Dietary fat quality influences the risk of type 2 diabetes and cardiovascular disease. A low-grade inflammation is suggested to contribute to the disease development, often accompanied by obesity. Whereas n-3 polyunsaturated fatty acids (PUFA) have been considered anti-inflammatory, n-6 PUFA have been proposed to act pro-inflammatory. Saturated fatty acids (SFA) act pro-inflammatory in vitro. This thesis aimed to investigate effects of different fatty acids on low-grade inflammation in observational and interventional studies. In Paper I and II, fatty acid composition in serum cholesterol esters was used as objective marker of dietary fat quality and related to serum C-reactive protein (CRP) and other circulating inflammatory markers in two population-based cohorts, conducted in middle-aged men and elderly men and women, respectively. In Paper III and IV, the impact of diets differing in fat quality on inflammation and oxidative stress was investigated in randomised controlled studies, in subjects with metabolic syndrome and abdominal obesity. In Paper I and II, a low proportion of linoleic acid (18:2 n-6) in serum was associated with higher CRP concentrations, indicating that a low intake of vegetable fats may be related to low-grade inflammation. High CRP concentrations were also associated with high proportions of palmitoleic (16:1) and oleic (18:1) acids and high stearoyl coenzymeA desaturase index, possibly reflecting altered fat metabolism and/or high SFA intake in this population. When comparing two high-fat diets rich in either saturated or monounsaturated fat, and two low-fat diets with or without long-chain n-3 PUFA supplementation during 12 weeks (Paper III), no differences in inflammation or oxidative stress markers were observed. Moreover, a 10-week intervention (Paper IV) with high linoleic acid intake showed no adverse effects on inflammation or oxidative stress. Instead, interleukin-1 receptor antagonist and tumor necrosis factor receptor-2 decreased after linoleic acid intake compared with a diet high in SFA. The results in this thesis indicate that dietary n-6 PUFA found in vegetable fats is associated with lower inflammation marker levels, and to some extent reduces systemic inflammation when compared with SFA. Supplementation of n-3 PUFA did not exert any systemic anti-inflammatory effects, maybe due to a relatively low dose.

Introdução. A Anemia das Doenças Crônicas (ADC) é uma desordem comum em idosos, freqüentemente multifatorial, e exacerbada por citocinas pró-inflamatórias. Nesta população, o diagnóstico de anemia por deficiência de ferro (ADF) é difícil utilizando-se os testes laboratoriais convencionais, devido à prevalência destes estados crônicos. Espécies reativas de oxigênio (ERO) são produzidas por fagócitos durante o burst oxidativo em defesa do hospedeiro, mas são também implicadas como agentes deletérios em um grande número de desordens inflamatórias. Objetivos. Verificar a eficiência do receptor de transferrina sérico (sTfR) e índice sTfR-log ferritina (sTfR-F) no diagnóstico da ADF e ADC; determinar os efeitos das anemias e de estados inflamatórios crônicos sem anemia no burst oxidativo, fagocitose, produção de óxido nítrico (NO) por monócitos, e produção de ácido hipocloroso (HOCl) por neutrófilos. Métodos. Participaram do estudo cinqüenta e três indivíduos (42 mulheres e 11 homens) idosos recrutados do departamento de Cardio-Geriatria da Rede Pública de Saúde de Ribeirão Preto-SP. A proteína C reativa (PCR), utilizada como marcador inflamatório foi analisada pela metodologia ultra-sensível. O status férrico foi estabelecido pelos níveis do sTfR (enzimaimunoensaio, kit Quantikine soluble transferrin receptor, R&D Systems, USA), ferritina sérica (ensaio quimioluminescente, kit Ferritin Immulite®- DPC, England) e índice sTfR-F. Foram compostos os seguintes grupos, com base nos valores de normalidade dos parâmetros PCR, concentração de hemoglobina, ferritina e sTfR: controle (n=15), ADC (n=12), inflamação (n=08), anemia inexplicável (n=06) e grupo ADF (n=12). Nenhum paciente apresentou função renal prejudicada, hemoglobinopatias, diabetes mellitus descompensada, doenças infecciosas ou malignas. Estudou-se o efeito da ADC, ADF, anemia inexplicável e inflamação no burst oxidativo, fagocitose de neutrófilos e monócitos por citometria de fluxo, produção de HOCl por neutrófilos purificados e de NO produzido em cultura de monócitos em meio RPMI 1640, estimulados por lipopolissacáride (LPS). Resultados. Os níveis de sTfR foram capazes de diagnosticar ADF em sete (58,34%) dos doze pacientes do grupo ADF. A intensidade de fluorescência (IF) do