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Obesity is a chronic disease caused by an imbalance of energy intake and expenditure resulting in excessive accumulation of adipose tissue (AT) either in major adipose depots like subcutaneous (SAT) or visceral (VAT), or ectopic lipid deposition in other organs and tissues such as liver or muscle. In 2019 obesity was ranked globally under the top five death causes and increases the risk for suffering from non-communicable diseases such as stroke, diabetes and various types of cancer. Current therapeutic strategies implement dietary interventions and increased physical activity, application of incretin-based drugs such as dual or triple agonists, or bariatric surgery. The recruitment and activation of brown adipose tissue (BAT) represents an intriguing therapeutic approach to combat obesity by increasing energy expenditure via thermogenesis. BAT is a highly metabolically active organ and its activity is induced by cold. To maintain body temperature, BAT is specialized in the dissipation of energy to produce heat by a high demand of oxygen and substrates such as lipids and glucose. Myoglobin (MB) expression was detected in BAT of cold-exposed rodents and is increased during brown adipocyte differentiation suggesting an unrecognized physiological role in BAT contributing to thermogenesis. Since BAT and muscle are both highly metabolic active organs and are derived from the same myogenic factor 5 positive progenitor, it is likely, that BAT MB might exert similar functions as in muscle tissue. In addition to facilitating oxygen supply, further contributions of MB have been assigned to scavenging ROS and regulating cellular NO levels. Furthermore, a role of MB as a lipid shuttle was proposed, as MB seems to enable energy production via beta-oxidation and prevent myocardial lipid accumulation. This project addressed the hypothesis that MB expression is upregulated in active (brown) AT to support thermogenesis by serving as lipid shuttle from the cytosol into the mitochondria, by contributing and sustaining oxygen supply and/ or by acting as ROS scavenger during thermogenesis. To investigate consequences of MB expression in BAT on mitochondrial function and thermogenesis in vitro and in vivo, MB overexpressing, knockdown or knockout adipocytes and global myoglobin-knockout (Mb-KO) mice were used. Initially, temperature- and differentiation-dependent changes in MB gene and protein expression were investigated in vivo and in vitro. MB expression was upregulated in BAT of cold-exposed C57BL/6N mice and during adipogenesis of brown adipocytes as confirmed in previous findings. Furthermore, BAT Mb gene expression correlated positively with Ucp1 suggesting MB in vivo being regulated by -adrenergic signaling. Surprisingly, in vitro Mb expression was inversely correlated to Ucp1 expression in immortalized and primary brown adipocytes after -adrenergic stimulation with norepinephrine or CL316,243. Since MB expression is increased during adipogenesis, the regulation by PPARG was investigated in immortalized brown adipocytes. Neither the stimulation by PPARG agonists such rosiglitazone or fatty acids nor cell-autonomous effects induced by hypothermia changed Mb gene expression concluding that other pathways regulate MB expression. Evaluating various MB expression levels (high, low, none) on mitochondrial respiration and responsiveness to adrenergic stimulation, Mb knockdown, knockout and overexpression experiments in immortalized and primary brown adipocytes were performed. Herein, a MB expression level dependent increase in maximal mitochondrial respiratory capacity and acute response to adrenergic stimulation, signaling and lipolysis was observed. Also in white adipocytes, metabolic activity was improved by MB overexpression. Since -adrenergic stimulation is accompanied with enhanced ROS production and MB acts as ROS scavenger in cardio myocytes, effects of MB expression on ROS and superoxide levels were determined. However, no impacts were detected, although cold-induced genes were found related to ROS in BAT of Mb-KO mice in vivo, thus a function as ROS scavenger in BAT cannot be excluded. MB binds fatty acids and acylcarnitines, therefore proposed lipid binding residues of MB were mutated. At first, the ability of MB’s lipid binding property was evaluated by dot blot lipid overlay assays. As a result, palmitic and oleic acid were bound by oxygen-carrying MB. The mutant instead showed a reduced binding capacity. In functional assays, the non-lipid binding property abolished the beneficial effects in substrate flux, mitochondrial respiration and thermogenesis of MB in immortalized brown adipocytes. At the end, this data clearly demonstrated that MB’s lipid binding is essential to augment substrate flux and permit increased mitochondrial respiration and thermogenesis. To investigate consequences of MB-deficiency on thermogenesis in vivo, whole-body Mb-KO mice models were exposed to thermoneutrality (30 °C), room temperature (23 °C, mild cold stress) and cold (8 °C) for seven days. Lack of MB resulted in impaired thermoregulation at temperatures below thermoneutrality and diminished the response to pharmacological BAT activation after intraperitoneal CL316,243 application. To address the translational potential, MB is differentially expressed in subcutaneous (SC) and visceral (VIS) depots of human adipose tissue (AT). In lean patients MB was significantly lower expressed in SC AT compared to VIS AT, whereas in obese patients the opposite was overserved. Further analyses revealed that MB expression was more pronounced in AT samples with higher thermogenic potential. As a conclusion, the present study demonstrates for the first time a functional relevance of MB’s lipid binding property and suggests MB as a previously unrecognized player in BAT biology that increases mitochondrial respiratory capacity, a crucial aspect for rapid adaptation to metabolic changes. The exact mechanisms how MB contributes to lipid transport, remain to be elucidated.

