Determinants of the intention to use performance-enhancing substances among Portuguese gym users

The present study examined the determinants of the intentions to use prohibited performance- enhancing substances (PES) and the hypothesis of gender and PES use influencing Theory of Planned Behavior (TPB) variables. A TPB approach was used. A convenience sample of Portuguese gym users (n = 453) completed an anonymous web-based survey. Variance-based structural equation modeling, multigroup analysis strategy, latent mean analysis approach and one-way ANOVA analysis were used. The findings showed that, at structural level, results support the TPB framework in terms of characterizing and predicting intentions to PES use in the gym users sample, and that subjective norms were the strongest predictor of PES use intentions. Female and male differed in intentions to use PES, subjective norms and beliefs. However, the predictive model in study remains invariable in both groups. Concerning PES use, results showed the existence of a significant difference, regarding all the TPB´s constructs of the PES users and nonusers’ groups, and that the predictive capacity of each predictor was different for each group. Psychological strategies should be based on subjective norms, alongside beliefs and attitudes towards PES use, since these variables influence the intention to use PES in that particular population

Optogenetics and biotechnology : production and in vitro characterization of Ab-Initio designed channelrhodopsin-2 mutants

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraNos últimos anos têm sido desenvolvidas várias ferramentas para permitir o controlo de neurónios específicos, possibilitando o estudo da sua função. Estas novas ferramentas superam a falta de selectividade e o fraco controlo temporal proveniente do uso de estimulação eléctrica no controlo de actividade neuronal. A optogenética refere-se á integração de óptica e genética para obter um ganho ou perda de função em eventos bem definidos dentro de células específicas em tecido vivo. A capacidade de “ligar” e “desligar” neurónios utilizando luz é de facto uma tecnologia inovadora que oferece uma solução para limitações passadas. A optogenética, considerada por vários especialistas como ‘’método do ano’’ e ‘’inovação da década’’, em 2010, é utilizada para hiperpolarizar ou despolarizar neurónios alvo, de uma forma menos invasiva, utilizando luz e usufruindo de uma alta resolução espacial e escala temporal na ordem dos milissegundos. Esta técnica tem permitido o mapeamento e estudo de redes neuronais com uma grande eficácia. A ‘’Channelrhodopsin-2’’ (ChR2) é um canal catiónico sensível à luz, derivado da microalga Chlamydomonas reinhardtii. Na última década, a ChR2 tornou-se o arquétipo central e a principal ferramenta da optogenética. Actualmente, a caixa de ferramentas optogenética está em contínua actualização, com contribuições de estratégias de engenharia protéica, tais como mutagénese dirigida e a construção de quimeras com troca de domínios de diferentes espécies de channelrhodopsin. No entanto, alguns aspectos da forma ‘’wild-type’’ da ChR2 ainda requerem atenção e melhoramento. Estes incluem o seu espectro de acção, cinética, níveis de expressão, inactivação, condutância e exactidão de pico de absorção. Em termos de propriedades espectrais, poucas variantes desta proteína têm sido geradas e completamente caracterizadas com sucesso. No entanto, o aprimorar do espectro de activação da ChR2 e do formato do respectivo pico de absorção são algumas das propriedades mais desejadas. A ChR2 é excitada preferencialmente com comprimentos de onda de luz azul (470nm), o que limita o seu uso em material biológico de alta taxa de difusão, tal como o cérebro. Luz de excitação com maiores comprimentos de onda diminui a difusão de luz produzida por tecidos biológicos, e não é absorvida pela hemoglobina, assim, formas da ChR2 ‘’red-shifted’’, a absorver luz vermelha ou mesmo perto de infravermelha, são ferramentas desejáveis para a excitação de tecidos profundos. Alem disto, variantes ‘’blue-shifted’’ são também ferramentas atrativas para desenvolver, XXI dado que a combinação de várias ChR2 que apresentem sensibilidades a diversos comprimentos de onda permitiriam a estimulação de diferentes populações neuronais sem interferência entre si. Neste projecto, realizámos um desenho ab-initio para produzir quatro novas variantes de ChR2, usando uma abordagem de mutagénese dirigida no ambiente do cromófero da ChR2 alterando de forma radical os resíduos alvo. As mutações foram selecionadas com a aplicação de Time Dependent – Density Functional Theory (TDDFT) para prever o espectro de absorção dos mutantes selecionados da ChR2. O ‘’colour tuning’’ da ChR2 foi alcançado em quatro novas variantes criadas. Em particular, fomos capazes de gerar três variantes ‘’red-shifted’’ e uma ‘’blue-shifted’’. Após caracterização espectral, as variantes F217D e F269D apresentaram um ‘’red-shift’’ significativo de 90nm, a variante L221D apresentou um ‘’red-shift’’ de 180nm, a variante F269H apresentou um ‘’blue-shift’’ de 20nm. Apesar dos nossos resultados, é necessária uma caracterização protéica adicional, tal como a avaliação do tráfego membranar em neurónios e as características electrofisiológicas destes novos