β2-glicoproteína I (β2GPI) é encontrada (200µg/mL) no plasma, 60% livre e 40% em lipoproteínas. Esta proteína de fase aguda, com afinidade por superfícies negativas pode ser clivada pela plasmina. Fragmentos são purificados como dímeros ou multímeros de β2GPI. Formas monomérica e dimérica de β2GPI foram purificadas de soro humano e identificadas por SDS-PAGE, imunoblot e ELISA Somente a forma monomérica foi detectada no teste ELISA Os efeitos de ambas as formas sobre o burst respiratório de polimorfonucleares humanos (PMN), estimulados in vitro com zymosan opsonizado, foram estudados por quimioluminescência amplificada por luminol ou lucigenina e citometria de fluxo, pela oxidação do OCFH. A forma monomérica inibiu a quimioluminescência amplificada por luminol (-43.6 x -7.33 AEU/s), mas não por lucigenina, e aumentou a oxidação de DCFH e a produção de Óxido Nítrico (•NO). É provável que o •No, via peroxinitrito, medeie os efeitos de β2GPI sobre o burst respiratório de PMN. / Circulating blood contains approximately 200µg/mL of β 2-glycoprotein I (β2GPI), either free (60%) or lipoprotein bound (40%). This acute phase protein, with affinity for negative surfaces, can be cleaved by plasmin. Fragments purify as dimeric or multimeric (β2GPI. Both (β2GPI forms were purified from human sera and identified by SDS-PAGE, immunoblotting and ELISA. ELISA reactivity was dependent on the monomeric status of (β2GPI. The effects of dimeric and monomeric (β2GPI upon respiratory burst of human polimorphonuclear neutrophils (PMN) stimulated in vitro with opsonized zymosan were studied. Respiratory burst was evaluated by luminol- or lucigenin-amplified chemiluminescence, or by DCFH oxidation (flow-cytometry assay). The monomeric, but not the dimeric form, inhibited the luminol chemiluminescence of zymosan-stimulated PMNs (-43.6 x -7.33 AEU/s). Lucigenin chemiluminescence was insensitive to (β2-GPI. Monomeric (β2GPI increases both DCFH oxidation and nitric oxide production. Nitric oxide, probably through peroxynitrite reactions, mediates (β2GPI effects upon PMNs respiratory burst.

Electrophoretic microsystems (EM) are powerful tools for the separation of species of microsystems analyzes which can easily be combined with electrochemical detection (ECD) and therefore making it ideal for a method of detection. However, the influence of high voltage at the working electrode used for the separation is a problem to be overcome due to the increased signal/noise ratio and possible damage of the electrode and/or the potentiostat. Thus, it was proposed in this thesis one EM hybrid PDMS / glass configuration with dual-channel potentiostat coupled to an electrically isolated in order to minimize the influence of high potential in the separation channel and improve the separation efficiency of the species and subsequently, improve detection limits. The EM contains two separate parallel channels 200 microns and a channel separation and another reference, and each containing a platinum electrode 15 or 50 μm placed about 1 to 4 μm in the channel. An electrode served as the working electrode, positioned in the separation channel, and another electrode as reference electrode, placed in the reference channel. This configuration associated with the electrically isolated potentiostat allowed the amperometric signals were measured without any change or potential interference arising from the high voltage applied separation. Aiming to evaluate the effectiveness of the methodology proposed in this thesis, samples nitrite, tyrosine and peroxynitrite (reactive nitrogen species – RNS), hydrogen peroxide (reactive oxygen species – ROS), ascorbic acid, glutathione and cysteine were injected into the channel containing the working electrode, while simultaneously boric acid buffer pH 11 containing TTAB was injected into the reference channel containing the reference electrode. From this configuration, we obtained a significant reduction in noise level (about 0.94 pA) and a relative improvement in the resolution ratified by electropherograms, compared with using single channel configuration. The limits of detection (LOD) for the chemical species mentioned above were 0.58 μM, 0.14 μM, 0.75 μM, 0.21 μM, 0.82 μM, was not obtained for cysteine and 1.63 μM, respectively. The efficiency can also be seen by analyzing nitrite performed on samples of perfusate blood of sheeps and rats, where have been detected a