Les nanomatériaux posent de nouvelles questions en termes de toxicologie humaine et environnementale et représentent une nouvelle interface avec le milieu biologique aux propriétés spécifiques. De nombreuses inconnues demeurent, en particulier à l’échelle moléculaire, pour permettre d’expliquer certains mécanismes de toxicité. Lorsqu’elles entrent en contact avec le milieu biologique, les nanoparticules se couvrent d’une couche de protéines adsorbées. Celle-ci leur confère une nouvelle « identité biologique » qui contrôle la réponse cellulaire et leur devenir au sein de l’organisme. Nous avons étudié l’adsorption de protéines modèles sur la silice nanostructurée. Après avoir caractérisé la silice nanoporeuse et les nanoparticules de silice utilisées, l’adsorption de la myoglobine, de l’hémoglobine et des protéines d’un extrait cellulaire de levure a été étudiée afin de déterminer les paramètres physico-chimiques et thermodynamiques de l’adsorption des protéines sur la silice. Un enrichissement en résidus basiques, regroupés en clusters de charge, favorise l’adsorption des protéines grâce à la formation d’interactions électrostatiques avec la surface chargée de la silice, indépendamment de la charge globale de la protéine. A l’inverse, un enrichissement en résidus aromatiques est défavorable à l’adsorption car ces résidus forment des interactions π-π qui rigidifient la structure de la protéine. L’identification des protéines adsorbées et non adsorbées à partir d’un milieu complexe pourrait également être utilisée pour les études de toxicité cellulaire. A partir de l’étude de la structure, de la dynamique et de l’activité de la myoglobine et de l’hémoglobine adsorbées sur les nanoparticules de silice, nous avons cherché à définir l’état d’une protéine adsorbée. L’étude de la structure, réalisée par dichroïsme circulaire, spectroscopie UV-visible, d’absorption X, infrarouge, fluorescence et microcalorimétrie, montre une perte partielle de structure importante des protéines adsorbées associée à une grande hétérogénéité de conformations, sans modification majeure de la structure de l’hème. Deux sites potentiels d’interaction entre myoglobine et nanoparticules de silice ont été identifiés à l’aide d’une technique de cartographie de surface par irradiation. L’étude de la dynamique de la myoglobine adsorbée par diffusion élastique et inélastique de neutrons a permis de montrer que l’adsorption s’accompagnait d’une diminution importante de la flexibilité de la protéine. Malgré la perte de structure, la metmyoglobine adsorbée conserve une activité de fixation de ligands très proche de celle de la protéine libre. L’hémoglobine adsorbée présente de façon inattendue une augmentation de son affinité pour l’oxygène et une diminution de sa coopérativité, sans dissociation du tétramère. Cet effet est reproductible lors de l’adsorption de l’hémoglobine humaine, de l’hémoglobine pontée DCL et de l’hémoglobine mutée S. Deux effecteurs permettent par ailleurs de moduler l’affinité de l’hémoglobine adsorbée. Aussi importantes soient-elles, les modifications de structure et d’activité observées sont entièrement réversibles après désorption dans des conditions douces. L’adsorption des hémoprotéines sur les nanoparticules de silice représente véritablement un nouveau type de stress avec résilience pour les protéines en termes de relations entre structure, dynamique et activité. / Nanomaterials raise new questions in environmental and human toxicology and represent a novel interface with specific properties with the biological medium. Several unknown remain to explain all the mechanisms of toxicity, especially at the molecular lever. When they enter the biological medium, nanoparticles get covered by a protein corona. This corona yields to a new “biological identity” that controls the cellular response to nanoparticles and their fate in the organism. We studied the adsorption of model proteins on nanostructured silica. The first part is dedicated to the characterization of nanoporous silica and silica nanoparticles that we used. Then the