mutantes para determinar as proriedades cinéticas do canal. Neste trabalho, também conseguimos definir e descrever com sucesso a expressão e purificação da ChR2 ‘’wild-type’’ e de todas as quatro novas variantes no sistema eucariótico de expressão heteróloga - Pichia pastoris. Por fim, o nosso estudo valida as previsões de Time- Dependent Density Functional Theory e revela que abordagens de simulação biofísica podem ser utilizadas com vista à criação de variantes de ChR2 inteligentemente desenhadas. O desenho de novas variantes ChR2, seguindo a lógica racional aplicada, é uma abordagem poderosa e fiável para obter proteínas optimizadas para estratégias biotecnológicas. Os resultados originais obtidos com este trabalho demonstram potential para aplicações futuras, já que novas e melhoradas variantes de ChR2 continuarão a desempenhar um papel central no desenvolvimento e implementação da optogenéticaOver the last few years, several tools have been developed to allow the control over specific types of neuron to enable the study of their function. These novel tools aim to overcome the lack of selectivity and the poor temporal control that derives from trying to control neuronal activity with electrical stimulation. Optogenetics refers to the integration of optics and genetics to obtain gain or loss of function in well-defined events and within specific cells in living tissue. The capacity to turn neurons “on and off” using light is indeed a groundbreaking technology that has become a solution for past limitations. Considered by many, “method of the year” and “breakthrough of the decade”, in 2010, optogenetics is used to hyperpolarize or depolarize specific targeted neurons using light in a less invasive manner, with high spatial resolution and a temporal resolution on the scale of milliseconds. This technique has allowed the mapping and study of neuronal networks with demonstrated efficacy. Channelrhodopsin-2 (ChR2) is a light-gated cation channel, derived from the microalga Chlamydomonas reinhardtii. In the last decade, ChR2 has become the central archetype and the main tool of optogenetics. Presently, the optogenetic toolbox is under continuous update, with contributions from protein engineering strategies, such as site-directed mutagenesis and construction of chimeras with domain swaps between channelrhodopsins of different species. However, some aspects of the wild-type form of ChR2 still require attention and enhancement. These include its action spectra, kinetics, expression levels, inactivation, conductance and absorption peak sharpness. In terms of spectral properties, few variants of this protein have been successfully generated and fully characterized. Nevertheless, tuning of ChR2 activation spectra and absorption peak sharpness are one of the most sought after properties. ChR2 is optimally excitable at a wavelength of blue light (470nm), which limits its use in high light-scattering biologic material, such as the brain. However, long-wavelength excitation light decreases the scattering of light produced by biological tissues and is not absorbed by haemoglobin. Thus, a red-shifted form of ChR2, absorbing red or even near infrared light would be a desirable tool for the excitation of relatively deep tissues. Furthermore, blue-shifted variants would also be attractive tools to develop, since the combination of ChR2 proteins with well separate wavelength sensitivities, combined with multicoloured optics, would permit the stimulation of different neuronal populations with no XXIII interference between them. In this project, we performed ab-initio design to produce four new ChR2 variants, using a radical site-directed mutagenesis approach on target residues in the environment of the ChR2 chromophore. The mutations were selected with the application of Time Dependent – Density Functional Theory (TDDFT) to predict the absorption spectra of ChR2 selected mutants. We achieved successful colour tuning of ChR2 with our four newly created variants. In particular, we were able to generate three red-shifted and one blue–shifted variant. After spectral characterization, the F217D and F269D variants presented a significant 90nm red shift, the L221D variant had a 180nm red shift and the F269H variant presented a 20nm blue shift. Despite our results, additional protein characterization is needed, such as the assessment of membrane trafficking in neurons and an electrophysiological characterization to determine channel kinetic proprieties for each of the variants. In this work, we were also able to define and describe the successful expression and purification of wild type ChR2 and of all the new four variants using the eukaryotic Pichia pastoris heterologous expression system. Finally, our study validates Time-Dependent Density Functional Theory predictions and reveals that biophysical simulation approaches may be used towards the creation of intelligently designed ChR2 variants. The design of new ChR2 variants, following our applied rationale, is a powerful and reliable approach to obtain enhanced proteins for biotechnological strategies. The original output obtained here shows potential for future optogenetic application, as new and improved ChR2 variants will continue to play a central role in the development and implementation of optogenetic

