Parmi les différents minéraux argileux, la sépiolite, qui est un silicate fibreux naturel, est un potentiel nano-transporteur prometteur pour le transfert non-viral de biomolécules. Il a en effet été montré que la sépiolite interagissait avec des molécules biologiques telles que les lipides, les polysaccharides et les protéines. Dans ce travail, nous démontrons que la sépiolite interagit également efficacement avec différents types de molécules d'ADN (génomique, plasmidique, oligonucléotides simple et double brin), et nous présentons la première étude détaillée sur les mécanismes d'interaction entre la sépiolite et l'ADN, ainsi qu’une caractérisation physico-chimique de bionanocomposites ADN-sepiolite. Une analyse spectroscopique a montré tout d’abord que l’interaction de l'ADN avec la sépiolite était plus forte en présence de polycations, la valence de ces derniers accroissant le rendement d’absorption, et deuxièmement, que l'ADN ainsi adsorbé pouvait être récupéré avec un rendement modulé par la présence d’EDTA, la structure de l'ADN et son activité biologique étant conservées. Par spectroscopie infrarouge à transformée de Fourier (FTIR) nous avons identifié les groupes silanol externes comme les principaux sites d'interaction avec l'ADN. Nous avons ensuite prouvé qu'il est possible d'utiliser la sépiolite pour extraire l'ADN de bactéries, pour la purification de l'ADN et pour la purification de toute contamination bactérienne. En combinant la microscopie à fluorescence, la microscopie électronique à transmission (MET), la vidéo-microscopie et l’analyse par cytométrie en flux (FACS), nous avons montré que la sépiolite peut être spontanément internalisée dans des cellules de mammifère par le biais de deux voies, l’endocytose et la macropinocytose. En tant que preuve de concept, nous montrons que la sépiolite est capable de transférer de manière stable l'ADN de plasmide dans des bactéries et des cellules de mammifères. Il a également été prouvé qu’en incubant des bactéries avec des bionanocomposites ADN-sepiolite, initialement préparés en présence d'une faible concentration en cations divalents et avec de la sépiolite traitée aux ultrasons (sSep), il était possible d'augmenter l'efficacité de la transformation bactérienne 20 à 30 fois par rapport aux méthodes basées sur l'«effet Yoshida». En outre, nous montrons que l'efficacité du transfert de gènes par la sépiolite peut être optimisée : l'utilisation de sSep et l'exposition à la chloroquine augmentent d’un facteur 100 et 2, respectivement, l’efficacité de transfection. Ces résultats ouvrent la voie à l'utilisation de bionanocomposites à base de sépiolite comme de nouveaux potentiels nano-transporteurs hybrides potentiels, à la fois pour la thérapie génique et le développement de nouveaux modèles biologiques en sciences fondamentales et appliquées. / Among the various clay minerals, sepiolite, which is a natural fibrous silicate, isa potential promising nanocarrier for the non-viral transfer of bio-molecules. Indeed,sepiolite has been shown to interact with biological molecules such as lipids,polysaccharides and proteins. Here, we show that sepiolite efficiently binds differenttypes of DNA molecules (genomic, plasmid, single strand and double strandoligonucleotides), introducing the first detailed study on the interaction mechanismsbetween sepiolite and DNA, as well as the physicochemical characterization of theresulting DNA-sepiolite bionanocomposites. The interaction mechanisms aresuggested to be electrostatic interactions, van der Waals forces, cation bridges, andhydrogen bonding. Spectroscopy analysis showed that the binding of DNA to sepiolitewas increased by polycations with valence dependent efficiency, and the DNApreviously adsorbed could be recovered with an efficiency that could be modulatedusing a chelating agent (EDTA), preserving the DNA structure and biological activity.Fourier-transform infrared spectroscopy identified the external silanol groups as themain sites of interaction with the DNA. It was proved that it is possible to use sepiolitefor extracting DNA from bacteria, for DNA purification and for purification from bacterialcontamination. By combining fluorescence microscopy, transmission electronmicroscopy (TEM), time-lapse video microscopy and flow cytometry analysis (FACS),we show that sepiolite can be spontaneously internalized into mammalian cells throughboth endocytic and non-endocytic pathways. As a proof of concept, we show thatsepiolite is able to stably transfer plasmid DNA into bacteria and mammalian cells. Itwas also proved that with the incubation of bacteria with the Sep/DNAbionanocomposite initially prepared in the presence of a low concentration of divalentcation, and using sonicated sepiolite (sSep), it is possible to increase the bacterialtransformation efficiency from 20 to 30-fold compared to previously reported methodswhich are based in the “Yoshida effect”. Additionally, we show that the efficiency ofsepiolite-mediated gene transfer can be optimized: the use of sSep and the exposureto the endosome disrupter chloroquine 100-fold and 2-fold stimulated DNA transfectionefficiency, respectively. These results open the way to the use of sepiolite-basedbionanocomposites as a novel class of hybrid nanocarriers for both potential genetherapy and the development of novel biological models of interest for academic andapplied sciences.

