Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.

Studies to gain mechanistic understanding of heart dysfunction based on animal and traditional cell culture models have significant limitations. Animal models are low throughput and fail to recapitulate many aspects of human cardiac biology, and 2D culture models utilizing human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) are higher throughput but fail to incorporate one or more in vivo parameters, such as 3D architecture, electrical pacing and mechanical constraint. High throughput 3D tissue platforms could better recapitulate the in vivo microenvironment of cardiac tissue. Previous work from our group demonstrated an approach to build 3D cardiac microtissues based on photolithography-based fabrication of a MEMS device, but design limitations prevented further iterations. In this work, we used a 3D printing approach to engineer iPSC-CM-derived cardiac microtissues with different form factors. Microtissues generated in this platform increased in lifespan compared to the first-generation platform by more than 100%. When modeling mutations associated with genetic cardiomyopathy, functional and structural differences were observed between tissues composed of wild-type and mutant iPSC-CMs. These findings suggest that this micro-device platform can be potentially used for both mechanistic and drug discovery studies. / 2020-07-02T00:00:00Z

Inflammation plays a key role during pathological hypertrophy and heart failure. Whilst the roles of pro-inflammatory cytokines are relatively well understood, little is known about the anti-inflammatory cytokines in the heart. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine that is expressed in the heart and may play a crucial role during cardiac remodelling. IL-10 exerts its function by binding to the IL-10 receptor (IL-10R). The primary aim of the PhD study was to investigate the effects of the ubiquitous ablation of IL-10R1 gene during pressure overload induced hypertrophy and to characterise the downstream pathway regulated by IL-10R1 in the heart following pressure overload. The second aim was to investigate the effects of cell specific ablation of IL-10R1 in both the macrophages and cardiomyocytes during pressure overload induced hypertrophy and to identify the specific site where IL-10R1 regulates hypertrophy in the heart. During this study three mouse lines were used: IL-10R1 global knockout (IL-10R1-/-), IL-10R1 macrophage-specific knockout (IL-10R1mKO) and IL-10R1 cardiomyocyte-specific knockout (IL-10R1cKO).Mice with systemic ablation of IL-10 receptor1 (IL-10R1-/-) displayed a significant increase in hypertrophy following two weeks of transverse aortic constriction (TAC) as indicated by heart weight/tibia length ratio (HW/TL). This was accompanied by a significant increase in cardiomyocyte surface area as well as expression of hypertrophic markers such as brain natriuretic peptide (BNP) and Atrial natriuretic peptide (ANP). The IL-10R1-/- mice also had a significant increase in cardiac fibrosis when compared to the WT TAC littermates. Importantly, ejection fraction (EF) and fractional shortening (FS) were significantly reduced in IL-10R1-/- mice compared with WT littermates following TAC. The STAT3 pathway is known as the major downstream signalling pathway regulated by the IL-10R via the activation of the JAK1/STAT3 pathway.  Western blot analysis showed that activation of the STAT3 signalling pathway was significantly reduced in IL-10R1-/- mice following TAC, indicating the possible involvement of this pathway. Furthermore, expression of STAT3 target genes: suppressor of cytokine signalling (SOCS3), tissue inhibitor of metalloproteinases3 (TIMP-3) and heme oxygenase (HO-1) were downregulated in the IL-10R1-/- mice following TAC. Overall the data obtain from the IL-10R1-/- mice indicate that IL-10R1 signalling plays a protective role in reducing pathological hypertrophy in the heart. Interestingly, IL-10R1mKO mice showed no difference in the hypertrophic response following TAC. Analysis of cardiac function and STAT3 activation also showed no difference between IL-10R1mKO and WT controls. This indicated that the protective effects of IL-10R1 mediated signalling during cardiac pressure overload was unlikely due to the effects in residential macrophages. In contrast, IL-10R1cKO mice displayed an elevated hypertrophic response, reduction of cardiac function and less STAT3 activation after TAC. This phenotype resembled those of IL-10R1 global knockout mice. In conclusion, this PhD study has shown that IL-10R1 mediated signalling in the heart is important in controlling pressure-overload hypertrophy. Using cell-specific knockout mice I have shown that IL-10R signalling in cardiomyocytes and not in macrophages is important in this process. These results will open a new insight in targeting IL-10 receptor in the treatment of myocardial hypertrophy in future.