Highlighting the Role of DC-NK Cell Interplay in Immunobiology and Immunotherapy

Dendritic cells (DCs) and natural killer (NK) cells are both part of the innate immune system, also playing crucial functions in the regulation of adaptive immune responses. In recent years, numerous works have demonstrated that DCs and NK cells mutually influence each other with major consequences in the type and effectiveness of elicited immune responses. Among other effects, DC-NK crosstalk can result in NK cell activation and DC maturation or deletion, depending on its activation status. In this chapter and after a brief overview of DCs and NK immunobiology, we focus on the process of DC-NK crosstalk, highlighting the relevance of rationally exploring this interplay in the development of more effective cancer immunotherapies

Ultimate frisbee players:characteristics according to their competitive level and spirit of the game

In Ultimate Frisbee, players are responsible for administering and reinforcing adherence to the rules of the game. As a self-refereed sport, UF relies upon the Spirit of the Game (SOTG). This study aimed to profile the experience, to ascertain the training history of the sport, the participation and goal orientations of international Ultimate Frisbee players in the European Beach Ultimate Championship, and to evaluate the contribution of these variables in a discriminant function to classify players according to SOTG level and competitive level. The sample consisted of 160 players [females 33.8% (29.3 ± 7.2 years); males 66.2% (37.6 ± 9.7 years)] who competed in the European Beach Ultimate Championship, 2019. Factorial ANOVA was used to test the effect of sex, competitive level and SOTG level (measured by the sum of the scores obtained in five questions addressing the following domains: (1) Knowledge and use of the rules; (2) Fouls and body contact; (3) Fair-mindedness; (4) Positive attitude and self-control; (5) Communication. The results revealed that sex was not a consistent source of SOTG level variation among players. In each competitive level, those with high SOTG levels obtained lower European Beach Ultimate Championship classification (t = 5.73 to 6.55, p < 0.001, d = 1.28 to 2.06), higher SOTG classification (t = −13.21 to −7.04, p < 0.001, d = 1.28 to 2.85), and better evaluation for fouls and body contact (t = 2.76 to 9.23, p < 0.05, d = 0.86 to 1.99) and for positive attitude (t = 5.88 to 7.43, p < 0.001, d = 2.06 to 2.57), than regular SOTG level players. Players of different competitive levels demonstrated similar SOTG results. These findings provide important information to coaches, physical education teachers and sport consultants

Concretos normais após incêndio

Este artigo resume trabalhos de investigação sobre à resistência mecânica à compressão e o módulo de elasticidade residuais dos concretos normais usados na construção civil com e sem carregamento durante o processo de aquecimento e arrefecimento. De acordo com os resultados, observa-se que a resistência à compressão e o módulo de elasticidade do concreto diminuem com o incremento da temperatura a que estiveram sujeitos e que o carregamento desde que não excessivo limita essa diminuição

Comparative photodynamic inactivation of bioluminescent E. coli by pyridinium and inverted pyridinium chlorins

Photodynamic inactivation (PDI) is a therapeutic approach in study due to the ability to reduce or completely eliminate the bacterial strains without the development of resistance mechanisms. In this therapeutic methodology the cationic chlorins (Chls) with pyridinium or inverted pyridinium moieties are one of the photosensitizers exploited in our biological approaches. In this context, we synthesized and characterized new free-base and zinc(II) complexes of pyridinium or inverted pyridinium Chl derivatives (1b, 2, 2a and 2b, respectively) for the inactivation of Escherichia coli (E. coli). The PDI assay was performed with white light irradiation delivered at a fluence rate of 25 mW cm−2. The obtained results of this study demonstrate high PDI efficiency of the zinc(II) metallated Chl 1b, reaching the detection limit of the bioluminescent method (5.2 log reduction) in 45 min of irradiation.publishe

