New roles of Rab GTPases on eye diseases: targeting VEGF secretion

RESUMO: A retina �� composta, entre outras estruturas, pelo epit��lio pigmentar da retina (EPR)e pela cor��ide. A regi��o central da retina denomina-se m��cula, e �� a zona mais afetada na degeneresc��ncia macular relacionada com a idade, a forma mais comum de degeneresc��ncia da retina. Nesta doen��a, a secre����o de fatores de crescimento pelo EPR �� afetada, nomeadamente a do fator de crescimento vascular endotelial (VEGF), e pouco se sabe ainda sobre os mecanismos moleculares conducentes a esta condi����o. A fam��lia de prote��nas Rab GTPases est�� envolvida nas vias intracelulares de sinaliza����o e tr��fego membranares, essenciais na transdu����o de sinais extracelulares em respostas biol��gicas. A sua crucial import��ncia nestes mecanismos levou-nos a considerar o seu potencial envolvimento nas vias de secre����o do VEGF, e a questionar-nos se teriam algum papel regulador sobre as mesmas. O principal objetivo deste trabalho �� identificar Rab GTPases importantes para as vias de secre����o e endocitose do VEGF no EPR. Essa identifica����o ajudar�� a esclarecer a patog��nese da degeneresc��ncia macular da retina, e poder�� servir para uma procura mais direcionada de novos agentes terap��uticos. A caracteriza����o de dois modelos in vitro do EPR, c��lulas prim��rias isoladas de murganho e a linha celular B6-RPE07,levou-nos a concluir que s��o ambos semelhantes. Contudo, a linha celular foi escolhida como prot��tipo do EPR por permitir o acesso a um n��mero ilimitado de c��lulas. No decurso deste trabalho, desenvolvemos e caracteriz��mos uma biblioteca de ferramentas moleculares que nos permitiram reduzir os n��veis proteicos das prote��nas Rab GTPases, com base na tecnologia de ��cido ribonucleico (ARN) de interfer��ncia. O papel das prote��nas Rab GTPases na secre����o do VEGF no EPR foi estudado com base no silenciamento de apenas uma prote��na, ou combinando v��rias, segundo a sua localiza����o e fun����es intracelulares descritas. Este trabalho permitiu-nos concluir que as prote��nas Rab GTPases s��o importantes intervenientes no processo de secre����o de VEGF pelo EPR, e confirmar dados anteriores que relatam o envolvimento de algumas Rab GTPases endoc��ticas no processo. Propomos ainda um novo modelo para a intera����o destas prote��nas no EPR, e sugerimos que a Rab10 e a Rab14 atuam negativamente sobre a Rab8, controlando o seu funcionamento. Os nossos resultados evidenciam a import��ncia das prote��nas Rab GTPases na secre����o do VEGF pelas c��lulas do EPR, e servem de base a futuros estudos que melhor procurem compreender este mecanismo e de que modo a sua altera����o se relaciona com a degeneresc��ncia da retina.--------ABSTRACT: Retinal pigment epithelium (RPE) and choroid are components of the mammalian retina, of which the central region is called macula. The most common form of retinaldegeneration, age-related macular degeneration (AMD), involves primarily deregulation of growth factors secretion by the RPE. Very little is known about the molecular mechanisms that lead to impairment of RPE���s homeostatic intracellular processes, namely the secretion of vascular endothelial growth factor (VEGF). Rab GTPases��� family regulates membrane targeting and traffic, being essential in the transduction of signal pathways. Given Rab proteins��� role in intracellular trafficking, we propose to identify key regulatory Rab proteins involved in either the secretory or the recycling pathways of VEGF in RPE. Understanding how Rab proteins��� function disruption could lead to retinal and choroidal pathology would ultimately contribute to find new therapeutic agents. Here, we characterized two mouse RPE in vitro cell models, primary cells and B6-RPE07 cell line, and concluded that both display important epithelial features as the RPE presents in vivo. Considering unlimited cell number and results reproducibility, we chose B6-RPE07 cells to further study Rab proteins��� function. To scrutinize the consequences of Rab proteins��� absence or diminished levels, we have developed novel molecular tools to achieve silencing of these key proteins using miRNA technology. We further addressed the effect of Rab proteins��� absence on VEGF secretion by performing an extensive screening where different Rab proteins were silenced, both individually and in multiple combinations considering their cellular/ compartment location. We conclude that Rab GTPases are important intervenients in VEGF secretion by RPE cells, confirming endocytic Rab proteins��� role in regulation of VEGF biology. We also propose a novel model for Rab proteins��� interaction in RPE. Our results suggest that Rab10 and Rab14 might influence Rab8 in a negative feedback mechanism, important for controlling VEGF secretion. Our achievements��� unravel Rab proteins��� role in VEGF secretion by RPE cells and are the basis for future studies to better understand RPE molecular secretory machinery.