concentration of 68.05 μM and 22.04 μM, respectively, by the proposed method. It was also proposed in this thesis, microfabrication and evaluation of a PMMA electrophoretic microsystem with single channel configuration coupled to a base made of the same material to fix the microchip with electrochemical detection using a carbon paste electrode. The purpose of the construction of the base was to obtain, by fixing, reproducibility of events. And the microfabrication of PMMA EM aimed the viability of its use in analysis perspective as having the lowest cost per unit made due to the use of CO2 laser for microfabrication, which has a value considerably lower, compared with photolithographic processes. The evaluation of this system was performed through the analysis standards of serotonin and acetaminophen, which proved that the microfabrication of this system showed good reproducibility and repeatability of events, making it viable processing. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os microssistemas eletroforéticos (MSE) são ferramentas poderosas para a separação de espécies em microssistemas de análises, onde pode ser facilmente combinada com detecção eletroquímica (DEQ) e tornando-se, portanto, um método de detecção ideal. No entanto, a influência da alta tensão no eletrodo de trabalho utilizada para a separação é um problema a ser contornado devido o aumento da relação sinal/ruído e possíveis danificações do eletrodo e/ou do potenciostato. Assim, foi proposto nesta tese um MSE híbrido de PDMS/vidro com configuração de duplo-canal acoplado a um potenciostato eletricamente isolado com objetivo de minimizar a influência do elevado potencial no canal de separação e melhorar a eficiência de separação das espécies e, subsequentemente, melhorar os limites de detecção. O MSE contém dois canais paralelos separados 200 μm, sendo um canal de separação e outro de referência, e cada um deles contendo um eletrodo de platina de 15 ou 50 μm colocados cerca de 1 a 4 μm dentro do canal. Um eletrodo serviu como eletrodo de trabalho, posicionado no canal de separação, e o outro eletrodo como eletrodo de referência, posicionado no canal de referência. Essa configuração associado ao potenciostato eletricamente isolado permitiu que os sinais amperométricos fossem medidos sem qualquer mudança de potencial ou de interferência oriunda da alta tensão de separação aplicada. Objetivando avaliar a eficiência da metodologia proposta nessa tese, amostras de nitrito e peroxinitrito (espécies reativas de nitrogênio – ERN), tirosina, peróxido de hidrogênio (espécie reativa de oxigênio – ERO), ácido ascórbico, glutationa e cisteína foram injetadas no canal contendo o eletrodo de trabalho, enquanto que simultaneamente o tampão de ácido bórico contendo TTAB pH 11 foi injetado no canal de referência contendo o eletrodo de referência. A partir desta configuração, obteve-se uma significativa diminuição no nível de ruído (cerca de 0,94 pA) e uma relativa melhora na resolução ratificadas pelos eletroferogramas, se comparado com a configuração que utiliza canal único. Os limites de detecção (LOD) para as espécies químicas supracitados foram de 0,58 μM, 0,14 μM, 0,75 μM, 0,21 μM, 0,82 μM, não foi obtida para a cisteína, e 1,63 μM, respectivamente. A eficiência também pode ser vista através das análises de nitrito realizadas em amostras de perfusato de sangue de ovelhas e ratos, onde foram detectados uma concentração de 68,05 μM e 22,04 μM, respectivamente, através da metodologia proposta. Foi proposto também nessa tese, a microfabricação e avaliação de um microssistema eletroforético de PMMA com configuração de canal único acoplado a uma base feita do mesmo material para fixar o microchip, com detecção eletroquímica usando eletrodo de pasta de carbono. O objetivo da construção da base foi obter, através da fixação, reprodutibilidade de eventos. E a microfabricação do MSE de PMMA objetivou a viabilidade do seu uso em análises tendo como perspectiva o baixo custo por unidade confeccionada devido ao uso de laser de CO2 para a microfabricação, o qual possui um valor agregado consideravelmente menor, se comparado com os processos fotolitográficos. A avaliação desse sistema foi feita através das análises de padrões de serotonina e acetaminofeno, onde comprovou-se que a microfabricação desse sistema apresentou boa reprodutibilidade e repetitividade de eventos, tornando-se viável o seu processamento.

