucleotides and interference RNA. In both cases, the particularity of their chemical structure is taken into account as soon as they can bind in a complementary manner to the messenger RNA and inhibit its translation. The great difference between these techniques is related to the proteases involved in the process, while for interference RNA the RISC machinery acts, for antisense oligodeoxyribonucleotides RNase H cleaves the RNA in the duplex DNA-RNA. Although these tools to edit the translation process are relevant to the treatment and even cure of metabolic disorders and cancers, it is still not effective when employed without a coating to protect the sequences before it reaches the destiny in vivo. Efforts have been made in developing modified bases to be more stable, but they show some toxicity. In this dissertation, chitosan, a natural cationic polyssacharide, is used to produce nanoparticles to protect the antisense oligodeoxyribonucleotide (ASO). For this reason, the commercial chitosan was modified, depolymerized, trimetilated or PEGlated and the nanoparticles were synthesized with sodium tripolyphosphate (TPP) by ionotropic gelation method. The stability along time, in different pHs and temperatures was assessed. The toxicity of nanoparticles without ASO was quantified by MTT tests in NB-4, HepaRG, HTC and BHK-570 cell lines. A green fluorescent protein (GFP) expressed by NB-4 cells was the target to evaluate the delivery efficiency of the ASO, and its fluorescence was measured by confocal microscopy. Results showed that nanoparticles were stable over time as well as in temperatures ranging from 22 to 45&deg;C and in acidic pH. Each cell line responded in a different manner to the treatment, with the health cell BHK-570 showing higher resistance. Furthermore, all of them presented lower viability when treated with trimetilated chitosan nanoparticles in the highest concentrations (ca 1011 nanoparticles/mL). NB-4 cells presented a decrease in fluorescence in 18 hours of treatment followed by an increase after 42 hours. We conclude that chitosan nanoparticles are a good alternative to the delivery of genetic material even more in the gastro intestinal tract due to its great stability in acid pH values.

A habilidade das células-tronco de se diferenciar em diferentes tipos celulares faz delas fortes candidatas para serem utilizadas em terapias celulares como tratamento de diversas doenças. No entanto, para explorar este potencial e necessário o estabelecimento de estratégias efetivas para modificações genéticas nessas células. Associado a outras tecnologias, o sistema de vetores lentivirais tem sido usado como um método atrativo para entrega de transgenes de interesse por possuírem uma alta eficiência de transdução. Além disso, eles permitem a transdução em células em proliferação ou quiescentes. A eficácia desses vetores para expressão de transgenes depende do uso de promotores corretos que possam garantir uma alta expressão genica e sustentável. Assim, o objetivo deste projeto foi comparar a eficiência dos promotores de citomegalovírus (CMV) e do fator de elongação-1<font face=\"Symbol\">a (EF1<font face=\"Symbol\">a) no sistema de entrega genica lentiviral em células-tronco mesenquimais de pele humana (HU). Para isso, foram construídos vetores lentivirais que possuem o promotor de CMV ou o promotor de EF1<font face=\"Symbol\">a, além do gene-repórter eGFP (\'\'enhanced green fluorescence protein\'\'). Para avaliar a eficiência de transfecção das construções, os vetores contendo os promotores e o gene-repórter (pLVCMVeGFP e pLVEF-1<font face=\"Symbol\">a-eGFP) foram utilizadas células 293T (produtora de partículas virais) e verificamos a presença da proteína fluorescente verde por microscopia de fluorescência, indicando a funcionalidade dos promotores. Visando analisar a eficiência das construções, células HU foram transduzidas e observamos a presença da proteína fluorescente nas transduções, demonstrando que os promotores encontram-se ativos em ambas as células. Após a verificação da eficácia das construções plasmidiais, foram feitas analises para avaliar a eficiência da transdução das construções em células HU. Para tal, as células foram transduzidas com lentivirus contendo um dos promotores analisados em diferentes multiplicidades de infecção (1, 5 e 10) e analisadas através da intensidade do sinal de fluorescencia e pela porcentagem de células eGFP positivas. Detectamos mais células eGFP positivas com a transdução do gene a partir do promotor PCMV do que com a do promotor PEF-1<font face=\"Symbol\">a. Assim, comparamos a eficiência destes promotores em MOI 1. Os resultados mostraram que a transdução com o promotor PEF-1<font face=\"Symbol\">a foi superior quando comparado ao promotor PCMV, portanto sendo a melhor escolha para esse sistema de entrega genica em células-tronco mesenquimais de pele humana. / The hability of stem cells to form a wide source spectrum of cell type makes them an interesting source for cell therapies. However, to exploit their remarkable potentials, the development of effective strategies for genetic modifications of MSCs is required. Lentiviral based vectors, with other techniques, offer an attractive system for efficient gene delivery in stem cells. These vectors efficiently transduce stem cells, and can infect dividing and nondividing cells. However, the efficiency of this vectors to genetic manipulation depends on the use of corrects cellular promoters for driving a high and stable expression of exogenous genes. In this study, we have chosen the cytomegalovirus (CMV) and the elongation factor 1-<font face=\"Symbol\">a promoters. We have compared the efficiency of this promoters in drive expression of the transgene in human dermal mesenchymal stem cells. The lentiviral vectors were constructed with the CMV promoter or the EF-1<font face=\"Symbol\">a promoter, together with the eGFP gene (enhanced green fluorescence protein). To evaluate the efficiency of the lentiviral vectors, 293T cells were transfected and analyzed for eGFP expression using fluorescence microscopy. We were able to observe the expression of eGFP, indicating that the vectors were working. Dermal mesenchymals stem cells were transduced with CMV- and EF-1<font face=\"Symbol\">a lentiviral vectors to evaluate the efficiency of the transductions. Efficiency of transductions was measured by flow cytometry (FACS) as percentage eGFP+ cells and signal intensity with different MOIs (multiplicity of infections).

Calcium plays important roles in both eukaryotic and prokaryotic cells. Its actions help to stabilize cell synthesis, growth and development. In this thesis, studies have been completed to determine effects of calcium and pH on bacterial cell growth and protein expression using the bacterial cell strain E.coli BL21(DE3). Our studies demonstrated the addition of calcium addition in the media does not affect growth but increases protein expression, while reducing the pH from 7 to 4 through the addition of 10mM EGTA in LB media inhibits both. Additionally, we report studies on the design, expression, and purification of fluorescent mCherry variants and their differences in their optical properties, including: extinction coefficients , quantum yields and pKa values. Also, we report progress in the crystallization of two GFP calcium sensors: G1 and D1, using 13 and15% PEG 4000 and 3350 respectively in 50mM HEPES buffer (pH 6.8-7.0) in an effort to optimize crystallization.

Soft sensors have been suggested as potent tools for on-line estimations of critical bioprocess variables to be able to control the biological process in an as high extent as possible. The formation of inhibitory by-products in the form of organic acids, caused by an overflow of glucose, is a problem in most bioprocesses expressing recombinant proteins.    In this project a new method of controlling the glucose feeding in an Escherichia coli fed-batch cultivation expressing the green fluorescent protein (GFP) was investigated. The new controller system implemented in the software controlled the feed rate based on on-line HPLC measurements of the concentration of organic acids.      The results showed that the controller managed to down-regulate the inhibitory organic acids to a low level as it tried to keep the glucose uptake rate at an optimum for maximum cell growth. The results suggested that the controller could be a powerful tool to create a more secure reproducibility and to generate high product yields in recombinant protein productions.