burst oxidativo de neutrófilos, que reflete a atividade celular, no grupo ADF foi significativamente menor que a do grupo controle (p<0,05), mas a IF de monócitos não foi estatisticamente diferente. As percentagens de monócitos e neutrófilos realizando burst oxidativo e fagocitose dos grupos ADF, anemia inexplicável e inflamação não foram estatisticamente diferentes do grupo controle. As percentagens de monócitos e neutrófilos realizando fagocitose do grupo ADC foram estatisticamente maiores que as do grupo controle (p<0,05). A produção de HOCl nos grupos ADC e inflamação foram estatisticamente menores comparadas ao grupo controle (p<0,05), enquanto houve uma superprodução de NO por monócitos no grupo ADC (p<0,05). Conclusão. No presente estudo, o sTfR foi uma ferramenta diagnóstica adicional à ferritina no diagnóstico da ADF, mas o valor do índice sTfR-F não aumentou a utilidade diagnóstica do sTfR em indivíduos idosos. NO parece modular a produção de HOCl, provavelmente por regular a atividade enzimática da mieloperoxidase (MPO). Estes resultados mostraram o papel importante do ferro na ADC e ADF em manter a resposta imunológica de fagócitos durante o processo de envelhecimento. / Background: Anemia of Chronic Disease (ACD) is a very common disorder in elderly and it is often multifatorial, exacerbated by pro-inflammatory cytokines. In these people, the diagnostic of iron deficiency anemia (IDA) or iron deficiency is difficult in these patients using the conventional iron status tests, because of the prevalence of these chronic states. Reactive oxygen species (ROS) are produced by phagocytic cells during the oxidative burst in defense of the host, but it has also been implicated as a very harmful agent in an increasing number of inflammatory-mediated disorders. Objectives: to elucidate the use of serum transferrin receptor (sTfR) and sTfR-log ferritin index (TfR-F index) to diagnose IDA and ACD; to determinate the effects of anemia and chronic inflammatory states without anemia in some function of oxidative stress, such as oxidative burst, phagocytosis, nitric oxide (NO) production by monocytes and the hypochlorous acid (HOCl) production by purified neutrophils. Methods: Fifty three (42 women and 11 men) elderly subjects recruited from geriatric department of healthy public system of Ribeirão Preto city were selected. The high sensitivity C-Reactive Protein (CRP) was analyzed as an inflammatory marker. The iron status was confirmed by sTfR (enzyme-linked immunosorbent assay, ELISA, kit Quantikine soluble transferrin receptor R&D Systems, USA), serum ferritin levels (chemiluminescence assay, kit Ferritin Immulite®- DPC, England) and TfR-F index. The groups were established according to the criteria CRP assay, hemoglobin analysis, ferritin and sTfR: control (n =15), ACD (n =12), inflammation (n =08), unexplained anemia (n=06), and IDA (n=12). Patients with impaired kidney function, hemoglobinopathy, decompensate diabetes mellitus or infectious and malignancies diseases were excluded. We also studied the effect of ACD, IDA, unexplained anemia and inflammation on oxidative burst, phogocytosis of neutrophils and monocytes by flow cytometry and HOCl generation of neutrophils and NO produced by the monocytic cell culture, cultured in RPMI 1640 medium, stimulated by lipopolysaccharide (LPS). Results: Our results showed that sTfR could diagnose IDA in seven (58.34%) of twelve patients in IDA group. Oxidative burst fluorescence intensity of neutrophil in IDA group was lower statistically compared to control group, p<0.05. Oxidative burst fluorescence intensity of monocytes did not differ significantly. The percentages of monocytes and neutrophils expressing oxidative burst and the phagocytosis did not differ significantly amongst control, IDA, unexplained anemia and inflammatory groups. However, the percentages of neutrophils and monocytes expressing phagocytosis in ACD group were statistically higher when compared to control group (p<0.05). The HOCl generation in ACD, and inflammation groups were lower significant statistically than control group, p<0.05, while there was an overproduction of NO by monocyte in ACD group (p<0.05). Conclusion: In the present study, the sTfR was an additional diagnostic tool to ferritin for the diagnosis of IDA, but the value of the sTfR-F index did not increase the utility diagnostic of sTfR in elderly patients. NO seems modulate the HOCl production, perhaps by regulates the myeloperoxidase (MPO) enzyme activity. These results showed the important role of iron in ACD and IDA to maintaining phagocyte immune defense of organism during the aging process.