adsorption of myoglobin, hemoglobin and protein mixture from yeast cells was studied to determine the thermodynamic and physical-chemical parameters of protein adsorption on silica. The enrichment of basic residues, gathered in charge clusters, favors the adsorption of proteins by the formation of electrostatic interactions with the charged surface of silica, independently of the global charge of the protein. On the contrary, the enrichment in aromatic residues is unfavorable to protein adsorption because they form π-π interactions that rigidify the protein structure. The identification of adsorbed and non-adsorbed proteins from a complex medium could also be used for cellular toxicity studies. From the study of the structure, the dynamics and the activity of myoglobin and hemoglobin adsorbed on silica nanoparticles, we tried to define the state of an adsorbed protein. The structural study, based on circular dichroism, fluorescence, infrared, X-ray and UV-visible spectroscopy and microcalorimetry, shows a substantial partial structure loss of adsorbed proteins together with a high conformational heterogeneity, without major modifications of the heme structure. Two potential interaction sites of myoglobin with silica nanoparticles have been identified by a footprinting technique. The study of adsorbed myoglobin dynamics by elastic and inelastic neutron scattering highlighted the important decrease of protein dynamics that occurs upon adsorption. However, despite the structure loss, adsorbed metmyoglobin retains almost all of its activity of ligand binding. Unexpectedly, adsorbed hemoglobin shows an increase of its oxygen affinity and a decrease of its cooperativity, without any dissociation of the tetramer. This effect can be reproduced on human hemoglobin, cross-linked DCL hemoglobin and variant S hemoglobin. Besides, two effectors allow modulating the affinity of adsorbed hemoglobin. Despite the extent of structural and activity changes, all these modifications are entirely reversible upon desorption in soft conditions. The adsorption of hemoproteins on silica nanoparticles depicts a new sort of stress with resilience for proteins in terms of structure, dynamics and activity relationship.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-02T19:23:04Z No. of bitstreams: 1 RitaCarvalho.pdf: 1268991 bytes, checksum: 9f30de20b2377b94837ba5ffe305debe (MD5) / Made available in DSpace on 2017-06-02T19:23:04Z (GMT). No. of bitstreams: 1 RitaCarvalho.pdf: 1268991 bytes, checksum: 9f30de20b2377b94837ba5ffe305debe (MD5) Previous issue date: 2016-07-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Cardiovascular diseases (CVDs) are considered as an important cause of morbidity and premature mortality worldwide. Rapid diagnosis of CVD is extremely important to ensure the survival of the patient. Myoglobin (Mb), for its low molecular weight is quickly released in the body, resulting in high sensitivity in order to be as valuable biomarker of acute myocardial infarction (AMI). Therefore, this study aims to develop an efficient platform, sensitive and stable for the immobilization of antibodies (Ab) myoglobin applicable to the monitoring of antigens (Ag) myoglobin using surface plasmon resonance (SPR). In this sense, the cyclic voltammetry, differential pulse voltammetry and impedance spectroscopy were used in selfassembled monolayers research (SAMs) simple and mixed acid 11-mercaptoundecanoic (MUA) and 3-mercaptopropionic acid (MPA). The mixed SAM MPA / MUA 3: 1 ratio showed low resistance to charge transfer and high surface coverage. In order to investigate the SAM capacity MPA / MUA opposite the locking Ac-MB were made comparative studies for the evaluation of the interaction of Ac-MB SAM MPA / MUA well as for SAM MPA / MUA modified polyamidoamine dendrimer of generation 5 (G5 PAMAM). Then, after choosing the more sensitive platform made up AC-Mb interaction studies with different concentrations of Ag-Mb (0,001; 0,5; 0,8; 1 and 500 µg.mL-1) through immunoassay in a "sandwich". The three-dimensional structure of the dendrimer