Cyanobacterial and Protozoan Flavodiiron Proteins are nitric oxide and/or dioxygen reductases

Dissertation presented to obtain the Ph.D degree in Biochemistry.

Peptidoglycan assembly machines: The Staphylococcus aureus Penicillin-Binding Proteins

Dissertation presented to obtain the Ph.D degree in Biology

Impact of viral mmunomodulatory proteins

Dissertation presented to obtain the Ph.D degree in Biology

Structural and functional characterization of ModA and TupA proteins belonging to the molybdate and tungstate transport system from desulfovibrio alaskensis G20

Funda����o para a Ci��ncia e Tecnologia - EXPL/BBB-BEP/0274/2012

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Funda����o para a Ci��ncia e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cec��la Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011)

Study of the electromyographic signal dynamic behavior in Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by motor neurons degeneration, which reduces muscular force, being very difficult to diagnose. Mathematical methods are used in order to analyze the surface electromiographic signal���s dynamic behavior (Fractal Dimension (FD) and Multiscale Entropy (MSE)), evaluate different muscle group���s synchronization (Coherence and Phase Locking Factor (PLF)) and to evaluate the signal���s complexity (Lempel-Ziv (LZ) techniques and Detrended Fluctuation Analysis (DFA)). Surface electromiographic signal acquisitions were performed in upper limb muscles, being the analysis executed for instants of contraction for ipsilateral acquisitions for patients and control groups. Results from LZ, DFA and MSE analysis present capability to distinguish between the patient group and the control group, whereas coherence, PLF and FD algorithms present results very similar for both groups. LZ, DFA and MSE algorithms appear then to be a good measure of corticospinal pathways integrity. A classification algorithm was applied to the results in combination with extracted features from the surface electromiographic signal, with an accuracy percentage higher than 70% for 118 combinations for at least one classifier. The classification results demonstrate capability to distinguish members between patients and control groups. These results can demonstrate a major importance in the disease diagnose, once surface electromyography (sEMG) may be used as an auxiliary diagnose method.

Crystallographic studies of proteins involved in the mRNA localization mechanisms in Drosophila melanogaster and amidation of the peptidoglycan residues in Staphylococcus aureus

Part of the work described in this chapter, was the subject of the following publication: D. Vieira, T. a. Figueiredo, A. Verma, R. G. Sobral, A. M. Ludovice, H. de Lencastre, and J. Trincao, ���Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan,��� Acta Crystallogr. Sect. F Struct. Biol. Commun., vol. 70, no. 5, pp. 1���4, Apr. 2014.

Functional characterization and directed engineering of redox proteins for a rational improvement of Bioelectrochemical systems

In recent years, new methods of clean and environmentally friendly energy production have been the focus of intense research efforts. Microbial fuel cells (MFCs) are devices that utilize naturally occurring microorganisms that feed on organic matter, like waste water, while producing electrical energy. The natural habitats of bacteria thriving in microbial fuel cells are usually marine and freshwater sediments. These microorganisms are called dissimilatory metal reducing bacteria (DMRB), but in addition to metals like iron and manganese, they can use organic compounds like DMSO or TMAO, radionuclides and electrodes as terminal electron acceptors in their metabolic pathways.(...)

Establishment and evaluation of flexible insect cell lines for rapid production of recombinant proteins

Funda����o para a Ci��ncia e a Tecnologia (FCT) - (PTDC/EBB-EBI/102266/2008 and SFRH/BD/43830/2008, respectively) and by European Community���s FP7/2007-2013 (grant agreement n�� 270089)

Electrohysterogram signal component cataloging with spectral and time-frequency methods

The Electrohysterogram (EHG) is a new instrument for pregnancy monitoring. It measures the uterine muscle electrical signal, which is closely related with uterine contractions. The EHG is described as a viable alternative and a more precise instrument than the currently most widely used method for the description of uterine contractions: the external tocogram. The EHG has also been indicated as a promising tool in the assessment of preterm delivery risk. This work intends to contribute towards the EHG characterization through the inventory of its components which are: ��� Contractions; ��� Labor contractions; ��� Alvarez waves; ��� Fetal movements; ��� Long Duration Low Frequency Waves; The instruments used for cataloging were: Spectral Analysis, parametric and non-parametric, energy estimators, time-frequency methods and the tocogram annotated by expert physicians. The EHG and respective tocograms were obtained from the Icelandic 16-electrode Electrohysterogram Database. 288 components were classified. There is not a component database of this type available for consultation. The spectral analysis module and power estimation was added to Uterine Explorer, an EHG analysis software developed in FCT-UNL. The importance of this component database is related to the need to improve the understanding of the EHG which is a relatively complex signal, as well as contributing towards the detection of preterm birth. Preterm birth accounts for 10% of all births and is one of the most relevant obstetric conditions. Despite the technological and scientific advances in perinatal medicine, in developed countries, prematurity is the major cause of neonatal death. Although various risk factors such as previous preterm births, infection, uterine malformations, multiple gestation and short uterine cervix in second trimester, have been associated with this condition, its etiology remains unknown [1][2][3].