An important element of metabolic regulation of cardiac and skeletal muscle energetics is the interaction of mitochondria with cytoskeleton. Mitochondria are in charge of supplying the cells with energy, adjusting its functional activity under conditions of stress or other aspects of life. Mitochondria display a tissue-specific distribution. In adult rat cardiomyocytes, mitochondria are arranged regularly in a longitudinal lattice at the level of A band between the myofibrils and located within the limits of the sarcomeres. In interaction with cytoskeleton, sarcomeres and sarcoplasmic reticulum they form the functional complexes, the intracellular energetic units (ICEUs). The ICEUs have specialized pathways of energy transfer and metabolic feedback regulation between mitochondria and ATPases, mediated by CK and AK. The central structure of ICEUs is the mitochondrial interactosome (MI) containing ATP Synthasome, respiratory chain, mitochondrial creatine kinase and VDAC, regulated by tubulins. The main role of MI is the regulation of respiration and the intracellular energy fluxes via phosophotransfer networks. The regulation of ICEUs is associated with structural proteins. The association of mitochondria with several cytoskeletal proteins described by several groups has brought to light the importance of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To purvey a better understanding of these findings, the present work investigated the mechanism of energy fluxes control and the role of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To show these complex associations in adult cardiac cells several proteins were visualized by confocal microscopy: α-actinin and β-tubulin isotypes. For the first time, it was showed the existence of the specific distribution of β-tubulin isotypes in adult cardiac cells. Respiratory measurements were performed to study the role of tubulins in the regulation of oxygen consumption. These results together confirmed the crucial role of cytoskeletal proteins -i.e. tubulins, α-actinin, plectin, desmin, and others- for the normal shape of cardiac cells as well as mitochondrial arrangement and regulation. In addition, in vivo - in situ mitochondrial dynamics were studied by the transfection of GFP-α-actinin, finding that fusion phenomenon does not occur as often as it is believed in healthy adult cardiac cells. / Un élément important de la régulation du métabolisme énergétique des muscles cardiaque et squelettiques est l'interaction des mitochondries avec le cytosquelette. Les mitochondries sont responsables de l'approvisionnement des cellules en énergie, elles sont capables d'ajuster leur activité fonctionnelle en fonction des conditions de stress ou d'autres aspects de la vie. Les mitochondries ont une distribution spécifique selon les tissus. Dans les cardiomyocytes de rats adultes, les mitochondries sont disposées régulièrement dans un entrelacement longitudinal au niveau des bandes A, entre les myofibrilles et dans les limites des sarcomères. En interaction avec le cytosquelette, le sarcomère et le réticulum sarcoplasmique, elles forment des complexes fonctionnels appelés unités énergétiques intracellulaires (ICEUs). Les ICEUs ont des voies spécialisées de transfert d'énergie et de régulation des feedback métaboliques entre les mitochondries et les ATPases, médiée par la CK et l'AK. La structure centrale des ICEUs est l'interactosome mitochondrial (MI) qui confient l'ATP synthasome, la chaîne respiratoire, la créatine kinase mitochondriale et VDAC, qui pourrait être régulé par les tubulines. Le rôle principal du MI est la régulation de la respiration et des flux d'énergie intracellulaires via les réseaux de phosphotransfert. La régulation des ICEUs est liée aux protéines structurales. L'association des mitochondries avec plusieurs protéines du cytosquelette, décrite par plusieurs groupes, a mis en évidence l'importance de la relation structure-fonction dans la régulation métabolique des cardiomyocytes de rats adultes. Pour fournir une meilleure compréhension de ces résultats, le présent travail étudie le mécanisme de contrôle des flux d'énergie et le rôle des relations structure-fonction dans la régulation métabolique de cardiomyocytes de rats adultes. Pour montrer ces associations complexes dans les cellules cardiaques adultes, plusieurs protéines ont été visualisées par microscopie confocale: l'α-actinine et les isoformes des β-tubulines. Pour la première fois, l'existence d'une distribution spécifique des isoformes de β-tubuline dans les cellules cardiaques adultes a été montré. Des mesures respiratoires ont été réalisées pour étudier le rôle des tubulines dans la régulation de la consommation d'oxygène. Ces résultats ont confirmé le rôle déterminant des protéines du cytosquelette -tubulines, α-actinine, plectine, desmine, et autres- pour le maintien de la forme normale des cellules cardiaques, ainsi que de l'arrangement et de la régulation mitochondrial. En outre, la dynamique mitochondriale a été étudiée in vivo et in situ par la transfection de la GFP-α-actinine, ceci permettant la mise en évidence du fait que le phénomène de fusion ne se produit pas aussi souvent qu'on ne le croit pour des cellules cardiaques adultes en bonne santé.