[pt] As doenças trasmitidas pelo sangue, assim como a necessidade de bancos de sangue para um pronto auxílio em casos de acidentes tem estimulado esforços para desenvolver substitutos do sangue. A albumina serica humana (HSA do ingles Human Serum Albumin) é a proteína mais abundante no plasma sanguíneo. A molécula heme é a transportadora de oxigênio no sangue. Portanto, um estudo detalhado da interação HSA/heme seria útil em pesquisas que visam tornar o complexo HSA-heme em um substituto do sangue. Nesta tese, foram usadas técnicas de dinâmica molecular e ferramentas estatísticas para estudar o sistema HSA-heme em solvente explícito. Tanto o ligante quanto a proteína foram também estudados separadamente em meio aquoso. Dentre outros resultados, nosso estudo revelou a organização da água circundante, os efeitos da ligação do heme na HSA, os mecanismos moleculares da ligação do heme, os movimentos coletivos da proteína livre e ligada, assim como também os aminoácidos que atuam como dobradiças moleculares na mudança conformacional que sofre a proteína ao ligar o heme. / [en] Diseases transmitted through the blood, as well as the need for blood banks to help in case of accidents, stimulated efforts to develop blood substitutes. The human serum albumin (HSA) is the most abundant protein in blood plasma. The heme molecule is the carrier of oxygen in the blood. Therefore, a detailed study of the interaction HSA/heme could give useful insights in the research aimed to convert the HSA-heme complex into a blood substitute. In this thesis, molecular dynamics techniques and statistical tools were applied to study the HSA-heme system in explicit solvent. Both ligand and protein were also studied separately in aqueous medium. Among other results, our study reveals the organization of the surrounding water, the effects of the heme upon its binding to HSA, the molecular mechanisms for heme binding, the collective motions of the protein with and without the heme, as well as the amino acids that act as molecular hinges in the conformational change between the free and bound forms of the protein.

The understanding of self-assembly processes is important for fabrication of well-defined structures with new functionalities for applications in the area of biomedical sciences, material sciences and electronics. In this thesis, two types of self-assembly processes are described: (1) self-assembly of fullerene derivatives in water and (2) self-assembly on surfaces using layer-by-layer (LbL) approach. The various interactions and parameters involved in the self-assembly are detailed in the introductory chapter 1. The various internal parameters like molecular geometry, intramolecular and intermolecular forces that guides the self-assembly process of amphiphiles in water are discussed. The experimental procedures used in the present thesis for the fabrication of nanostructures via self-assembly approach are also described. In the later part of the chapter, the LbL technique for fabrication of thin films and microcapsules is reviewed where various interactions involved in the growth of LbL assembly are discussed. The effect of ionic strength and pH on the growth and property of LbL assemblies is elaborated. A brief discussion of the materials used in the thesis ‒ fullerene, bovine serum albumin (BSA) and nanocrystalline cellulose (NCC) is also provided The self-assembly behaviour of amphiphilic fullerene derivatives are described in chapter 2. Fullerene is anisotropically substituted with five polar hydroxyl groups using organo-copper reagent. The derivative can interact in water via the van der Waals and hydrophobic interactions of the fullerene moiety as well as the intermolecular hydrogen bonding among the hydroxyl groups and also with water. The penta-hydroxy fullerene derivative self-assembles in water as vesicular structures. The size of these vesicles can be varied by modifying the kinetics of self-assembly which was done by changing the rate of addition of non-solvent (water) to the solution of the fullerene derivative. In the second derivative, the hydroxyl groups are substituted with less polar methoxy groups. The penta-methoxy fullerene derivative cannot participate in inter-molecular hydrogen bonding formation unlike the penta-hydroxy derivative but there is possibility of hydrogen bond formation with water where oxygens