Células de mamíferos são os hospedeiros mais frequentemente utilizados para a fabricação de proteínas biofarmacêuticas e para a produção de vacinas virais, A qualidade é um elemento-chave para o estabelecimento de um processo de bioconversão eficiente. No presente trabalho utilizamos a linhagem de células BHK- 21C13(Baby Hamster Kidney) adaptadas para cultivo em suspensão. O uso de Soro Fetal Bovino (SFB) é tradicionalmente utilizado, sendo considerado um suplemento universal, pois permite o crescimento em várias linhagens de células de mamíferos; porém, uso de SFB apresenta risco de infecção por prions, variabilidade entre lotes e aumento no custo em etapa de purificação (Downstream processing). O objetivo do presente trabalho foi comparar o cultivo de células BHK-21 C13 entre dois meios suplementados com SFB e sem SFB, através do estudo cinético para cultivo em suspensão estático e agitado com frascoT, frasco spinner e biorreator, respectivamente. Os parâmetros; Xmáx e µmáx, não foram significativamente influenciados pelo meio de cultura em cultivo estático, em cultivo com agitação em frasco spinner e também no cultivo em biorreator. O tempo de duplicação ficou próximo para todas as condições testadas. A produtividade alcançada foi: 0,032x106 cel/mL.h-1 para o meio com SFB e 0,031 X106 cel/mL.h-1 para o meio sem SFB. Ao final do processo foi possível obter uma concentração celular em torno de 4,7x106 cel/mL, tanto para o cultivo com SFB quanto para o cultivo sem SFB. Dessa forma, o uso de meio de cultivo sem SFB não alterou os principais parâmetros cinéticos, não apresentando as desvantagens do uso do SFB. / Mammalian cells are the most frequently used hosts for the production of biopharmaceutical proteins and viral vaccines. Quality is a key element for the establishment of an efficient bioconversion process. In this work, we used the cell line Baby Hamster Kidney C13 (BHK-21 C13) adapted to suspension culture was used. Fetal Bovine Serum (FBS) is traditionally used and it is considered a universal insert due to its power to increase cell growth in this kind of animal cells. However, the utilization of FBS introduces risks of infection from prions, variability between batches and increase in cost associated to purification stages (downstream processing). This work aimed to compare the kinetic behaviors of BHK-21 C13 cells in two media supplemented with FBS and without FBS using both one static and two suspension systems, T-flask, spinner flask and bioreactor respectively. The parameters; Xmax and µmax were not significantly influenced by the culture medium in T- flask culture static, in spinner flask cultivation and were neither significantly influenced by growing in culture media stirred bioreactor. The doubling time was close to all conditions tested. At the end of the growth phase it was possible to obtain a nearby cell concentration of 4.7 x 106 cells / ml, both for cultivation with FBS as for FBS without cultivation. Thus, the use of culture medium without FBS did not affect the main kinetic parameters. Besides, it does not show the disadvantages of culture media using FBS.