Influence of the meso-substituents of zinc porphyrins in dye-sensitized solar cell efficiency with improved performance under short periods of white light illumination

The sensitization activity of four zinc metalloporphyrin dyes: meso-tetrakis(4-pyridyl)porphyrinato Zn(II) (a), meso-triphenyl-(4-carboxyphenyl)porphyrinato Zn(II) (b), meso-tetrakis(4-carboxyphenyl)porphyrinato Zn(II) (c) and meso-tripyridyl(4-carboxyphenyl)porphyrinato Zn(II) (d) is reported here, in terms of current-potential curve, open-circuit potential, fill factor, and overall solar energy conversion efficiency which have been evaluated under 100 mW/cm2 light intensity and their performances compared to the benchmark N719 (di-tetrabutylammonium cis-bis(isothiocyanato)bis(2,2′-bipyridyl-4,4′-dicarboxylato) ruthenium(II)). This work focus the structural aspects of dyes with anchoring groups using TiO2-based Dye Sensitized Solar Cells (DSSCs), which includes pyridyl and carboxyphenyl acid groups and argue how the combination of both anchoring groups, in the same structure, may allow relevant optimization of DSSCs performance in the near future. Also, a noticeable improvement in the photovoltaic performance of all dyes, reaching a maximum increase from 25% to 69% in the overall DSSC efficiency under short periods of white light illumination is discussed.publishe

Reusable and highly sensitive SERS immunoassay utilizing gold nanostars and a cellulose hydrogel-based platform

This work was funded by FEDER funds through the COMPETE 2020 Programme and National Funds through the FCT---Fun-dação para a Ciência e a Tecnologia, I. P., under the scope of the project. This work also received funding from the European Community’s H2020 program under grant agreement No. 716510 (ERC-2016-STG TREND), 640598 (ERC-StG-2014, NEWFUN), and 685758 (1D-Neon). This work was supported by the Applied Molecular Biosciences Unit – UCIBIO and Associate Laboratory for Green Chemistry – LAQV which are financed by Portugal national funds from /2016 and SFRH/BD/132057/2017 from the FCT/MCTES and MIT Portugal PhD Program (to I. C. and M. J. O., respectively). Professor César Laia (LAQV-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Portugal) is acknowledged for permitting the use of DLS equipment. Professor Ludwig Krippahl (NOVA LINCS, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Portugal) is acknowledged for developing the gel analysis application eReuss. M. J. O. acknowledges Rocío Palomares Jurado PhD for visualisation/data presentation support and continued interest in the project. M. J. O. acknowledges David Peitinho MSc for help and advice in Python script. Diego Wiechers de Carvalho BSc is acknowledged for purifying the recombinant Plasmodium falciparum histidine-rich protein 2 sample and Daniela Gomes PhD for providing SEM images. Publisher Copyright: © The Royal Society of Chemistry 2021.The development of robust and sensitive point-of-care testing platforms is necessary to improve patient care and outcomes. Surface-enhanced Raman scattering (SERS)-based immunosensors are especially suited for this purpose. Here, we present a highly sensitive and selective SERS immunoassay, demonstrating for example the detection of horseradish peroxidase (HRP), in a sandwich format. The strength of our biosensor lies in merging: (i) SERS-immunotags based on gold nanostars, allowing exceptional intense SERS from attached Raman probes, covalent attachment of anti-HRP antibodies by a simple chemical method providing exceptional antigen binding activity; (ii) the ease of preparation of the capture platform from a regenerated cellulose-based hydrogel, a transparent material, ideal for microfluidics applications, with low background fluorescence and Raman signal, particularly suited for preserving high activity of the covalently bound anti-HRP antibodies. The sandwich complexes formed were characterised by atomic force microscopy, and by scanning electron microscopy coupled with electron diffraction spectroscopy; and (iii) the robustness of the simple Classical Least Squares method for SERS data analysis, resulting in superior discrimination of SERS signals from the background and much better data fitting, compared to the commonly used peak integral method. Our SERS immunoassay greatly improves the detection limits of traditional enzyme-linked immunosorbent assay approaches, and its performance is better or comparable to those of existing SERS-based immunosensors. Our approach successfully overcomes the main challenges of application at point-of-care, including increasing reproducibility, sensitivity, and specificity, associated with an environmentally friendly and robust design. Also, the proposed design withstands several cycles of regeneration, a feature absent in paper-SERS immunoassays and this opens the way for sensitive multiplexing applications on a microfluidic platform.preprintpublishe