Orientador: Joaquim Mansano Garcia / Banca: Lígia Veiga Pereira / Banca: Irina Kerkis / Resumo: O estudo dos processos de diferenciação em células tronco embrionárias (CTE) representa uma importante ferramenta para o entendimento das vias moleculares que os regem, apresentando grande aplicação tanto na ciência básica quanto na engenharia de tecidos e medicina regenerativa. Pouco é conhecido sobre as marcas epigenéticas existentes na cromatina destas células, e de que forma a regulação da expressão gênica ocorre no momento da diferenciação. O presente trabalho teve como objetivo o estudo dos efeitos da hiperacetilação das histonas causada pela droga tricostatina A (TSA), uma inibidora das enzimas histona desacetilases, sobre a diferenciação destas células em estádios iniciais e avançados. Para tanto, a hiperacetilação induzida pela droga foi estimada por reações de imunocitoquímica para AcLys9H3. Os efeitos anti-proliferativos da TSA foram mensurados pelo teste de TUNEL e contagem de células. Ainda, foram conduzidos experimentos de diferenciação in vitro de CTE e análise da expressão de proteínas características de linhagens celulares diferenciadas por reações de imunocitoquímica (Oct3/4, nestina, âIII tubulina, desmina e troponina I), em cultivos tratados com TSA em diferentes concentrações e em diferentes momentos. Desta forma, foi estimada a população de tipos celulares oriundos dos folhetos embrionários ectodérmico e mesodérmico, como neurônios, e células musculares, quando foi promovida a hiperacetilação das histonas nas CTE, em diferentes momentos da diferenciação celular in vitro. A TSA induziu apoptose em níveis superiores aos do grupo controle, e retardou/inibiu a divisão celular. Promoveu hiperacetilação dose-dependente nos períodos estudados, e estimulou a diferenciação de precursores mesodérmicos (50nM d5) e ectodérmicos (15nMd0-5 e 50nMd5), cardiomiócitos (50nMd5 e 100nMd13) e neurônios (15nMd0-5, 50nMd5, 100nMd5, 100nMd13). / Abstract: Studies on embryonic stem cells (ESC) differentiation represents an important tool leading to understanding of its molecular pathways, with many applications both on basic research and tissue engineering / regenerative medicine. Little is known about epigenetic marks on ESC chromatin, and how gene expression occurs at differentiation time. The aim of this work was to study effects of histone hiperacetylation, induced by cell treatment with trichostatin A (TSA), an histone deacetylase inhibitor, on both initial and late differentiation. For that, drug-induced hyperacetylation was studied by AcLys9H3 immunocitochemistry. TSA anti-proliferative effects were analysed by TUNEL test and cell counts. Experiments on ESC in vitro differentiation and immunocitochemistry for specific cell types proteins (Oct3/4, nestin, âIII tubulin, desmin and troponin I) were performed, in treated and control groups, at different moments. This analysis showed specific cell types populations derived from embryonic ectodermal and mesodermal, such as neurons and cardiomyocytes, when histone hyperacetylation were induced, on both initial and late diferentiation. Our results showed that TSA induces apoptosis and inhibits cellular proliferation. Also, TSA promoted dose-dependent histone hyperacetylation at studied moments, and stimulated mesodermal (50nM d5) and ectodermal (15nMd0-5 e 50nMd5) precursors, cardiomyocytes (50nMd5 e 100nMd13) and neurons (15nMd0-5, 50nMd5, 100nMd5, 100nMd13) differentiation. / Mestre

No description available.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Indivíduos obesos apresentam maior risco de morbidade e mortalidade atribuída às doenças cardiovasculares. A composição da dieta é um fator que prediz o fenótipo cardíaco em resposta a obesidade e, o tipo de ácido graxo pode afetar de forma diferencial a estrutura e a função do miocárdio. Estudos têm mostrado que a disfunção mitocondrial exerce um papel chave na patogênese da insuficiência e hipertrofia cardíaca, e as alterações mitocondriais observadas em falhas cardíacas apontam para defeitos em sítios específicos da cadeia transportadora de elétrons. Desta forma, o objetivo deste estudo foi avaliar a função contrátil ventricular em camundongos, alimentados com dieta hiperlipídica, rica em ácidos graxos