PAMAM G5 favored amplification of SPR signal as grants most accessible area for the immobilization of antibody Mb. The sandwich immunoassay amplified the interaction signal Ac / Ag Mb, with significant advantages in sensitivity immunosensor. The results show that it was possible to develop an immunosensor capable of detecting low concentrations (0.00067 μg.mL-1) Ag-Mb. In this sense, the proposed system has great potential alternative to early diagnosis, simple and fast IAM. / As doenças cardiovasculares (DCVs) são consideradas como importante causa de morbidade e mortalidade prematura em todo o mundo. O diagnóstico rápido das DCVs é extremamente importante para garantir a sobrevivência do paciente. A mioglobina (Mb), por seu baixo peso molecular, é rapidamente liberada no organismo, resultando em elevada sensibilidade de forma a se constituir como precioso biomarcador de infarto agudo do miocárdio (IAM). Portanto, este trabalho tem por objetivo o desenvolvimento de uma plataforma eficiente, sensível e estável para a imobilização de anticorpos (Ac) de mioglobina aplicável ao monitoramento de antígenos (Ag) de mioglobina empregando ressonância de plásmons de superfície (SPR). Neste sentido, as técnicas de voltametria cíclica, voltametria de pulso diferencial e impedância eletroquímica foram empregadas na investigação de monocamadas auto-organizadas (SAMs) simples e mistas de ácido 11-mercaptoundecanóico (MUA) e ácido 3-mercaptopropiônico (MPA). A SAM mista MPA/MUA de proporção 3:1 apresentou baixa resistência à transferência de carga e elevada cobertura de superfície. Com o propósito de investigar a capacidade da SAM de MPA/MUA frente à imobilização de Ac-Mb foram feitos estudos comparativos para a avaliação da interação da do Ac-Mb com a SAM de MPA/MUA bem como para a SAM de MPA/MUA modificada com dendrímero de poliamidoamina de geração 5 (PAMAM G5). Em seguida, após a escolha da plataforma mais sensível, fez-se os estudos da interação do Ac-Mb com diferentes concentrações do Ag-Mb (0,001; 0,5; 0,8; 1 e 500 µg mL-1) através de imunoensaio em formato “sanduíche”. A estrutura tridimensional do dendrímero PAMAM G5 favoreceu a amplificação do sinal de SPR, pois concede uma maior área acessível para a imobilização do anticorpo da Mb. O imunoensaio em sanduíche amplificou o sinal de interação Ac/Ag da Mb, apresentando vantagens significativas na sensibilidade do imunossensor. Os resultados mostram que foi possível desenvolver um imunossensor capaz de detectar baixas concentrações (0,00067 µg.mL-1) do Ag-Mb. Neste sentido, o sistema proposto apresenta grande potencial alternativo de diagnóstico precoce, simples e rápido do IAM.

Thesis (M.Sc. (Clinical Pathology))--Mahidol University, 1978.

An effective electrochemical sensor for myoglobin (Myb) detection was developed using a simple procedure of denaturing the protein with guanidine hydrochloride and detecting the released heme group by cyclic voltammetry. The concentration of denaturant was optimized to obtain maximum current response for the analyte (Myb). To improve the sensitivity of the sensor, the working electrode, glassy carbon electrode was modified with a layer of Titania nanotubes (TNT). The direct electrochemical behavior of the modified electrode (TNT-GCE) was studied using cyclic voltammetry (CV). The performance of the sensor was investigated and optimized and the system was evaluated by monitoring the Myb concentration. Despite the reduced current response for the modified electrode compared to bare GCE, the sensitivity of the system was improved significantly by overcoming the large background current due to denaturant. The developed TNT modified electrode improved the detection limit of Myb and showed good stability, sensitivity and reproducibility. Under optimal conditions, the catalytic currents are linearly proportional to the concentrations of Myb in the wide range from 50 nM to 6 M. This approach provides improved sensitivity in the given range, and may provide a novel and efficient platform for the fabrication of sensors for other heme proteins.