Primary cilia and the regulation of hedgehog signaling: link to cardiac development

RESUMO: As c��lulas eucari��ticas evolu��ram um sistema de sinaliza����o complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinaliza����o s��o essenciais para a sobreviv��ncia da c��lula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transdu����o de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a prote��na Smoothened (Smo) na aus��ncia dos seus ligandos, as prote��nas Sonic hedgehog (Shh). Estas prote��nas s��o reguladores fundamentais do desenvolvimento embrion��rio, como ilustrado pelas malforma����es dr��sticas observadas em embri��es humanos e de murganho com perturba����es da transdu����o de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malforma����es ��sseas. Igualmente importantes s��o as consequ��ncias da ativa����o inapropriada da via de sinaliza����o Hh na forma����o de tumores. Curiosamente, os componentes desta via localizam-se nos c��lios prim��rios. Al��m disso, demonstrou-se que esta localiza����o �� crucial para a sinaliza����o atrav��s da via Hh. Na presen��a dos ligandos, Ptch1 �� internalizado e destinado a degrada����o ou sequestrado num compartimento da c��lula de onde n��o pode desempenhar o seu papel inibit��rio. A prote��na Arl13b �� uma pequena GTPase pertencente �� fam��lia Arf/Arl da superfam��lia Ras de pequenas GTPases e foi implicada no s��ndrome de Joubert, uma ciliopatia caracterizada por ataxia cong��nita cerebelar, hipotonia, atrso mental e cardiopatia cong��nita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gesta����o (E13,5) e exibem anomalias morfol��gicas nos c��lios que levam �� interrup����o da sinaliza����o Hh. Al��m disso, a Arl13b est�� diretamente envolvida na regula����o da via Hh, controlando a localiza����o de v��rios componentes desta via nos c��lios prim��rios. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que s��o estruturas de actina envolvidas em macropinocitose e internaliza����o de recetores, e que regula a sua forma����o. Al��m disso, aprofund��mos o conhecimento do processo de ativa����o da via de sinaliza����o Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos ap��s ativa����o da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir da��, sugerindo uma indu����o cont��nua da reorganiza����o do citoesqueleto de actina quando a via est�� ativada. Observ��mos ainda que a inibi����o da forma����o de CDRs atrav��s do silenciamento de WAVE1, uma prote��na necess��ria para a forma����o destas estruturas, resulta na diminui����o da ativa����o da via de sinaliza����o Hh. Al��m disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, atrav��s do silenciamento de uma prote��na necess��ria para a cis��o de macropinossomas, nomeadamente a prote��na BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose s��o necess��rias para a ativa����o da via de sinaliza����o Hh e indicam que esta via de internaliza����o controla os n��veis de sinal Hh. Durante o desenvolvimento, as c��lulas proliferativas dependem do c��lio prim��rio para a transdu����o de v��rias vias de sinaliza����o. A via Hh induz a diferencia����o do m��sculo card��aco. Por conseguinte, os murganhos deficientes na via de sinaliza����o Hh exibem uma variedade de defeitos de lateralidade, incluindo altera����o do looping do cora����o, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investig��mos o papel da Arl13b no desenvolvimento do cora����o. Mostramos que a Arl13b �� altamente expressa no cora����o de embri��es de murganho e de murganhos adultos ao n��vel do mRNA e da prote��na. Al��m disso, o perfil de distribui����o da Arl13b no cora����o segue o dos c��lios prim��rios, que s��o essenciais para o desenvolvimento card��aco. Cora����es de murganhos hnn no estadio E12,5 mostram um canal ��trio-ventricular aberto, espessamento da camada compacta ventricular e aumento do ��ndice mit��tico no ventr��culo esquerdo. Al��m disso, um atraso de 1 a 2 dias no desenvolvimento �� observado em cora����es de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b �� necess��ria para o desenvolvimento embrion��rio do cora����o e que defeitos card��acos podem contribuir para a letalidade embrion��ria de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regula����o dos n��veis de superf��cie do recetor Ptch1, que envolve a remodela����o do citoesqueleto de actina e a forma����o de CDRs ap��s a ativa����o da via de sinaliza����o Hh. Este mecanismo permite um feedback negativo que evita a repress��o excessiva da via atrav��s da remo����o de Ptch1 da superf��cie da c��lula. Al��m disso, determinou-se que uma muta����o de perda de fun����o na Arl13b causa defeitos card��acos durante o desenvolvimento, possivelmente relacionados com a associa����o dos defeitos em c��lios prim��rios e na sinaliza����o Hh, existentes em murganhos deficientes para Arl13b. A via de sinaliza����o Hh tem tido um papel central entre as vias de sinaliza����o, uma vez que a sua regula����o �� crucial para o funcionamento apropriada da c��lula. Assim, a descoberta de um novo mecanismo de tr��fego atrav��s de macropinocitose e CDRs que controla a ativa����o e repress��o da via de sinaliza����o Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir �� identifica����o de novos alvos e estrat��gias terap��uticas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.