on methoxy group can act as hydrogen bond acceptor. The penta-methoxy fullerene does not show any vesicle formation in water. The computational simulation studies were carried out on the two fullerene derivatives to understand the self-assembly behaviour of these two derivatives. Furthermore, the vesicle structures formed by the penta-hydroxy fullerene derivative are used for entrapment of hydrophobic polymer, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) and also hydrophilic dye, Rhodamine B. In both the cases, fluorescence quenching is observed due to electron transfer reaction with fullerene and hence these fullerene vesicles can be used to study the effect of confinement on electron transfer reactions and other chemical dynamics. The layer-by-layer self-assembly approach for the fabrication of biopolymeric thin films and microcapsules is discussed in the chapters 3 to 6. The biocompatible nanoparticles and nanofibers were used as the components of the assembly. In chapter 3, we have described fabrication of thin film of bovine serum albumin (BSA) nanoparticles via LbL approach using biopolymer chitosan as the complementary polymer. The driving force for the assembly growth of the assembly was the electrostatic and complementary hydrogen bond formation between the two components. The idea of incorporating nanoparticles in the thin film was that the nanoparticles can act as reservoirs for functional materials. The films were loaded with anticancer drug doxorubicin and show pH dependent release of the drug. The various interactions involved in the LbL assembly of BSA nanoparticles and polymers were investigated towards understanding the growth mechanism of the assembly in chapter 4. The understanding of the interactions involved in the assembly formation is important in order to modify the conditions of the assembly for enhancing the growth. It is inferred from the study reported in this chapter that not only the interaction of nanoparticles with polymers but also the inter-particle interactions are important factors in determining the growth of LbL assembly of nanoparticles/polymers. The growth of the assembly is enhanced on minimizing the inter-particle repulsions, which was achieved in case of BSA nanoparticles by modifying the pH of the assembly. We also utilized the LbL self-assembly approach for the delivery of lipophilic drugs. The lipophilic drugs are difficult to administer in the body due to their poor water solubility and hence show poor pharmacokinetic profile. The methods for incorporating hydrophobic drugs in LbL assembled thin films and microcapsules are described in chapters 5 and 6. In chapter 5, hydrophobic molecules binding property of albumin has been exploited for solubilisation of a water-insoluble molecule, pyrene (model drug) and hydrophobic drug, curcumin, by preparation of non-covalent conjugates with BSA. The interaction with BSA provided negative zeta potential to the previously uncharged molecules and hence they can be incorporated in the LbL assembled thin films and microcapsules using electrostatic as well as hydrogen bonding interaction with biopolymer, chitosan. The fabrication of protein encapsulated stable microcapsules with hydrophobic molecules incorporated in the shell of the microcapsules has also been demonstrated. The microcapsules were further capable of loading hydrophilic molecules like Rhodamine B. Thus, this approach can be employed for fabrication of multi-agent carrier for hydrophobic and hydrophilic drugs as well as therapeutic macromolecules. In chapter 6, we have incorporated nanocrystalline cellulose (NCC) LbL assembled thin films and microcapsules. The assembly formed was porous in nature due to the nano-fibrous morphology of NCC. The nanoassemblies can act as potential drug delivery carrier, which has been demonstrated by loading anticancer drug doxorubicin, and a lipophilic drug, curcumin. Doxorubicin hydrochloride, the salt form of the drug, doxorubicin, has good water solubility and hence can be postloaded in the assembly by diffusion from its aqueous solution. In the case of curcumin, which has limited solubility in water, a stable aqueous dispersion of the drug was prepared via noncovalent interaction with NCC prior to incorporation in the LbL assembly. The interaction of various other lipophilic drugs with NCC was analysed computationally.