Akute Nierenschädigung (acute kidney injury, AKI) ist ein häufiger Grund für die Aufnahme auf eine Intensivstation und hat eine hohe Morbidität und Mortalität, insbesondere bei Patienten mit Indikation zur Nierenersatztherapie. In etwa 7-10% wird eine Rhabdomyolyse als mögliche Ursache angegeben. Die rasche Entfernung vom Myoglobin, als direkt renal schädigendes Molekül, erscheint sinnvoll. Allerdings gibt es bisher wenige Daten zur Wirksamkeit einer Nierenersatztherapie bei akuter Nierenschädigung im Rahmen einer Rhabdomyolyse. In dieser prospektiven, randomisierten, einfach verblindeten, monozentrischen Studie wurde erstmalig die Myoglobinclearance bei einer CVVHDF mit „Standard“ high-flux Filter im Postdilutionsmodus (Kontrollarm) mit einer CVVHD mit high-cutoff Filter (Interventionsarm) verglichen. In beiden Gruppen erfolgte die Antikoagulation des extrakorporalen Kreislaufs mit Citrat. Das Ziel der Studie bestand darin, die „Nicht-Unterlegenheit“ des CVVHD-HCO Arms gegenüber des CVVHDF Arms zu zeigen („Non-inferiority design“). Im Rahmen dieser Studie wurden 70 Intensivpatienten mit der Indikation zur kontinuierlichen Nierenersatztherapie eingeschlossen und zu gleichen Teilen in beide Therapiearme randomisiert. Die Patientencharakteristika beider Studienarme unterschieden sich nicht. Die Myoglobin-Clearance war im Interventionsarm durchschnittlich 5.5ml/min (4-7ml/min (95%-Konfidenzintervall), p< 0.0005) höher und damit dem Kontrollarm nicht unterlegen bzw. sogar signifikant überlegen. Ebenso war die Clearance von β2-Mikroglobulin im Interventionsarm signifikant höher. Die Kreatinin- und Harnstoffclearance in den beiden Studienarmen war nach Adjustierung anhand des Dialysegesamtumsatzes gleich. Die Clearance von Interleukin-6 und Albumin während des gesamten Beobachtungszeitraums unterschied sich nicht. Im Vergleich der einzelnen Zeitpunkte war allerdings die Il-6 Clearance nach 1, 6 und 24 Stunden signifikant höher im Interventionsarm. In beiden Studienarmen kam es zu keinem relevanten Albumin-verlust. Die Dialysefilterstandzeit war im Interventionsarm länger, so betrug diese im Kontrollarm: 57.0 [38.0, 72.0] Stunden und im Interventionsarm 70.0 [56.75, 72.0] Stunden (p = 0.029). Frühere Studien zu HCO-Filtern untersuchten häufig β2-Mikroglobulin als Markermolekül für mittleres Molekulargewicht. Die klinische Bedeutung erhöhter β2-Mikroglobulin Spiegel bei akuter Nierenschädigung ist allerdings unklar. Im Gegensatz dazu sind hohe Myoglobin Plasmaspiegel mit akuter Nierenschädigung und einer erhöhten Mortalität assoziiert. Eine rasche und effektive Entfernung dieses nephrotoxischen Moleküls erscheint sinnvoll und ist relevant. Aus diesem Grund wurde in dieser Studie die Myoglobin Clearance bewusst als primärer Endpunkt ausgewählt. Eine gesunde Niere kann deutlich mehr Myoglobin ausscheiden als ein extrakorporales System, weshalb immer der Versuch unternommen werden sollte, die Diurese mit ausreichender Flüssigkeitsgabe zu erhalten. Sollte dennoch eine Nierenersatztherapie notwendig werden, ist aktuell unklar, welches extrakorporale System dafür am besten geeignet ist und ob dies die Erholung der Nierenfunktion oder die Mortalität beeinflussen könnte. Eine bessere Il-6 Clearance im Interventionsarm konnte nur zu Beginn der Dialysebehandlung gezeigt werden. Die Beobachtung einer schlechter werdenden Clearance während zunehmender Behandlungsdauer ist vereinbar mit Ergebnissen anderen Studien. Als Ursache diskutiert werden Proteinablagerungen und Polarisierung am Dialysefilter. Die längere Dialysestandzeit im CVVHD-HCO Arm könnte mit der der Hämokonzentration am Dialysefilter im CVVHDF Arm zusammenhängen. Dieser Effekt wird durch den Postdilutionsmodus verstärkt. Durch das konzentrierte Blut steigt das Risiko der Gerinnselbildung und einer vorzeitigen Verstopfung der Dialysekapillaren. Eine weitere mögliche Erklärung der kürzeren Dialysestandzeit ist die komplexere technische Betreuung einer CVVHDF mit RCA, da unterschiedliche Dialysat- und Substituatlösungen verwendet werden müssen. Die Ergebnisse dieser Studie zeigen, dass durch den Einsatz einer CVVHD mit high cutoff Filter Myoglobin sicher aus der Zirkulation bei Patienten mit akuter Nierenschädigung entfernt werden kann. Die Myoglobin Clearance ist dabei sogar höher als beim Einsatz einer CVVHDF mit high-flux Filter. Besonders blutungsgefährdete oder blutende Patienten mit Rhabdomyolyse und akutem Nierenversagen können vom Einsatz einer CVVHD-HCO unter Nutzung einer regionalen Citratantikoagulation profitieren. Die signifikant längere Dialysefilterstandzeit stellt einen weiteren großen Vorteil des Einsatzes einer CVVHD-HCO dar. Die generierten Ergebnisse der vorliegenden Arbeit liefern die Basis für die Planung weiterer klinischer Studien. Perspektivisch könnte untersucht werden, ob der