Atualmente, o Brasil encontra-se na privilegiada posição de maior produtor e exportador mundial de carne bovina, tornando a pecuária uma das atividades nacionais mais importantes e rentáveis. Este dado enfatiza a importância de pesquisa e desenvolvimento em reprodução bovina, especialmente em hormônios estimuladores da ovulação, tais como a gonadotrofina coriônica equina (eCG). Os produtos comerciais à base de eCG comercialmente disponíveis são purificados a partir do sangue de éguas gestantes, apresentando variabilidade de lote para lote e presença de contaminantes. Estes fatos, juntamente com a limitação do material de partida (sangue equino), enfatizam a necessidade de haver um sistema de expressão de eCG recombinante passível de ser explorado comercialmente. Neste quesito, as células de mamíferos se mostram um sistema robusto para tal finalidade, visto que são capazes de adicionar modificações pós-traducionais às cadeias polipeptídicas, tais como a glicosilação, o que é essencial para o correto dobramento, maturação e montagem das duas subunidades, além de interferir diretamente com a meia-vida, o reconhecimento do receptor, a solubilidade e a atividade biológica das proteínas. No entanto, mesmo entre os sistemas de expressão heteróloga em células de mamífero, encontra-se muita variabilidade nos padrões de glicosilação adicionado. No presente trabalho, foi realizado um estudo comparativo através da clonagem e expressão de uma forma fusionada de eCG (reCGβα) em duas linhagens celulares diferentes: (1) CHO-DG44, um dos sistemas de expressão mais utilizados pelas indústrias farmacêuticas, capaz de adicionar N-glicanos complexos; e (2) 293T, uma linhagem humana capaz de produzir glicoproteínas carreando oligossacarídeos complexos e sialilados. Os resultados de atividade biológica (in vitro e in vivo) apontam uma maior atividade de reCG produzido por células CHO-DG44. O perfil de N-glicosilação de reCG produzido pelas células CHOD-G44 assemelhou-se mais à eCG selvagem, quando comparado a reCG produzido por células 293T. Por fim, estudos clínicos foram realizados com reCG produzido em meio livre de soro fetal bovino e parcialmente purificado, onde atividade específica de reCG produzido por células CHO-DG44 mostrou-se similar ao produto comercial selvagem. / Brazil is currently the major beef producer and exporter, rendering to livestock one of the country´s most economically relevant activities. This emphasizes the importance of research and development in bovine reproduction, especially at ovulation-stimulatory hormones, such as equine gonadotropin (eCG). The commercially available eCG-based products are purified from blood of pregnant heifers, presenting batch-to-batch variability and the presence of contaminants. These facts, together with the limitation of the bulk material (equine blood), emphasize the need of an eCG expression system able to be commercially explored. In this aspect, mammalian cells are a robust system, capable of add post-translational modifications to polypeptide chains, such as glycosylation, which is essential for the correct folding, maturation and assembly of both eCG subunits. In addition, glycosylation directly interferes with the protein half-life, receptor recognition, solubility and biological activity. In the present work, a comparative study was carried out by cloning and expressing a fusion form of eCG (reCGβα) in two different mammalian cell lines: (1) CHO-DG44, one of the most used by pharmaceutical companies expression systems, capable of add complex-type N-glycans; and (2) 293T, a human cell line capable of produce glycoproteins carrying complex and sialylated oligosaccharides. The in vitro and in vivo biological activity results show a higher potency of reCG produced by CHO-DG44 cells. The N-glycosylation pattern produced by CHO-DG44 cells was more similar to native eCG in comparison to the N-glycosylation produced by 293T cells. Finally, clinical studies were performed with serum absent media produced and partially purified reCG, showing that the specific activity of reCG produced by CHO cells was similar to the commercial wild type product.

O objetivo deste trabalho foi estudar o processo pelo qual hormônios e fatores de crescimento controlam a proliferação celular em mamíferos. O modelo experimental utilizado foi linhagens de células estabelecidas em cultura. Os estudos centraram-se em dois tipos básicos de células: fibroblastos e células adrenais e o ataque experimental foi feito sob dois pontos de vista: bioquímico e genético. O ataque bioquímico envolveu desenvolver estudos cinéticos da síntese de DNA não só durante o carenciamento de células para soro, como também durante a reestimulação de células carenciadas por:soro, hormônios e fatores de crescimento. Medidas do conteúdo intracelular de cAMP foram efetuadas com o intuito de adquirir informações à respeito do mecanismo de ação destes fatores. Um modelo de ciclo celular foi proposto no qual o controle do crescimento seria exercido através de reguladores positivos e negativos que agiriam estimulando ou inibindo a passagem de células do estado de repouso (Go) para a fase proliferativa. Entre os reguladores positivos (estimuladores) do sistema fibroblasto, encontra-se hormônios clássicos, como esteróides e insulina, e fatores de crescimento de natureza hormonal como EGF, PF (fator proteico extraído de glândulas pituitárias) e prostaglandina F2α. O esteróide hidrocortisona pode agir como regulador negativo, inibindo o crescimento de fibroblastos. Medidas do período de tempo transcorrido desde a estimulação de células carenciadas (Go), até o aparecimento da onda de síntese de DNA (período definido operacionalmente como Gl) foram feitas. Em fibroblastos 3T3 este período foi de 12 a 13 horas tanto para células estimuladas com soro como com hormônios clássicos (hidrocortisona, insulina) ou fatores de crescimento (EGF, PF) ou ainda com combinações deles (EGF + PF + insulina; PF + hidrocortisona; PF + hidrocortisona + insulina). No sistema células adrenais, adrenocorticotropina (ACTH) foi o único hormônio clássico que apresentou atividade sobre o crescimento destas células e também o único efetuador negativo encontrado. Neste sistema PF mostrou-se como o único fator com