Potenciação de Características Fenotípicas e Funcionais de Células Dendríticas para Aplicação em Imunoterapia Oncológica de Nova Geração

Tese de Doutoramento em Ciências Farmacêuticas, na especialidade de Farmacologia e Farmacoterapia, apresentada à Faculdade de Farmácia da Universidade de Coimbra.The field of cancer immunotherapy is growing at a fast pace, with new developments in this field leading to changes in cancer therapy. Dendritic cells (DCs) are immune cells with a key role at the interface of innate and adaptive immunity. DCs application in immunotherapy has been scrutinised and settled as highly desirable and full of translational and clinical potential, with around 400 clinical trials focused on anti-tumor DC vaccines. Notwithstanding the good safety profile of DC immunotherapy, the rate of success in inducing clear therapeutic outcomes is inconstant. In parallel with the growing knowledge of cancer biology, DC-based approaches must be optimized given that most of the protocols were already established twenty years ago. The production of clinical grade monocyte-derived DCs (mo-DCs) is the most frequent approach for antitumor vaccines production. Typically, mo-DCs are generated from culturing monocytes with variable concentrations of granulocyte-macrophage colony- stimulating factor (GM-CSF) and interleukin (IL)-4. However, mo-DCs do not possess the ideal functional and phenotypical characteristics required for the induction of a robust anti- tumor immune response. Hence, there is a large space for improvement of protocols and a clear need for the establishment of clinical standard operating procedures (CSOPs). Cancer stem cells (CSCs) are a recently identified small cell population present in the tumor, resistant to radio/chemotherapy and known to be responsible for disease recurrence. Therefore, developing a DC vaccine targeting CSC may represent a promising approach in cancer therapy. In this study, we aimed to (1) contribute to the standardization of CSOPs, (2) to target and eradicate CSCs by developing a DC-based immunotherapy targeting these cells, and to (3) develop novel cocktail combinations for tailoring DC differentiation into superior subsets with enhanced immunogenic properties in relation to mo-DCs. First, we performed an in-depth analysis on the impact of good manufacturing practice (GMP) media on the production of clinical-grade DCs. DCs generated in different media presented different phenotypical and functional capacities. Just by changing the nutritive support, we were able to shift DCs profile from immunogenic to a more tolerogenic outline. Importantly, DCs produced in GMP media presented highly different phenotypic and functional features from the ones generated in Roswell Park Memorial Institute medium (RPMI)/pre-clinical medium. These results highlight the gap between pre-clinical data and their translation into a clinical setting. Second, we loaded DCs with CSC lysates and compared the phenotypic profile, functionality and the capacity of these DCs to prime cytotoxic T-lymphocyte cells (CTLs) in relation to classical DCs, loaded with total tumor cell lysates. Our preliminary results demonstrated that DCs loaded with pancreatic CSC lysate were able to trigger T cells to an enhanced and specific elimination of CSCs. These results indicate that our strategy may be an effective approach to eliminate CSCs, a core tumor cell population. Third, from two high-throughput screenings comprising 168 initial combinations, we reached two novel cocktails for DC differentiation that may be alternatives to standard mo- DCs protocols, attaining DC differentiation into subsets with enhanced immunostimulatory properties. DCs differentiated in the presence of these cocktails, C91 and C102, presented a superior maturation profile by expressing higher levels of the co-stimulatory molecule CD80 and major histocompatibility complex (MHC) molecules, when compared to standard mo-DCs. Furthermore, these matured DCs also secreted higher levels of IL- 12p70, essential for an efficient priming and polarization of Th1 cells and subsequent anti- tumor response. Overall, our data provides a strong contribution to the field of DC-based cancer immunotherapy, potentiating several key steps in the production of DC vaccines. We provided insight knowledge on their culture methods, loading with cancer antigens and new protocols of differentiation, contributing to the development of next-generation DC vaccines, a key tool for reaching robust and efficient clinical therapeutic responses in the global fight against cancer.A área de imunoterapia oncológica cresce a um ritmo