Einsatz einer CVVHD-HCO die Prognose bei Patienten mit schwerer akuten Nierenschädigung und Rhabdomyolyse verbessern könnte und zu welchem Zeitpunkt die Nierenersatztherapie begonnen werden sollte.:1. EINFÜHRUNG 4 1.1. Akute Nierenschädigung beim kritisch kranken Patienten 4 1.2. Nierenersatztherapie bei akuter Nierenschädigung 5 1.3. Clearance von Molekülen mit mittlerem Molekulargewicht bei verschieden Nierenersatzverfahren 7 1.4. Renale Schädigung durch Myoglobin 10 1.5. Fragestellung und Studiendesign 12 2. PUBLIKATION 15 3. ZUSAMMENFASSUNG DER ARBEIT 16 4. LITERATURVERZEICHNIS 19 5. ANHANG 27 5.1. Supplemental Table 1: Dialysis protocol 27 5.2. Supplemental Table 2: Substance specific clearances (ml/min) after 1h and adjustment for TTR 28 5.3. Supplemental Table 3: Substance specific clearances (ml/min) at different time points 29 III. Nachweis über Anteile der Co-Autoren 30 IV. Erklärung über die eigenständige Verfassung der Arbeit 32

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos &#946;-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies &#945;-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 &#181;M) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no C&#945; (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-&#945; da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA&#8226; (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos &#945;-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos &#945;-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos &#945;-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb formada no ciclo peroxidático da proteína. Avaliando os níveis de lesão de Mb, os &#946;-cetoácidos diminuíram o nível de desorganização protéica na estrutura secundária e terciária elicitada por H2O2. Ainda, houve maior preservação da estrutura primária da proteína, sendo que MAA protegeu mais em comparação a AA, embora quando utilizado este último composto, foi mostrado que há acetilação dose-dependente de Mb. Acetoacetato aumentou a velocidade de descoramento da hemeproteína, provavelmente por ataque de espécies tripletes geradas no sistema. Músculos de rato, plantar e sóleo, expostos ex vivo a concentrações citotóxicas de glicose oxidase (GOX, gera H2O2 em fluxo), foram protegidas pelos ésteres etílicos AAE e MAAE. Foi detectado biacetilo no meio intracelular em músculos expostos a MAAE e GOX. A concentração deste composto &#945;-dicarbonílico é claramente relacionada à abundância de Mb em cada um dos tipos de músculos estudados. Em resumo, Mb tratada com metabólitos &#946;-cetoácidos (AA e MAA) gera radicais centrados em carbono e produtos &#945;-dicarbonílicos altamente reativos no estado triplete. Experimentos realizados com tecido muscular ex vivo sugerem que esta reação possivelmente ocorra in vivo. Levantamos a hipótese de que a geração de espécies carbonílicas reativas e seus adutos em condições de desbalanço metabólico possam contribuir para a compreensão das bases moleculares de desordens cetogênicas. / Acetoacetate (AA) and 2-methylacetoacetate (MAA) are &#946;-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet &#945;-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 &#181;M) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an &#945;-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The &#945;-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA&#8226; adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and &#945;-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the &#945;-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and &#945;-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both &#946;-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2. Therefore, Mb primary structure was more preserved, and MAA was more protective than AA. Moreover using the later compound, it was shown that Mb acetylation is dose-dependent. Acetoacetate increased the rate of the hemeprotein bleaching, probably due to the attack of triplet products generated in the system. Plantaris and soleous rat muscles exposed to damaging concentrations of glucose oxidase (GOX, generates H2O2 in flux), was cytoprotected by AAE and MAAE. Intracellular diacetyl was detected in muscle samples exposed to MAAE and GOX. The &#945;-dicarbonyl concentration is clearly related to the Mb abundance in the muscle types. In summary, Mb treated with peroxides reacts with &#946;-ketoacid metabolites (AA and MAA), yielding carbon-centered radicals and highly reactive &#945;-dicarbonyl products in the triplet state. Experiments carried out ex vivo with muscle tissue showed that this reaction possibly occurs in vivo. A new route for generation and accumulation of carbonyl reactive species and adducts is here proposed to occur in unbalanced metabolic situations, such as is the case of ketogenic disorders.