atividade estimulatória sobre o crescimento. Gl aqui foi de 11 horas tanto para células estimuladas com soro como com PF. Além disso os hormônios clássicos hidrocortisona e insulina não apresentaram atividade estimulatória por si só ou em combinação com PF. A análise da ação de hidrocortisona no sistema fibroblasto e de ACTH no sistema células adrenais estimuladas, forneceu evidências de que após deixar Go, em direção a S, numa certa altura de Gl as células tornam-se irreversivelmente comprometidas com o processo replicativo. Este comprometimento parece ocorrer 5 horas antes de S, sendo referido como Glc. Em face destes resultados foi proposto que os reguladores agem estimulando ou inibindo a transição Go → Glc. Na tentativa de obter maior definição do sistema de controle do crescimento, aproveitamo-nos das vantagens oferecidas pelo modelo experimental usado, para a busca de mutantes do tipo regulatório. Esta busca resultou no isolamento das linhagens ST1 e AR-1, derivadas, respectivamente, de fibroblastos 3T3 e células adrenais Y-l. Entre os vários aspectos interessantes da linhagem ST1 destaca-se: a) o dramático efeito de hidrocortisona causando mudança nas características das células as quais passam de um fenótipo tipicamente transformado para normal. Este fenômeno foi observado tanto \"in vitro\" (através de medidas de parâmetros de crescimento) como \"in vivo\" (através de ensaios de tumorogenicidade); b) as alterações morfológicas de caráter antagônico provocadas, por um lado, pela adição de hidrocortisona (causando achatamento) e, por outro, pela retirada do soro ou adição de cAMP ao meio de cultura (arredondamento). Através do estudo da ação de inibidores, obteve-se evidências do envolvimento de microtúbulos nestas alterações morfológicas. A análise do conteúdo intracelular de cAMP indicou que este nucleotídeo não atua como mediador da ação de hidrocortisona. Sua ação parece ser devida à indução de alterações no sistema superfície celular - membrana -citoesqueleto. Ao contrário de outros variantes de células Y-l resistentes à ACTH, células AR-1 mostraram-se também resistentes a cAMP. A utilidade destas células nos estudos da postulada mediação deste nucleotídeo na ação de ACTH, é óbvia. / The aim of this work was to study the process by which hormones and growth factors control proliferation of mammalian cells. Cell lines established in culture were used as the experimental model. The studies were centered on two basic types of cells: fibroblasts and adrenal cells and the experimental approach was made from two viewpoints: biochemical and genetic. The biochemical approach involved kinetic studies of the DNA synthesis process not only during serum starvation but also during restimulation of serum starved cells by serum, hormones and growth factors. Intracellular cyclic AMP determinations were made in order to gain informations on the mechanism of action of these factors. A cell cycle model was proposed in which cell growth control would be exerted by positive and negative regulators that would act by stimulating or inhibiting the flow of cells from a resting state (Go) to the proliferative phase. Among the positive regulators (stimulators) found for the fibroblast system are: classical hormones, like steroids and insulin, and growth factors of homonal nature, like EGF, PF (protein factor extracted from pituitary glands) and prostaglandin F2α. The steroid hydrocortisone can also act as a negative regulator, inhibiting fibroblast growth. Measurements of the time interval between stimulation of serum starved (Go) cells and the onset of DNA synthesis (period that is operationally defined as Gl) were made. In 3T3 fibroblasts this period was 12 to 13 hours for cells stimulated not only by serum but also by classical hormones (hydrocortisone, insulin) or growth factors (EGF, PF) or even by combinations of these factors (EGF + PF + insulin; PF + hydrocortisone; PF + hydrocortisone + insulin). In the adrenal system, adrenocorticotropin (ACTH) was the only classical hormone to present activity on the growth of these cells and also the only negative regulator found. In this system PF was shown to be the only factor with growth stimulatory activity. GL was estimated as 11 hours for cells stimulated with serum or PF. Moreover hydrocortisone and insulin had no stimulatory activity \"per si\" or in combination with PF. The analysis of hydrocortisone action on the fibroblast system on one hand and of that of ACTH on the adrenal system, on the other, indicated that upon leaving Go, towards S, at a certain point in Gl, cells become irreversibly committed to the replicative process. This commitment seems to occur 5 hours before S and is referred to as Glc. In view of these data we proposed that regulators act by stimulating or inhibiting the transition Go → Glc. In an attempt to obtain a better definition of the growth control system and taking advantage of the experimental model utilized, we searched for mutants of the regulatory type. This search resulted in the isolation of the lines ST1 and AR-l from 3T3 fibroblasts and Y-l adrenal cells, respectively. Among several interesting aspects of the STl cell line, we point out: a) the dramatic effect of hydrocortisone changing the characteristics of these cells from a typically transformed phenotype to a normal pattern. This phenomenon was observed both \"in vitro\" (by measuring a number of growth parameters) and \"in vivo\" (by tumorogenicity assays). b) morphological alterations of antagonistic nature caused by hydrocortisone (flattening) on one hand, and by the removal of serum or cAMP addition to the culture medium (rounding) on the other. Evidence for the involvement of microtubules in these alterations were obtained through studies on the action of several inhibitors. Quantitative analysis of intracellular cAMP indicated that this nucleotide does not act as a mediator of hydrocortisone action. Rather, this action seems to be due to the induction of alterations on the cell surface-membrane-cytoskeleton system. Contrary to other variants of the Y-1 line which are resistant to ACTH, AR-1 cells are also resistant to cAMP. The usefulness of these cells in studies of the postulated mediation by cAMP of the ACTH action, is obvious.