A glicação é uma reação não enzimática que ocorre entre proteínas e açúcares redutores e, é responsável pela formação de adultos e de ligações cruzadas entre proteínas, como por exemplo: a pentosidina, produto final de glicação avançada que se acumula em vários tecidos ao longo do tempo. A glicação é deletéria para o organismo e está associada a modificações estruturais em proteínas e alterações de suas funções específicas, tais como: atividade enzimática, capacidade de ligação e tempo de vida de proteínas, além de ser responsável pela produção de espécies reativas de oxigênio (EROS). O mecanismo de formação da pentosidina envolve reações oxidativas e, uma das estratégias para minimizá-Ia é o aumento da atividade antioxidante nos tecidos. A pariparoba (Pothomorphe umbellata (L.) Miq) demonstrou atividade antioxidante in vitro e in vivo quando aplicada sobre a pele. Essa atividade foi atribuída ao 4-nerolidilcatecol (4-NC), que se mostrou 10 vezes mais potente que o &#945;-tocoferol. Os extratos de pariparoba também inibiram a lipoperoxidação espontânea da pele em camundongos sem pelo. Neste trabalho empregou-se o modelo de glicação de albumina de soro bovino (BSA) frente à D-ribose, com avaliação da fluorescência produzida pela pentosidina formada na reação. Avaliou-se igualmente a atividade do 4-NC em diferentes concentrações sobre a reação de glicação da BSA em presença de D-ribose após 24 horas, empregando-se a aminoguanidina como controle positivo. Nas condições experimentais o 4-NC não foi capaz de inibir a reação de glicação, ao contrário da aminoguanidina. Foi também utilizado modelo para avaliação da propriedade contrátil de fibroblastos em matriz tridimensional de gel de colágeno, glicado e não glicado com D-ribose. O 4-NC na concentração de 100 &#181;M permitiu a manutenção da propriedade contrátil de fibroblastos em gel colágeno glicado. Estudos de glicação em maiores períodos de tempo devem ser realizados visando a confirmar a possível atividade anti-glicação deste composto. / Glycation is a non enzymatic reaction which occurs between proteins and reductor sugars, responsible for the formation of adducts and crosslinkers between proteins, such as, pentosidine, an advanced glycation end-product (AGE) which accumulates in many tissues during aging. AGEs accumulation is deleterious to the body and is associated with structural modifications in proteins and imbalance in their specific functions, such as: enzymatic activity, binding capacity, protein turnover and also responsible for the production of reactive oxygen species (ROS). The mechanism of pentosidine formation involves oxidative reactions. One of the strategies to reduce pentosidine formation is by increasing antioxidant activity in tissues. Pariparoba (Pothomorphe umbellata (L.) Miq. has showed antioxidant activity in vitro and in vivo when applied on the skin. This activity was attributed to 4-nerolydilcatechol (4-NC), which is 10 times more potent than &#945;-tocopherol. Extracts of Pariparoba also inhibited the spontaneous lipid peroxidation in the skin of hairless mice. In this work, the bovine serum albumin (BSA) model for glycation with D-ribose, evaluated by pentosidine fluorescence spectroscopy was employed. The activity of 4¬NC was evaluated in different concentrations in this model after 24 hours. Aminoguanidine was used as positive control. In this experimental condition, 4-NC was not capable to inhibit the BSA glycation. We also evaluated the contractile properties of fibroblasts on tridimensional matriz of collagen gel glycated or not with D-ribose. 4-NC (100 &#181;M) was able to keep the contractile capacity of fibroblasts in glycated collagen. Studies of glycation in longer periods of time should be made in order to further evaluate the possible anti-glycation activity of this compound.