O ferro (Fe) é um oligoelemento capaz de aceitar e doar elétrons. Tal propriedade o torna extremamente útil em diversos componentes importantes ao bom funcionamento do organismo e da célula. O Fe está associado a algumas proteínas, está presente em citocromos, em moléculas que se ligam ao oxigênio (hemoglobina e mioglobina) e em uma grande variedade de enzimas. O aumento e a diminuição da sua oferta levam a alterações na expressão de RNAs mensageiros e proteínas responsáveis pela sua própria homeostase. Sabe-se que a expressão de vários genes envolvidos no metabolismo do Fe é regulada pós-transcricionalmente, por meio de mecanismo que é desencadeado por sua ligação em regiões não traduzíveis presentes em mRNAs específicos, o que interfere no seu grau de poliadenilação, e por conseguinte, na estabilidade e na tradução do transcrito. A Mb é uma heme-proteína de 18,8 kDa, altamente expressa no tecido muscular esquelético e cardíaco, e que pertence a mesma família da hemoglobina. Sabendo-se que cerca de 15% do Fe existente no organismo está presente nos músculos, no presente trabalho avaliamos se a suplementação e restrição de Fe, a curto e longo prazo, alteram a expressão gênica da Mb no músculo oxidativo Soleus (S), glicolítico Extensor Digital Longo (EDL) e no cardíaco. Observamos que a restrição de Fe, a longo prazo, provocou um aumento na expressão gênica e protéica da Mb, apenas no músculo Soleus, sem alterar o grau de poliadenilação do transcrito, enquanto a suplementação não alterou os parâmetros avaliados em nenhum dos tecidos. A administração aguda de Fe não alterou a expressão gênica e protéica da Mb, nem o grau de poliadenilação do transcrito em nenhum dos tecidos estudados. Estes resultados sugerem que a regulação da expressão da Mb pelo Fe se dá apenas transcricionalmente, e de maneira tecido específica. / Iron is a trace element that can accept and donate electrons. This property makes iron extremely important to several components involved with the proper functioning of the organism and cells. Iron is associated with some proteins, is present in cytochromes, molecules that bind to oxygen (hemoglobin and myoglobin) and a variety of enzymes. The increase and decrease of its offer lead to changes in the expression of mRNAs and proteins responsible for their own homeostasis. It is known that the expression of several genes involved in the metabolism of iron is regulated post-transcriptionally through a mechanism that is triggered by its binding in non-translatable regions of specific mRNAs, which interferes with their polyadenylation, and as a consequence, with the stability and translation of the transcripts. Mb is a heme-protein with 18,8 kDa, highly expressed in skeletal and cardiac muscle, and it belongs to the same family of hemoglobin. About 15% of iron in the body is present in muscle tissue. Thus, this study aimed to investigate if long- and short-term Fe supplementation and restriction affect Mb gene expression in the oxidative Soleus (S), glycolitic Extensorum Digitalis Longus (EDL), and cardiac muscles. It was shown that long- term Fe restriction increased Mb mRNA and protein expression only in S muscle, without interfering in the transcript polyadenylation, whereas Fe supplementation did not alter any parameter evaluated in the three tissues. The short-term iron administration did not change the Mb mRNA, polyadenylation and protein expression in any of the tissues studied. The present results indicate that the regulation of Mb gene expression by iron occurs only at transcriptional level and in a tissue specific manner.