β2-glicoproteína I (β2GPI) é encontrada (200µg/mL) no plasma, 60% livre e 40% em lipoproteínas. Esta proteína de fase aguda, com afinidade por superfícies negativas pode ser clivada pela plasmina. Fragmentos são purificados como dímeros ou multímeros de β2GPI. Formas monomérica e dimérica de β2GPI foram purificadas de soro humano e identificadas por SDS-PAGE, imunoblot e ELISA Somente a forma monomérica foi detectada no teste ELISA Os efeitos de ambas as formas sobre o burst respiratório de polimorfonucleares humanos (PMN), estimulados in vitro com zymosan opsonizado, foram estudados por quimioluminescência amplificada por luminol ou lucigenina e citometria de fluxo, pela oxidação do OCFH. A forma monomérica inibiu a quimioluminescência amplificada por luminol (-43.6 x -7.33 AEU/s), mas não por lucigenina, e aumentou a oxidação de DCFH e a produção de Óxido Nítrico (•NO). É provável que o •No, via peroxinitrito, medeie os efeitos de β2GPI sobre o burst respiratório de PMN. / Circulating blood contains approximately 200µg/mL of β 2-glycoprotein I (β2GPI), either free (60%) or lipoprotein bound (40%). This acute phase protein, with affinity for negative surfaces, can be cleaved by plasmin. Fragments purify as dimeric or multimeric (β2GPI. Both (β2GPI forms were purified from human sera and identified by SDS-PAGE, immunoblotting and ELISA. ELISA reactivity was dependent on the monomeric status of (β2GPI. The effects of dimeric and monomeric (β2GPI upon respiratory burst of human polimorphonuclear neutrophils (PMN) stimulated in vitro with opsonized zymosan were studied. Respiratory burst was evaluated by luminol- or lucigenin-amplified chemiluminescence, or by DCFH oxidation (flow-cytometry assay). The monomeric, but not the dimeric form, inhibited the luminol chemiluminescence of zymosan-stimulated PMNs (-43.6 x -7.33 AEU/s). Lucigenin chemiluminescence was insensitive to (β2-GPI. Monomeric (β2GPI increases both DCFH oxidation and nitric oxide production. Nitric oxide, probably through peroxynitrite reactions, mediates (β2GPI effects upon PMNs respiratory burst.

[pt] A pandemia da COVID-19 instaurou inúmeras pesquisas sobre a doença, a qual foi atribuída à tempestade de citocinas. Embora, as vacinas sejam eficazes para combater a disseminação da COVID-19 e, recentemente, tenha sido aprovado pela ANVISA (Agência Nacional de Vigilância Sanitária) o fármaco antiviral Paxlovid (Trade Mark), no tratamento da COVID-19, estudos sobre formas de tratamento ainda são necessários. Dentre as novas estratégias terapêuticas, a utilização de nanopartículas de ouro (AuNPs) tem se destacado devido o potencial efeito antiinflamatório. O objetivo deste trabalho foi estudar as eventuais propriedades antiinflamatórias das AuNPs, sintetizadas por ablação a laser pulsado (PLA) em água, em macrófagos RAW 264.7. Ademais, o presente trabalho visou investigar a possível ligação entre o efeito imunomodulador das nanopartículas e os produtos secundários obtidos através da reação de redução da CO2 (CO2RR) ativada por PLA em água. Nesse sentido, foram analisados a proliferação, a citotoxicidade, os níveis das espécies reativas de oxigênio (EROs) e das espécies reativas de nitrogênio (ERNs) nos macrófagos RAW 264.7 submetidas ao tratamento com as AuNPs e expostas ao lipopolissacarídeo (LPS), o qual foi utilizado como agente precursor do processo inflamatório. Em conclusão, os resultados apontaram que AuNPs na concentração de 10 (micro)g/mL não tiveram caráter citotóxico e diminuíram as EROs e ERNs. As AuNPs demonstraram promissora atividade antiinflamatórias com reduzidos valores de citotoxicidade, o que aponta para uma futura aplicação clínica. / [en] With the advent of the COVID-19 pandemic, numerous studies on the disease were initiated, a disease originated from a cytokine storm. Although vaccines are effective in combating the spread of COVID-19 and, more recently, the antiviral drug Paxlovid (Trade Mark) has been approved by ANVISA (Brazilian Health Regulatory Agency) for the treatment of COVID-19, studies on forms of treatment are still needed. Among the new therapeutic strategies, the use of gold nanoparticles (AuNPs) has been in the spotlight due to their potential antiinflammatory effect. This work aims to study the potential anti-inflammatory properties of AuNPs, synthesized by pulsed laser ablation (PLA) in water, in RAW 264.7 macrophages. Furthermore, the present work aimed to investigate the possible connection between the immunomodulatory effect of the nanoparticles and the secondary products obtained through the CO2 reduction reaction (CO2RR) activated by PLA in water. In this sense, we analyzed the proliferation, cytotoxicity, levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in RAW 264.7 macrophages subjected to treatment with AuNPs and exposed to lipopolysaccharide (LPS), which was used as a precursor agent of the inflammatory process. In conclusion, the results showed that the AuNPs at a concentration of 10 (micro)g/mL did not have a cytotoxic character and reduced ROS and RNS. AuNPs demonstrated promising anti-inflammatory activity with reduced cytotoxicity values, which points to future clinical applications.