acelerado, com novos desenvolvimentos neste campo a levarem alterações de grande impacto no tratamento do cancro. Células dendríticas (DCs) são células do sistema imune com um papel fundamental na interface entre a imunidade inata e adquirida. A utilização de DCs em imunoterapia tem sido amplamente estudada, apresentando um enorme potencial translacional e clínico, com cerca de 400 ensaios clínicos focados em vacinas de DCs anti-tumorais. Apesar do excelente perfil de segurança da imunoterapia com DCs, a taxa de sucesso em induzir resultados terapêuticos objectivos é inconstante. Paralelamente ao crescente conhecimento da biologia celular e molecular num contexto oncológico, as abordagens baseadas em DCs deverão ser otimizadas, tendo em conta que maioria dos protocolos usados foram estabelecidos há mais de vinte anos. A produção de grau clínico de DCs derivadas de monócitos (mo-DCs) é a abordagem mais frequente no desenvolvimento de vacinas antitumorais. Normalmente, mo-DCs são diferenciadas a partir da cultura de monócitos com concentrações variáveis de GM-CSF e IL-4. No entanto, as mo-DCs não possuem as características funcionais e fenotípicas ideais necessárias para a indução de uma resposta imune antitumoral robusta. Pelo exposto, existe um grande espaço para o aprimoramento de protocolos e uma clara necessidade de estabelecimento de procedimentos clínicos operativos normalizados (CSOPs). As células estaminais cancerígenas (CSCs) são uma pequena população de células recentemente identificada e presentes no tumor, resistentes à rádio/quimioterapia e conhecidas por serem responsáveis pela recorrência da doença. Ou seja, o desenvolvimento de uma vacina de DCs direcionada contra CSCs poderá representar uma abordagem promissora na terapia oncológica. Neste estudo, pretendemos (1) contribuir para a padronização de CSOPs, (2) direcionar e erradicar CSCs através do desenvolvimento de uma abordagem de imunoterapia baseada em DCs direcionadas para essas células e (3) desenvolver novas combinações de cocktails para optimizar a diferenciação de DCs em subpopulações com características imunogénicas superiores relativamente às mo-DCs. Numa primeira abordagem, realizámos uma análise aprofundada sobre o impacto de meios de cultura de boas práticas de fabrico (GMP) na produção de DCs de grau clínico. DCs diferenciadas em meios de cultura distintos apresentaram diferentes capacidades fenotípicas e funcionais. A simples mudança do suporte nutritivo alterou o perfil das DCs de imunogénico para tolerogénico. É de realçar que as DCs produzidas em meios GMP apresentaram características diferenciadoras das geradas em RPMI/ meio pré-clínico. Estes resultados destacam a lacuna existente entre resultados pré-clínicos e a sua transposição para a clínica Numa segunda abordagem, carregamos as DCs com lisados de CSC e comparamos o perfil fenotípico e a capacidade destas DCs fazerem o priming de linfócitos T citotóxicos relativamente a DCs clássicas, carregadas com lisados tumorais totais. Os resultados preliminares obtidos demonstraram que as DCs carregadas com lisados de CSC pancreáticas foram capazes de desencadear uma efectiva e específica eliminação de CSCs. Estes resultados indicam que a nossa estratégia poderá ser eficaz para eliminar CSCs, uma população-chave de células tumorais. Por fim, a partir de duas análises de alto rendimento que incluíram 168 combinações iniciais, definimos dois novos cocktails de diferenciação que poderão ser alternativos aos protocolos convencionais de obtenção de mo-DCs, proporcionando a diferenciação de DCs em subpopulações com características aprimoradas. DCs diferenciadas com estes cocktails, C91 e C102, apresentaram um perfil de maturação superior por expressarem níveis mais elevados da molécula co-estimuladora CD80 e de moléculas do complexo major de histocompatibilidade (MHC), relativamente às mo-DCs padrão. Adicionalmente, estas DCs maduras também produziram níveis superiores de IL-12p70, citocina fundamental para a activação e expansão de linfócitos T citotóxicos e Th1 e, consequentemente, para uma resposta antitumoral eficaz. Em conclusão, os resultados obtidos constituem um contributo relevante na área da imunoterapia oncológica baseada em DCs, potenciando várias etapas importantes na produção de vacinas de DCs. Especificamente, produzimos conhecimento aprofundado sobre os seus métodos de cultura, carregamento com antigénio tumorais e diferenciação, contribuindo para o desenvolvimento de vacinas de DCs de próxima geração, uma ferramenta-chave para alcançar respostas clínicas terapêuticas robustas na luta global contra o cancro.Tecnimede Group, ToxFinde