Células de mamíferos são os hospedeiros mais frequentemente utilizados para a fabricação de proteínas biofarmacêuticas e para a produção de vacinas virais, A qualidade é um elemento-chave para o estabelecimento de um processo de bioconversão eficiente. No presente trabalho utilizamos a linhagem de células BHK- 21C13(Baby Hamster Kidney) adaptadas para cultivo em suspensão. O uso de Soro Fetal Bovino (SFB) é tradicionalmente utilizado, sendo considerado um suplemento universal, pois permite o crescimento em várias linhagens de células de mamíferos; porém, uso de SFB apresenta risco de infecção por prions, variabilidade entre lotes e aumento no custo em etapa de purificação (Downstream processing). O objetivo do presente trabalho foi comparar o cultivo de células BHK-21 C13 entre dois meios suplementados com SFB e sem SFB, através do estudo cinético para cultivo em suspensão estático e agitado com frascoT, frasco spinner e biorreator, respectivamente. Os parâmetros; Xmáx e µmáx, não foram significativamente influenciados pelo meio de cultura em cultivo estático, em cultivo com agitação em frasco spinner e também no cultivo em biorreator. O tempo de duplicação ficou próximo para todas as condições testadas. A produtividade alcançada foi: 0,032x106 cel/mL.h-1 para o meio com SFB e 0,031 X106 cel/mL.h-1 para o meio sem SFB. Ao final do processo foi possível obter uma concentração celular em torno de 4,7x106 cel/mL, tanto para o cultivo com SFB quanto para o cultivo sem SFB. Dessa forma, o uso de meio de cultivo sem SFB não alterou os principais parâmetros cinéticos, não apresentando as desvantagens do uso do SFB. / Mammalian cells are the most frequently used hosts for the production of biopharmaceutical proteins and viral vaccines. Quality is a key element for the establishment of an efficient bioconversion process. In this work, we used the cell line Baby Hamster Kidney C13 (BHK-21 C13) adapted to suspension culture was used. Fetal Bovine Serum (FBS) is traditionally used and it is considered a universal insert due to its power to increase cell growth in this kind of animal cells. However, the utilization of FBS introduces risks of infection from prions, variability between batches and increase in cost associated to purification stages (downstream processing). This work aimed to compare the kinetic behaviors of BHK-21 C13 cells in two media supplemented with FBS and without FBS using both one static and two suspension systems, T-flask, spinner flask and bioreactor respectively. The parameters; Xmax and µmax were not significantly influenced by the culture medium in T- flask culture static, in spinner flask cultivation and were neither significantly influenced by growing in culture media stirred bioreactor. The doubling time was close to all conditions tested. At the end of the growth phase it was possible to obtain a nearby cell concentration of 4.7 x 106 cells / ml, both for cultivation with FBS as for FBS without cultivation. Thus, the use of culture medium without FBS did not affect the main kinetic parameters. Besides, it does not show the disadvantages of culture media using FBS.

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.