poliinsaturados, buscando elucidações através da bioenergética mitocondrial. Após desmame, camundongos machos C57Bl/6 passaram a receber dieta manipulada contendo 7% (C) ou 19% (HF) de óleo de soja, até os 135 dias de idade. A ingestão alimentar e a massa corporal foram monitoradas e foi realizado teste de tolerância à glicose. No final do período experimental, os animais foram anestesiados e submetidos à avaliação da composição corporal por Absortimetria de Raios X de Dupla Energia (DXA), e em seguida, sacrificados por exsanguinação. No plasma foram determinados o perfil lipídico e a insulina. O coração, o tecido adiposo intra-abdominal e o subcutâneo foram coletados, pesados, processados para análise histomorfológica. Fibras cardíacas do ventrículo esquerdo foram utilizadas para análise da respiração mitocondrial através de oxígrafo. O coração também foi utilizado para a técnica de perfusão de coração isolado de Langendorff, e para análise da expressão de proteínas relacionadas à bioenergética de cardiomiócitos, através de Western Blotting. O índice de HOMA e de adiposidade foram calculados. O grupo HF apresentou maior adiposidade, sem alteração na ingestão alimentar. Foi observada intolerância a glicose, hiperinsulinemia e resistência à insulina, além de alterações desfavoráveis no perfil lipídico. Foi observado alteração na morfologia cardíaca e quadro de cardiomiopatia hipertrófica, refletindo em alteração hemodinâmica, determinando maior contratilidade, maior pressão ventricular e função diastólica prejudicada. Em relação à atividade mitocondrial dos cardiomiócitos foi observada menor oxidação de carboidratos (-47%) e de ácidos graxos (-60%). Porém, sem alteração na expressão de proteínas relacionadas à bioenergética de cardiomiócitos, CPT1, UCP2, GLUT1, GLUT4, AMPK e pAMPK. A partir desses resultados, concluímos que o tipo e a quantidade de ácidos graxos predizem o fenótipo cardíaco na obesidade, promovendo alteração na capacidade oxidativa mitocondrial, na morfologia e na hemodinâmica cardíaca / Obese individuals have a higher risk of morbidity and mortality attributed to cardiovascular disease. The diet composition is one factor that predicts the cardiac phenotype in response to obesity and the type of fatty acid differentially influences the myocardial structure and function. Studies have showed that mitochondrial dysfunction is considered to play a key role in the pathogenesis of cardiac hypertrophy and failure, also the mitochondrial alterations present in heart failure indicate to defects at specific sites in electron transport chain. Thus, the aim of the study was evaluated the ventricular contractile function in mice fed high fat diet, rich in polyunsaturated fatty acids, looking through mitochondrial bioenergetics. After weaning, mouse C57Bl/6 received manipulated diet containing 7% (C) or 19% (HF) of soybean oil, until 135 days of age. The food intake and the body mass were monitored, and the glucose tolerance test was realized. At the end of the experimental period, the animals had their body composition evaluated by Dual-energy X-ray Absorptiometry (DXA), after, were sacrificed by exsanguination. In plasma was determined the lipid profile and insulin. The heart, intraabdominal and subcutaneous adipose tissue were collected, weighted and processed to morphological analysis. The left ventricular myocardial fibers were used to analyze mitochondrial respiration by technique of high resolution respirometry. The heart was used to the Langendorff technique of isolated heart perfusion, and to analyze the expression of proteins related to the cardiomyocytes bioenergetics, through of Western Blotting. The HOMA-IR and the adiposity index were calculated. The group HF showed higher adiposity, but did not differ about food intake. Was observed glucose intolerance, hiperinsulinemia, insulin resistance and also unfavorable alterations in lipid profile. Was observed alterations in cardiac morphology and hypertrophic cardiomyopathy, reflecting in hemodinamic alterations with increase of contractility, higher ventricular pressure and impaired diastolic function. About the mitochondrial activity of cardiomyocytes was observed lower oxidation of carbohydrates (-47%) and fatty acids (-60%). However, the expression of proteins related to the cardiomyocytes bioenergetics, CPT1, UCP2, GLUT1, GLUT4, AMPK e pAMPK, did not differ between the groups. From these results, we conclude that the type and amount of fatty acids predict the cardiac phenotype in obesity, promoting the impairment of mitochondrial oxidative capacity, alterations in cardiac morphology and hemodynamics