Optogenetics and biotechnology : production and in vitro characterization of Ab-Initio designed channelrhodopsin-2 mutants

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraNos últimos anos têm sido desenvolvidas várias ferramentas para permitir o controlo de neurónios específicos, possibilitando o estudo da sua função. Estas novas ferramentas superam a falta de selectividade e o fraco controlo temporal proveniente do uso de estimulação eléctrica no controlo de actividade neuronal. A optogenética refere-se á integração de óptica e genética para obter um ganho ou perda de função em eventos bem definidos dentro de células específicas em tecido vivo. A capacidade de “ligar” e “desligar” neurónios utilizando luz é de facto uma tecnologia inovadora que oferece uma solução para limitações passadas. A optogenética, considerada por vários especialistas como ‘’método do ano’’ e ‘’inovação da década’’, em 2010, é utilizada para hiperpolarizar ou despolarizar neurónios alvo, de uma forma menos invasiva, utilizando luz e usufruindo de uma alta resolução espacial e escala temporal na ordem dos milissegundos. Esta técnica tem permitido o mapeamento e estudo de redes neuronais com uma grande eficácia. A ‘’Channelrhodopsin-2’’ (ChR2) é um canal catiónico sensível à luz, derivado da microalga Chlamydomonas reinhardtii. Na última década, a ChR2 tornou-se o arquétipo central e a principal ferramenta da optogenética. Actualmente, a caixa de ferramentas optogenética está em contínua actualização, com contribuições de estratégias de engenharia protéica, tais como mutagénese dirigida e a construção de quimeras com troca de domínios de diferentes espécies de channelrhodopsin. No entanto, alguns aspectos da forma ‘’wild-type’’ da ChR2 ainda requerem atenção e melhoramento. Estes incluem o seu espectro de acção, cinética, níveis de expressão, inactivação, condutância e exactidão de pico de absorção. Em termos de propriedades espectrais, poucas variantes desta proteína têm sido geradas e completamente caracterizadas com sucesso. No entanto, o aprimorar do espectro de activação da ChR2 e do formato do respectivo pico de absorção são algumas das propriedades mais desejadas. A ChR2 é excitada preferencialmente com comprimentos de onda de luz azul (470nm), o que limita o seu uso em material biológico de alta taxa de difusão, tal como o cérebro. Luz de excitação com maiores comprimentos de onda diminui a difusão de luz produzida por tecidos biológicos, e não é absorvida pela hemoglobina, assim, formas da ChR2 ‘’red-shifted’’, a absorver luz vermelha ou mesmo perto de infravermelha, são ferramentas desejáveis para a excitação de tecidos profundos. Alem disto, variantes ‘’blue-shifted’’ são também ferramentas atrativas para desenvolver, XXI dado que a combinação de várias ChR2 que apresentem sensibilidades a diversos comprimentos de onda permitiriam a estimulação de diferentes populações neuronais sem interferência entre si. Neste projecto, realizámos um desenho ab-initio para produzir quatro novas variantes de ChR2, usando uma abordagem de mutagénese dirigida no ambiente do cromófero da ChR2 alterando de forma radical os resíduos alvo. As mutações foram selecionadas com a aplicação de Time Dependent – Density Functional Theory (TDDFT) para prever o espectro de absorção dos mutantes selecionados da ChR2. O ‘’colour tuning’’ da ChR2 foi alcançado em quatro novas variantes criadas. Em particular, fomos capazes de gerar três variantes ‘’red-shifted’’ e uma ‘’blue-shifted’’. Após caracterização espectral, as variantes F217D e F269D apresentaram um ‘’red-shift’’ significativo de 90nm, a variante L221D apresentou um ‘’red-shift’’ de 180nm, a variante F269H apresentou um ‘’blue-shift’’ de 20nm. Apesar dos nossos resultados, é necessária uma caracterização protéica adicional, tal como a avaliação do tráfego membranar em neurónios e as características electrofisiológicas destes novos mutantes para determinar as proriedades cinéticas do canal. Neste trabalho, também conseguimos definir e descrever com sucesso a expressão e purificação da ChR2 ‘’wild-type’’ e de todas as quatro novas variantes no sistema eucariótico de expressão heteróloga - Pichia pastoris. Por fim, o nosso estudo valida as previsões de Time- Dependent Density Functional Theory e revela que abordagens de simulação biofísica podem ser utilizadas com vista à criação de variantes de ChR2 inteligentemente desenhadas. O desenho de novas variantes ChR2, seguindo a lógica racional aplicada, é uma abordagem poderosa e fiável para obter proteínas optimizadas para estratégias biotecnológicas. Os resultados originais obtidos