Les thérapies anticancéreuses basées sur des principes physiques (radiofréquences, ultrasons, laser, électroporation...) ont considérablement augmenté lors de la dernière décennie. Leurs objectifs sont de détruire directement les cellules cancéreuses, de favoriser l'entrée ciblée de molécules thérapeutiques ou encore de stimuler le système immunitaire du patient afin d'éliminer la tumeur. Le plasma froid suscite l'intérêt dans le domaine de l'oncologie grâce à sa capacité à générer des espèces réactives oxygénées (ROS) et azotées (RNS) qui peuvent être génotoxiques et cytotoxiques pour les cellules cancéreuses. Deux approches d'utilisation du plasma sont étudiées : soit l'exposition directe de cellules au jet plasma, soit l'exposition indirecte via l'utilisation d'un Milieu Activé par Plasma (PAM). Le PAM étant plus facile à délivrer par injection dans la tumeur, c'est cette approche qui est choisie lors de ces travaux. Le travail de thèse présenté consiste à étudier l'effet génotoxique et cytotoxique du PAM, obtenu après exposition du milieu au jet de plasma d'hélium, sur des tumeurs in vitro et in vivo. Pour les études in vitro, nous avons choisi d'utiliser un modèle 3D : le sphéroïde (MCTS - MultiCellular Tumor Spheroid). Ce modèle présente des caractéristiques proches du modèle in vivo grâce à son organisation en sphéroïde. Les MCTS présentent en effet des gradients de pénétration d'oxygène, de nutriments et de prolifération cellulaire. La première partie de la thèse concerne l'identification et la quantification des espèces générées dans le PAM. Les méthodes d'analyses utilisées sont la résonance paramagnétique électronique, la fluorimétrie, la colorimétrie, la chromatographie en phase liquide et la spectrométrie de masse. Ces analyses ont mis en évidence que la toxicité du PAM était due à plusieurs facteurs : d'un côté la génération de ROS et RNS mais aussi à la dégradation des nutriments pour les cellules contenues dans le milieu via par exemple l'oxydation et la nitrosylation des acides aminés. La deuxième partie est dédiée à l'étude des effets du PAM sur les MCTS HCT-116 (cancer du côlon).[...] / Cancer therapies based on physical principles (radiofrequency, ultrasound, laser, electroporation...) have considerably increased in the last decade. Their objectives are to directly destroy cancer cells, to favor the targeted entry of therapeutic molecules or to stimulate the patient's immune system in order to eliminate the tumor. Cold plasma still arouses interest in the field of oncology through its ability to generate reactive oxygen species (ROS) and nitrogen species (RNS) which can be genotoxic and cytotoxic for cancer cells. Two approaches to the use of plasma are studied: either direct exposure of cells to the plasma jet, or indirect exposure via the use of a Plasma Activated Medium (PAM). The PAM being easier to deliver by injection into the tumor, this approach was chosen in this work. The work presented consists in studying the genotoxic and cytotoxic effects of PAM resulting from exposure of the medium to the helium plasma jet on in vitro and in vivo tumors. For in vitro studies, we chose to use a 3D model: the spheroid (MCTS - MultiCellular Tumor Spheroid). This model has similar characteristics to the in vivo model thanks to its spheroidal organization. The spheroids have indeed gradients of oxygen penetration, nutrients and cell proliferation. The first part of the thesis concerns the identification and quantification of the species generated in PAM. The analytical methods used are paramagnetic electronic resonance, fluorimetry, colorimetry, liquid chromatography and mass spectrometry. These analyses revealed that the toxicity of PAM was due to several factors: on the one hand to the generation of ROS and RNS and on the other hand to the degradation of cell nutrients contained in the medium via, for example, the oxidation and nitrosylation of the amino acids. The second part is dedicated to the study of the effects of PAM on HCT-116 (colon cancer) spheroids[...]