Les structures d'acto-myosine sont impliquées dans de nombreuses fonctions cellulaires. Comprendre leur organisation et leur comportement collectif est toujours difficile. Nous avons étudié l'anneau cytokinétique dans les cellules de mammifères et dans les levures de fission, en orientant les cellules dans les microcavités, ce qui permet de voir l'anneau dans un seul plan focal. Avec cette configuration, nous révélons de nouvelles structures et des dynamiques distinctes pour les deux systèmes cellulaires. Dans les cellules de mammifères, nous trouvons des motifs réguliers de la myosine et la formine. Les caractéristiques de ces motifs sont stables tout au long de sa fermeture et leur apparition coïncide avec la constriction. Nous proposons que ce phénomène est une propriété inhérente du réseau d'acto-myosine et que la formation de ces motifs entraîne une augmentation du stress. Ces hypothèses sont confirmées par notre modèle en champ moyen. Par contraste, l'anneau de levure de fission montre des inhomogénéités tournantes de l'actine, de la myosine, des protéines de la construction de la paroi (Bgs) et d'autres protéines. La dynamique des inhomogénéités de myosine est inchangée, si la croissance de la paroi est inhibée. Cependant, l'inhibition du mouvement des inhomogénéités conduit à l'arrêt de la fermeture. Nous proposons que la fermeture de l'anneau est entraînée par la rotation de l'actine et de la myosine qui tirent des protéines Bgs, lesquelles construisent ainsi le septum. Cette hypothèse est confirmée par nos calculs et par des simulations numériques. Nous suggérons que la transition entre les états de différents ordres et dynamiques pourrait être une façon de réguler in vivo les systèmes d'acto-myosine. / Actomyosin structures are involved in many cell functions. Understanding their organization and collective behavior is still challenging. We study the cytokinetic ring in mammalian cells and in fission yeasts, by orienting cells in microcavities. This allows seeing the ring in a single plane of focus. With this setup, we reveal new structures and distinct dynamics for both cellular systems. In mammalian cells we find a pattern of regular clusters of myosin and formin. The characteristics of this pattern are stable throughout closure and its formation coincides with the onset of constriction. We propose that its characteristic is an inherent property of the actomyosin network and that its formation leads to an increase in stress generation. These hypotheses are supported by our theoretical mean field model. In contrast, fission yeast rings show rotating inhomogeneities (speckles), i.e. rotations of actin, myosin, cell wall building proteins (Bgs) and other proteins. Myosin speckles dynamic is unchanged, if wall growth is inhibited. However, the inhibition of speckle motion leads to stalled closure. We propose that the ring closure is driven by the rotation of actin and myosin, which pull Bgs thereby building the septum. This model is supported by our calculations and by numerical simulations. We suggest that the transition between states of different orders and dynamics might be a way to regulate actomyosin systems in vivo.

O objetivo deste trabalho foi estudar o processo pelo qual hormônios e fatores de crescimento controlam a proliferação celular em mamíferos. O modelo experimental utilizado foi linhagens de células estabelecidas em cultura. Os estudos centraram-se em dois tipos básicos de células: fibroblastos e células adrenais e o ataque experimental foi feito sob dois pontos de vista: bioquímico e genético. O ataque bioquímico envolveu desenvolver estudos cinéticos da síntese de DNA não só durante o carenciamento de células para soro, como também durante a reestimulação de células carenciadas por:soro, hormônios e fatores de crescimento. Medidas do conteúdo intracelular de cAMP foram efetuadas com o intuito de adquirir informações à respeito do mecanismo de ação destes fatores. Um modelo de ciclo celular foi proposto no qual o controle do crescimento seria exercido através de reguladores positivos e negativos que agiriam estimulando ou inibindo a passagem de células do estado de repouso (Go) para a fase proliferativa. Entre os reguladores positivos (estimuladores) do sistema fibroblasto, encontra-se hormônios clássicos, como esteróides e insulina, e fatores de crescimento de natureza hormonal como EGF, PF (fator proteico extraído de glândulas pituitárias) e prostaglandina F2α. O esteróide hidrocortisona pode agir como regulador negativo, inibindo o crescimento de fibroblastos. Medidas do período de tempo transcorrido desde a estimulação de células carenciadas (Go), até o aparecimento da onda de síntese de DNA (período definido operacionalmente como Gl) foram feitas. Em fibroblastos 3T3 este período foi de 12 a 13 horas tanto para células estimuladas com soro como com hormônios clássicos (hidrocortisona, insulina) ou fatores de crescimento (EGF, PF) ou ainda com combinações deles (EGF + PF + insulina; PF + hidrocortisona; PF + hidrocortisona + insulina). No sistema células adrenais, adrenocorticotropina (ACTH) foi o único hormônio clássico que apresentou atividade sobre o crescimento destas células e também o único efetuador negativo encontrado. Neste sistema PF mostrou-se como o único fator com atividade estimulatória sobre o crescimento. Gl aqui foi de 11 horas tanto para células estimuladas com soro como com PF. Além disso os hormônios clássicos hidrocortisona e insulina não apresentaram atividade estimulatória por si só ou em combinação com PF. A análise da ação de hidrocortisona no sistema fibroblasto e de ACTH no sistema células adrenais estimuladas, forneceu evidências de que após deixar Go, em direção a S, numa certa altura de Gl as células tornam-se irreversivelmente