Um estudo sistemático a respeito da utilização de padrão interno em determinações multielementares por espectrometria de absorção atômica (ETAAS) foi desenvolvido. O objetivo principal do presente trabalho foi verificar a possibilidade de melhorar a precisão e a exatidão dos resultados analíticos, que são obtidos na análise de fluidos biológicos. O pré-tratamento dessas amostras foi simplificado e reduzido a uma única etapa de diluição com surfactante (Triton X-100) e ácido (HNO3). Conseqüentemente, a complexidade da solução diluída de amostra, a ser introduzida no tubo de grafite, apresenta uma elevada quantidade de concomitantes que podem provocar interferências químicas. A seleção preliminar dos elementos a serem testados como padrão interno considerou a semelhança de parâmetros físico-químicos relacionados com o processo de atomização. Desta forma, Ag, Bi, In e Tl foram testados como padrão interno para a determinação simultânea de Cd/Pb em sangue e urina, enquanto Bi, Ge, In, Sb, Sn e Te foram os elementos selecionados para a determinação de Mn/Ni/Se em soro sangüíneo. A melhoria da qualidade dos resultados analíticos obtidos na determinação simultânea de Cd e Pb em sangue foi observada quando Ag foi utilizada como padrão interno, na presença de NH4H2PO4 como modificador químico. Verificou-se uma melhoria na exatidão dos resultados obtidos para Cd e Pb, após a correção com padrão interno. Por outro lado, os resultados obtidos na análise de urina não foram corrigidos por nenhum dos elementos testados. Os melhores resultados para a determinação simultânea de Mn, Ni e Se foram obtidos com a utilização de Bi, Sn e Te como padrão interno. Entretanto, verificou-se que a correção de todos os resultados não seria viável com o uso de um único padrão interno. O melhor desempenho nos testes realizados na presença de soro sangüíneo foi obtido com Bi, que melhorou discretamente a precisão dos resultados obtidos para Se. Desta forma, a padronização interna visando a determinação simultânea de Mn, Ni e Se não foi eficiente. A padronização interna em ETAAS, com a finalidade de melhorar a precisão e a exatidão dos resultados analíticos, é uma estratégia tão complexa, quanto os efeitos interferentes que se pretende corrigir: são necessários mais estudos para compreender melhor como a utilização de uma condição de compromisso afeta os processos de atomização, bem como mais informações a respeito das interferências físicas e químicas causadas por amostras complexas, analisadas por ETAAS após uma simples etapa de diluição. Deve-se considerar com especial atenção o modificador químico e as temperaturas das etapas de pirólise e de atomização empregadas, que são parâmetros críticos para o desempenho de um elemento como padrão interno. / A systematic study involving the use of internal standard for multielement determinations by electrothermal atomic absorption spectrometry was developed. The main objective of this work was evaluate the possibility of improving precision and accuracy of the analytical results for biological fluids. The sample pre-treatment was reduced to a single dilution step with surfactant (Triton X-100) and acid (HNO3), increasing the amount of concomitant introduced into the atomizer. The preliminary selection of the elements to be tested as internal standard considered the resemblance of physico-chemical parameters related with the atomization process. Thus, Ag, Bi, In and Tl were tested as internal standard for the simultaneous determination of Cd/Pb in blood and urine, and Bi, Ge, In, Sb, Sn and Te were the selected elements for the determination of Mn/Ni/Se in blood serum. The correction of the results obtained for the simultaneous determination of Cd and Pb in blood was achieved when Ag was used as internal standard, in presence of NH4H2PO4 as chemical modifier. An improvement for the accuracy of the results was observed for both analytes after their correction with the internal standard. On the other hand, the results obtained for the urine analysis were not corrected by using the tested elements. The best results for the simultaneous determination of Mn, Ni and Se were observed when Bi, Sn and Te were used as internal standard. However, the correction for the results for all analytes was not possible by using only one internal standard. The best performance in presence of the serum was obtained for Bi, which improves slightly the precision for the Se results. Thus, the internal standardization for the simultaneous determination of Mn, Ni and Se was not efficient. The internal standardization in ETAAS, aiming the improvement of precision and accuracy of the analytical results, is a strategy as complex as the interference effects to be corrected: more studies are required in order to better understand how the adoption of a compromised condition disturbs the atomization processes, as well as to get more information about the physical and chemical interference caused by complex samples, analyzed by ETAAS after a single dilution step. The chemical modifier and the selected temperatures for the pyrolysis and atomization steps are critical parameters for the performace of an internal standard and they should be carefully considered.

Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação. / Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices.