com este trabalho demonstram potential para aplicações futuras, já que novas e melhoradas variantes de ChR2 continuarão a desempenhar um papel central no desenvolvimento e implementação da optogenéticaOver the last few years, several tools have been developed to allow the control over specific types of neuron to enable the study of their function. These novel tools aim to overcome the lack of selectivity and the poor temporal control that derives from trying to control neuronal activity with electrical stimulation. Optogenetics refers to the integration of optics and genetics to obtain gain or loss of function in well-defined events and within specific cells in living tissue. The capacity to turn neurons “on and off” using light is indeed a groundbreaking technology that has become a solution for past limitations. Considered by many, “method of the year” and “breakthrough of the decade”, in 2010, optogenetics is used to hyperpolarize or depolarize specific targeted neurons using light in a less invasive manner, with high spatial resolution and a temporal resolution on the scale of milliseconds. This technique has allowed the mapping and study of neuronal networks with demonstrated efficacy. Channelrhodopsin-2 (ChR2) is a light-gated cation channel, derived from the microalga Chlamydomonas reinhardtii. In the last decade, ChR2 has become the central archetype and the main tool of optogenetics. Presently, the optogenetic toolbox is under continuous update, with contributions from protein engineering strategies, such as site-directed mutagenesis and construction of chimeras with domain swaps between channelrhodopsins of different species. However, some aspects of the wild-type form of ChR2 still require attention and enhancement. These include its action spectra, kinetics, expression levels, inactivation, conductance and absorption peak sharpness. In terms of spectral properties, few variants of this protein have been successfully generated and fully characterized. Nevertheless, tuning of ChR2 activation spectra and absorption peak sharpness are one of the most sought after properties. ChR2 is optimally excitable at a wavelength of blue light (470nm), which limits its use in high light-scattering biologic material, such as the brain. However, long-wavelength excitation light decreases the scattering of light produced by biological tissues and is not absorbed by haemoglobin. Thus, a red-shifted form of ChR2, absorbing red or even near infrared light would be a desirable tool for the excitation of relatively deep tissues. Furthermore, blue-shifted variants would also be attractive tools to develop, since the combination of ChR2 proteins with well separate wavelength sensitivities, combined with multicoloured optics, would permit the stimulation of different neuronal populations with no XXIII interference between them. In this project, we performed ab-initio design to produce four new ChR2 variants, using a radical site-directed mutagenesis approach on target residues in the environment of the ChR2 chromophore. The mutations were selected with the application of Time Dependent – Density Functional Theory (TDDFT) to predict the absorption spectra of ChR2 selected mutants. We achieved successful colour tuning of ChR2 with our four newly created variants. In particular, we were able to generate three red-shifted and one blue–shifted variant. After spectral characterization, the F217D and F269D variants presented a significant 90nm red shift, the L221D variant had a 180nm red shift and the F269H variant presented a 20nm blue shift. Despite our results, additional protein characterization is needed, such as the assessment of membrane trafficking in neurons and an electrophysiological characterization to determine channel kinetic proprieties for each of the variants. In this work, we were also able to define and describe the successful expression and purification of wild type ChR2 and of all the new four variants using the eukaryotic Pichia pastoris heterologous expression system. Finally, our study validates Time-Dependent Density Functional Theory predictions and reveals that biophysical simulation approaches may be used towards the creation of intelligently designed ChR2 variants. The design of new ChR2 variants, following our applied rationale, is a powerful and reliable approach to obtain enhanced proteins for biotechnological strategies. The original output obtained here shows potential for future optogenetic application, as new and improved ChR2 variants will continue to play a central role in the development and implementation of optogenetic