comprometidas com o processo replicativo. Este comprometimento parece ocorrer 5 horas antes de S, sendo referido como Glc. Em face destes resultados foi proposto que os reguladores agem estimulando ou inibindo a transição Go → Glc. Na tentativa de obter maior definição do sistema de controle do crescimento, aproveitamo-nos das vantagens oferecidas pelo modelo experimental usado, para a busca de mutantes do tipo regulatório. Esta busca resultou no isolamento das linhagens ST1 e AR-1, derivadas, respectivamente, de fibroblastos 3T3 e células adrenais Y-l. Entre os vários aspectos interessantes da linhagem ST1 destaca-se: a) o dramático efeito de hidrocortisona causando mudança nas características das células as quais passam de um fenótipo tipicamente transformado para normal. Este fenômeno foi observado tanto \"in vitro\" (através de medidas de parâmetros de crescimento) como \"in vivo\" (através de ensaios de tumorogenicidade); b) as alterações morfológicas de caráter antagônico provocadas, por um lado, pela adição de hidrocortisona (causando achatamento) e, por outro, pela retirada do soro ou adição de cAMP ao meio de cultura (arredondamento). Através do estudo da ação de inibidores, obteve-se evidências do envolvimento de microtúbulos nestas alterações morfológicas. A análise do conteúdo intracelular de cAMP indicou que este nucleotídeo não atua como mediador da ação de hidrocortisona. Sua ação parece ser devida à indução de alterações no sistema superfície celular - membrana -citoesqueleto. Ao contrário de outros variantes de células Y-l resistentes à ACTH, células AR-1 mostraram-se também resistentes a cAMP. A utilidade destas células nos estudos da postulada mediação deste nucleotídeo na ação de ACTH, é óbvia. / The aim of this work was to study the process by which hormones and growth factors control proliferation of mammalian cells. Cell lines established in culture were used as the experimental model. The studies were centered on two basic types of cells: fibroblasts and adrenal cells and the experimental approach was made from two viewpoints: biochemical and genetic. The biochemical approach involved kinetic studies of the DNA synthesis process not only during serum starvation but also during restimulation of serum starved cells by serum, hormones and growth factors. Intracellular cyclic AMP determinations were made in order to gain informations on the mechanism of action of these factors. A cell cycle model was proposed in which cell growth control would be exerted by positive and negative regulators that would act by stimulating or inhibiting the flow of cells from a resting state (Go) to the proliferative phase. Among the positive regulators (stimulators) found for the fibroblast system are: classical hormones, like steroids and insulin, and growth factors of homonal nature, like EGF, PF (protein factor extracted from pituitary glands) and prostaglandin F2α. The steroid hydrocortisone can also act as a negative regulator, inhibiting fibroblast growth. Measurements of the time interval between stimulation of serum starved (Go) cells and the onset of DNA synthesis (period that is operationally defined as Gl) were made. In 3T3 fibroblasts this period was 12 to 13 hours for cells stimulated not only by serum but also by classical hormones (hydrocortisone, insulin) or growth factors (EGF, PF) or even by combinations of these factors (EGF + PF + insulin; PF + hydrocortisone; PF + hydrocortisone + insulin). In the adrenal system, adrenocorticotropin (ACTH) was the only classical hormone to present activity on the growth of these cells and also the only negative regulator found. In this system PF was shown to be the only factor with growth stimulatory activity. GL was estimated as 11 hours for cells stimulated with serum or PF. Moreover hydrocortisone and insulin had no stimulatory activity \"per si\" or in combination with PF. The analysis of hydrocortisone action on the fibroblast system on one hand and of that of ACTH on the adrenal system, on the other, indicated that upon leaving Go, towards S, at a certain point in Gl, cells become irreversibly committed to the replicative process. This commitment seems to occur 5 hours before S and is referred to as Glc. In view of these data we proposed that regulators act by stimulating or inhibiting the transition Go → Glc. In an attempt to obtain a better definition of the growth control system and taking advantage of the experimental model utilized, we searched for mutants of the regulatory type. This search resulted in the isolation of the lines ST1 and AR-l from 3T3 fibroblasts and Y-l adrenal cells, respectively. Among several interesting aspects of the STl cell line, we point out: a) the dramatic effect of hydrocortisone changing the characteristics of these cells from a typically transformed phenotype to a normal pattern. This phenomenon was observed both \"in vitro\" (by measuring a number of growth parameters) and \"in vivo\" (by tumorogenicity assays). b) morphological alterations of antagonistic nature caused by hydrocortisone (flattening) on one hand, and by the removal of serum or cAMP addition to the culture medium (rounding) on the other. Evidence for the involvement of microtubules in these alterations were obtained through studies on the action of several inhibitors. Quantitative analysis of intracellular cAMP indicated that this nucleotide does not act as a mediator of hydrocortisone action. Rather, this action seems to be due to the induction of alterations on the cell surface-membrane-cytoskeleton system. Contrary to other variants of the Y-1 line which are resistant to ACTH, AR-1 cells are also resistant to cAMP. The usefulness of these cells in studies of the postulated mediation by cAMP of the ACTH action, is obvious.