To address the challenge of accurate, low cost and robust biosensors for diabetes management and early detection of diabetes complications, we have developed novel, robust sensing chemistry (or receptors) for electrochemical POC biosensors. The biosensors have been developed for the bio-markers associated with diabetes management such as glycated haemoglobin (HbA1c), glycated albumin, glucose, biomarkers associated with diabetes complications such as microalbuminuria, urine creatinine and albumin-to-creatinine ratio (ACR) and biomarkers associated with anaemia and malnutrition conditions such as haemoglobin and serum albumin. For haemoglobin detection, a new POC bio sensing technique has been developed based on Aza-heterocyclic chemicals. The repeatability and accuracy of the biosensor have been tested on real pathology samples. The glycated form of haemoglobin, called glycated haemoglobin or HbA1c, is the gold standard test in diabetes management as it gives the 90-days average blood glucose value. We demonstrate a simple method for electrochemical detection of HbA1c by combining bosonic affinity principle along with aza-heterocyclic receptors. The technique has been verified on the real clinical patient samples. Albumin is the most abundant protein in the human blood. Human serum albumin (HSA) is either alone or an associative biomarker in several chronic diseases like necrosis, nephrosis, hepatitis, malnutrition, arthritis, immune disorders, cancer, diabetes and in some severe infections. In pathology laboratories, the serum albumin is usually tested on serum samples and not in whole blood samples. Since albumin is not a metalloproteinase, it is very difficult to develop electrochemical POC biosensor. We have developed a novel technique for the electrochemical detection of serum albumin in whole blood samples, by exploiting its binding property with redox active copper salts. The accuracy of technique has been verified on both real human blood plasma as well as whole blood samples. Glycated albumin, which is the glycated form of serum albumin, is emerging as a novel biomarker for diabetes management, as it gives the average blood glucose value of 15-20 days. It is also extremely useful in chronic kidney disease patients and patients with hemoglobinopathies where HbA1c can give the erroneous results. By combining the copper chemistry along with bosonic affinity principle, we present the first ever demonstration of glycated albumin sensing. Instant blood glucose monitoring is an integral part of diabetes management. Most of the glucometers available in the market are based on glucose oxidase enzyme. We have demonstrated a low cost non-enzymatic electrochemical technique for blood glucose detection using alkaline methylene blue chemistry. The accuracy of the technique has been verified on real human blood plasma samples. Glucometer is one of the most easily available POC biosensor and a useful tool for diabetes population. India has second largest diabetes population in the world. To analyse the accuracy of the POC glucometers which are available in Indian market, a comprehensive study was conducted. The results were compared with clinical accuracy guidelines using exhaustive statistical analysis techniques. The shortcomings of the commercial glucometers are elucidated, regarding different international standards. Diabetic nephropathy is one of the major diabetes complications and is the primary cause of chronic kidney disease (CKD). The presence of albumin in urine is a well-established biomarker for the early detection of diabetic nephropathy. We have developed a technique for electrochemical detection of microalbuminuria for point of care applications by exploring the binding property of human albumin with electrochemically active molecules like copper and hemin. Methylene blue mediated sensing technique has also been proposed. Urine Albumin-to creatinine ratio (ACR) is another variant of the microalbumuria test that can be done any time and does not suffer from the dilution factor of urine. Iron binding property of creatinine is exploited to develop creatinine biosensor, thus enabling POC ACR tests.

Diploma thesis deals of nanostructured surfaces, nanoparticles and electrochemical characterization methods such as cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. The aim of this thesis is a theoretical research issues of production and characterization nanostructured modified electrodes. The practical part is the production of biomodified nanostructured electrodes by anodi- zation W/Al layers with galvanic deposition of gold or deposition of mercury, a modifi- cation of the electrodes by 11-mercaptoundecanoic acid and by bovine serum albumin (BSA). The thesis includes SEM images of nanostructured electrodes contact angle mea- surements of these electrodes and form an electrical circuit with subsequent simulation waveforms.

Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA), is a membrane metallopeptidase overexpressed on most prostate cancer cells. Additionally, GCPII also attracted neurologists' attention because it cleaves neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG). Since NAAG exhibits neuroprotective effects, GCPII may participate in a number of brain disorders, which were shown to be ameliorated by GCPII selective inhibitors. Therefore, GCPII has become a promising target for imaging and prostate cancer targeted therapy as well as therapy of neuronal disorders. Globally, prostate cancer represents the second most prevalent cancer in men. With the age, most men will develop prostate cancer. However, prostate tumors are life threatening only if they escape from the prostate itself and start to spread to other tissues. Therefore, considerable efforts have been made to discover tumors earlier at more curable stages as well as to target aggressive metastatic cancers that have already invaded other tissues and become resistant to the standard treatment. Since patients undergoing a conventional therapy (a combination of chemotherapy and surgery) suffer from severe side effects, more effective ways of treatment are being searched for. Novel approaches include selective...