Molecular targeting of proteins by homocysteine: implications in familial and clinical hypercholesterolemia

Cardiovascular diseases (CVDs) are one of the leading causes of death and disability worldwide and one of its underlying causes is hypercholesterolemia. Hypercholesterolemia can have genetic (familial hypercholesterolemia, FH) and non-genetic causes (clinical hypercholesterolemia, CH), the first much more severe, with occurrence of premature atherosclerosis. While the pathophysiological role of homocysteine (Hcy) on CVD is still controversial, molecular targeting of protein by S and N-homocysteinylation offers a new paradigm to be considered in the vascular pathogenesis of hypercholesterolemia. On this regard, the present study aims to give new insights on protein targeting by Hcy in both CH and FH conditions. A total of 187 subjects were included: 65 normolipidemic and 122 hypercholesterolemic. Total (tHcy) and free (fHcy) fractions were quantified in serum samples after validation of an HPLCFD method, to assess S-homocysteinylation. Also, the lactonase (LACase) activity of paraoxonase-1 (PON1) was quantified by a colorimetric assay, as a surrogate of N-homocysteinylation. tHcy does not differ among groups. Nevertheless, fHcy declines in the hypercholesterolemic groups, with more evidence to the FH population. Consequently, there seems to be an increase of Shomocysteinylation, regardless of lipid lowering therapy (LLT). Also, despite of LLT use, LACase activity is lower in FH, thus the risk for protein N-homocysteinylation seems to be higher. Moreover, the decrease in LACase/ApoA1 and LACase/HDL ratios in FH, shows that HDL is dysfunctional in this population, despite its normal concentration values. Data supports that the pathophysiological role of Hcy on hypercholesterolemia may reside in its ability to post-translationally modify proteins. This role is particularly evident in FH condition. In the future, it will be interesting to identify which target proteins are modified and thus involved in vascular pathology progression.

Study of in vivo interactions between penicillin-binding proteins of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a major human pathogen that has acquired resistance to practically all classes of ��-lactam antibiotics, being responsible of Multidrug resistant S. aureus (MRSA) associated infections both in healthcare (HA-MRSA) and community settings (CA-MRSA). The emergence of laboratory strains with high-resistance (VRSA) to the last resort antibiotic, vancomycin, is a warning of what is to come in clinical strains. Penicillin binding proteins (PBPs) target ��-lactams and are responsible for catalyzing the last steps of synthesis of the main component of cell wall, peptidoglycan. As in Escherichia coli, it is suggested that S. aureus uses a multi-protein complex that carries out cell wall synthesis. In the presence of ��-lactams, PBP2A and PBP2 perform a joint action to build the cell wall and allow cell survival. Likewise, PBP2 cooperates with PBP4 in cell wall cross-linking. However, an actual interaction between PBP2 and PBP4 and the location of such interaction has not yet been determined. Therefore, investigation of the existence of a PBP2-PBP4 interaction and its location(s) in vivo is of great interest, as it should provide new insights into the function of the cell wall synthesis machinery in S. aureus. The aim of this work was to develop Split-GFPP7 system to determine interactions between PBP2 and PBP4. GFPP7 was split in a strategic site and fused to proteins of interest. When each GFPP7 fragment, fused to proteins, was expressed alone in staphylococcal cells, no fluorescence was detectable. When GFPP7 fragments fused to different peptidoglycan synthesis (PBP2 and PBP4) or cell division (FtsZ and EzrA) proteins were co-expressed together, fluorescent fusions were localized to the septum. However, further analysis revealed that this positive result is mediated by GFPP7 self-association. We then interpret the results in light of such event and provide insights into ways of improving this system.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor ��-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to ���in-cell��� Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.