In Anbetracht des Kompromisses zwischen der Erzielung des höchsten Gewinns und der geringsten Umweltbelastung ist ein tiefes Verständnis der ökonomischen Folgen der Stickstoff (N) Düngung erforderlich. Die vorliegende Doktorarbeit liefert umfassende Einblicke in (i) die Auswirkungen des standortspezifischen N-Managements (SSNM) auf die Rentabilität und Risikominderung, (ii) die Auswirkungen von Unsicherheiten und Risikoeinflüssen auf optimale N-Düngergaben und (iii) das Potenzial und die Kosten der Vermeidung von Treibhausgas (THG) Emissionen durch N-Düngereduktion. Ein Modellierungsansatz wurde entwickelt, um die Wirkung von Ertrag und Proteingehalt, Wirtschafts- und Risikoauswirkungen sowie THG-Emissionen auf die N-Düngung zu simulieren. Die Ergebnisse der Arbeit zeigen, dass SSNM die Wirtschaftlichkeit verbessert, indem es eine höhere Weizenqualität und damit Preisprämien erzielt. SSNM reduziert das Risiko, die Backqualität nicht zu erreichen, und es gibt keine wesentlichen Nachteile beim Verlustrisikomanagement im Vergleich zum einheitlichen Management. Preisprämien für eine höhere Weizenqualität bieten Anreize für höhere N-Düngergaben. Prämien verflachen die Gewinnfunktion weiter, was unzureichende Argumente für eine Absenkung des N-Inputs aus der Wirtschaftlichkeitssicht liefert, selbst bei einer hohen Risikoaversion der Landwirte. Eine moderate Reduzierung der mineralischen N-Düngung kann die THG-Emissionen bei moderaten Opportunitätskosten mindern. Die THG-Vermeidung durch N-Düngereduktion in einer bestimmten Region kann unter Berücksichtigung kultur- und ertragszonenspezifischer Ertragswirkungen optimiert werden. Insgesamt liefert diese Arbeit wichtige Erkenntnisse über die Chancen und Nachteile der Anpassung der N-Düngergaben. Darüber hinaus leistet sie einen direkten Beitrag zur Identifizierung von kosten- und risikoeffizienten N-Managementoptionen und bildet die Grundlage für effektive politische Ansätze zur THG-Vermeidung durch selektive N-Düngereduktion. / Considering the tradeoff between achieving the highest profit and causing the lowest environmental impact, there is a need for a profound understanding of the economic consequences of nitrogen (N) fertilizer application. The present doctoral research provides comprehensive insights into (i) effects of site-specific N management (SSNM) on profitability and risk mitigation; (ii) impacts of uncertainties and risk implications on optimal N fertilizer rates; and (iii) potential and costs of mitigating greenhouse gas (GHG) emissions by N fertilizer reduction. A modelling approach was developed to simulate the response of yield, protein, economic and risk implications, and GHG emissions to N fertilizer application. Findings of the thesis show that SSNM improves profitability by achieving higher grain quality, thus, price premiums. SSNM reduces the risk of not reaching the baking grain quality and poses no considerable disadvantage on downside risk management compared to uniform management. Price premiums for higher wheat quality provide incentives for higher N input rates. Premiums further flatten the profit function, giving insufficient arguments for lowering N input from a farm profitability perspective, even in presence of high risk aversion of farmers. Moderate reduction of mineral N fertilizer can mitigate GHG emissions at moderate opportunity costs. GHG mitigation by N fertilizer reduction in a given region can be optimized considering crop and yield-zone-specific yield responses. Overall, this thesis provides important insights on chances and drawbacks of adjusting N fertilizer rates. Moreover, it makes a direct contribution in identifying cost- and risk-efficient N management options and provides a basis for effective policy approaches to reduce GHG emissions by selective N fertilizer reduction.