Atualmente, o Brasil encontra-se na privilegiada posição de maior produtor e exportador mundial de carne bovina, tornando a pecuária uma das atividades nacionais mais importantes e rentáveis. Este dado enfatiza a importância de pesquisa e desenvolvimento em reprodução bovina, especialmente em hormônios estimuladores da ovulação, tais como a gonadotrofina coriônica equina (eCG). Os produtos comerciais à base de eCG comercialmente disponíveis são purificados a partir do sangue de éguas gestantes, apresentando variabilidade de lote para lote e presença de contaminantes. Estes fatos, juntamente com a limitação do material de partida (sangue equino), enfatizam a necessidade de haver um sistema de expressão de eCG recombinante passível de ser explorado comercialmente. Neste quesito, as células de mamíferos se mostram um sistema robusto para tal finalidade, visto que são capazes de adicionar modificações pós-traducionais às cadeias polipeptídicas, tais como a glicosilação, o que é essencial para o correto dobramento, maturação e montagem das duas subunidades, além de interferir diretamente com a meia-vida, o reconhecimento do receptor, a solubilidade e a atividade biológica das proteínas. No entanto, mesmo entre os sistemas de expressão heteróloga em células de mamífero, encontra-se muita variabilidade nos padrões de glicosilação adicionado. No presente trabalho, foi realizado um estudo comparativo através da clonagem e expressão de uma forma fusionada de eCG (reCGβα) em duas linhagens celulares diferentes: (1) CHO-DG44, um dos sistemas de expressão mais utilizados pelas indústrias farmacêuticas, capaz de adicionar N-glicanos complexos; e (2) 293T, uma linhagem humana capaz de produzir glicoproteínas carreando oligossacarídeos complexos e sialilados. Os resultados de atividade biológica (in vitro e in vivo) apontam uma maior atividade de reCG produzido por células CHO-DG44. O perfil de N-glicosilação de reCG produzido pelas células CHOD-G44 assemelhou-se mais à eCG selvagem, quando comparado a reCG produzido por células 293T. Por fim, estudos clínicos foram realizados com reCG produzido em meio livre de soro fetal bovino e parcialmente purificado, onde atividade específica de reCG produzido por células CHO-DG44 mostrou-se similar ao produto comercial selvagem. / Brazil is currently the major beef producer and exporter, rendering to livestock one of the country´s most economically relevant activities. This emphasizes the importance of research and development in bovine reproduction, especially at ovulation-stimulatory hormones, such as equine gonadotropin (eCG). The commercially available eCG-based products are purified from blood of pregnant heifers, presenting batch-to-batch variability and the presence of contaminants. These facts, together with the limitation of the bulk material (equine blood), emphasize the need of an eCG expression system able to be commercially explored. In this aspect, mammalian cells are a robust system, capable of add post-translational modifications to polypeptide chains, such as glycosylation, which is essential for the correct folding, maturation and assembly of both eCG subunits. In addition, glycosylation directly interferes with the protein half-life, receptor recognition, solubility and biological activity. In the present work, a comparative study was carried out by cloning and expressing a fusion form of eCG (reCGβα) in two different mammalian cell lines: (1) CHO-DG44, one of the most used by pharmaceutical companies expression systems, capable of add complex-type N-glycans; and (2) 293T, a human cell line capable of produce glycoproteins carrying complex and sialylated oligosaccharides. The in vitro and in vivo biological activity results show a higher potency of reCG produced by CHO-DG44 cells. The N-glycosylation pattern produced by CHO-DG44 cells was more similar to native eCG in comparison to the N-glycosylation produced by 293T cells. Finally, clinical studies were performed with serum absent media produced and partially purified reCG, showing that the specific activity of reCG produced by CHO cells was similar to the commercial wild type product.