abstract: Myocardial infarction (MI) remains the leading cause of mortality and morbidity in the U.S., accounting for nearly 140,000 deaths per year. Heart transplantation and implantation of mechanical assist devices are the options of last resort for intractable heart failure, but these are limited by lack of organ donors and potential surgical complications. In this regard, there is an urgent need for developing new effective therapeutic strategies to induce regeneration and restore the loss contractility of infarcted myocardium. Over the past decades, regenerative medicine has emerged as a promising strategy to develop scaffold-free cell therapies and scaffold-based cardiac patches as potential approaches for MI treatment. Despite the progress, there are still critical shortcomings associated with these approaches regarding low cell retention, lack of global cardiomyocytes (CMs) synchronicity, as well as poor maturation and engraftment of the transplanted cells within the native myocardium. The overarching objective of this dissertation was to develop two classes of nanoengineered cardiac patches and scaffold-free microtissues with superior electrical, structural, and biological characteristics to address the limitations of previously developed tissue models. An integrated strategy, based on micro- and nanoscale technologies, was utilized to fabricate the proposed tissue models using functionalized gold nanomaterials (GNMs). Furthermore, comprehensive mechanistic studies were carried out to assess the influence of conductive GNMs on the electrophysiology and maturity of the engineered cardiac tissues. Specifically, the role of mechanical stiffness and nano-scale topographies of the scaffold, due to the incorporation of GNMs, on cardiac cells phenotype, contractility, and excitability were dissected from the scaffold’s electrical conductivity. In addition, the influence of GNMs on conduction velocity of CMs was investigated in both coupled and uncoupled gap junctions using microelectrode array technology. Overall, the key contributions of this work were to generate new classes of electrically conductive cardiac patches and scaffold-free microtissues and to mechanistically investigate the influence of conductive GNMs on maturation and electrophysiology of the engineered tissues. / Dissertation/Thesis / Supplementary Videos / Doctoral Dissertation Biomedical Engineering 2018

Made available in DSpace on 2014-07-29T15:07:10Z (GMT). No. of bitstreams: 1 Laylla Silva Ramalho.pdf: 1874808 bytes, checksum: 1da12c8c62ee1a2f9ad9bb50e4e4890b (MD5) Previous issue date: 2012-05-11 / Magnetic fluids consist of surface-coated magnetic nanoparticles dispersed in a liquid carrier. These nanostructures have attracted a lot of attention of the biomedical community because of its possible applications as drug carriers, disease detection, and also on the treatment of several diseases, including cardiovascular ones. This work had the following objectives: (i) evaluate the effect ex-vivo of biocompatible magnetic nanoparticles in the rat heart function and, in-vivo, in the arterial blood pressure and heart rate of the rats, as well as, (ii) investigate the endocytosis and exocytosis of the nanoparticles through a magnetophoresis technique. The samples were characterized by X-ray diffraction (XRD), Dynamic light sacttering (DLS) and Vibrating sample magnetometer (VSM). The cardiac function was evaluated by the Langendorf technique under constant flow. On the other hand, in order to evaluate the effect of nanoparticles in the cardiovascular parameters, femoral artery and vein were cannulated and arterial pressure and heart rate were measure after 24 hs. The magnetic fluid infusion in the isolated heart showed a tiny increase of the intraventricular diastólic pressure and a decrease of the intraventricular systolic pressure. No changes were observed in perfusion pressure. The infusion of the magnetic nanoparticles in the rats had not promoted any significant variations of the artery pressure or the heart rate. These results suggest that magnetic nanoparticles can be used on clinical trials. In addition, magnetophoresis experiments were preformed in order to investigate phenomenon associated to nanoparticles and dissociated cardiomyocytes interactions from rat heart. Different samples containing distinct particle sizes and coating layers were evaluated as function of incubation time. It was observed that, besides endocytosis (or adsorption), an exocytosis (or desorption) mechanism start to occur above a critical time. A mathematical model that takes into account both mechanisms were developed which, together with other works from the literature, allowed us to estimate the individual wrapping time of the nanoparticles. The results show a strong dependence upon nanoparticle diameter and corroborate with theoretical models of receptor-mediated endocytosis of nanoparticles. / Fluidos magnéticos (FM) consistem de nanopartículas magnéticas formadas por ferritas dispersas em um líquido carreador formando uma composição coloidal. Estas nanoestruturas têm atraído a atenção da comunidade biomédica já que possuem aplicações na área de carreadores de fármacos, detecção de doenças, e podem, ainda, contribuir para o tratamento de diversas doenças, inclusive cardiovasculares. Este trabalho teve como objetivos: (i) avaliar o efeito ex-vivo de nanopartículas magnéticas biocompatíveis no funcionamento de corações de ratos e, in-vivo, na pressão arterial e freqüência cardíaca de ratos wistar e, também, (ii) investigar o processo de endocitose/adsorção e exocitose/dessorção de nanopartículas magnéticas biocompatíveis em cardiomiócitos via técnica de magnetoforese. As amostras foram sintetizadas e caracterizadas por Difração de Raio-X (DRX), Espalhamento de Luz Dinâmico (DLS) e Magnetometria de Amostra Vibrante (VSM). A função cardíaca foi avaliada pela técnica de Langendorff com fluxo constante. Por outro lado, para avaliar o efeito das nanopartículas nos parâmetros cardiovasculares in vivo, artéria e veia femoral foram canuladas e o registro da pressão arterial e frequência cardíaca foi realizado após 24 hs. A infusão do fluido magnético nos corações isolados promoveu ligeiro aumento na pressão intraventricular diastólica e uma queda na pressão intraventricular sistólica. O fluido promoveu uma leve diminuição na pressão de perfusão, o que representa uma vasodilatação coronariana, sendo esse efeito reversível durante a lavagem. A infusão das nanopartículas magnéticas não promoveu alterações significativas na pressão arterial ou frequência cardíaca. Estes resultados sugerem a viabilidade para a utilização clínica das nanopartículas magnéticas. Adicionalmente, experimentos de magnetoforese foram realizados para avaliar fenômenos associados à interação de nanopartículas com cardiomiócitos dissociados do coração de ratos. Diferentes amostras de nanopartículas de ferrita de manganês, contendo diâmetros distintos e diferentes camadas de cobertura, foram avaliadas em função do tempo de incubação. Observou-se, além de um processo endocitótico (ou adsortivo), um mecanismo exocitótico (ou dessortivo) a partir de um tempo crítico. Um modelo matemático que incluiu ambos os mecanismos foi desenvolvido e, conjuntamente com outros da literatura, permitiu estimar o tempo de wrapping de cada nanopartícula. Os resultados mostraram-se fortemente dependente do diâmetro das nanopartículas e, inclusive, corroboram com modelos teóricos de endocitose de nanopartículas mediada por receptores.

A Cardiomiopatia Chagásica Crônica (CCC) é o elemento mórbido mais importante da Doença de Chagas e sua elucidação se tornou fundamental. Estudos da imunologia da CCC demonstram que o sistema imune desempenha um papel duplo no curso da doença, agindo de forma a controlar as formas parasitárias e ainda promovendo lesão tissular. Porém, pouco se sabe do papel das células estruturais, tais como os cardiomiócitos, no curso da doença. Sabe-se que, em outras patologias cardíacas, o IFN-γ, citocina produzida em abundância no coração dos pacientes com CCC, determina o priming de diversas populações celulares, modulando positivamente a sua resposta. Cardiomiócitos HL-1 e animais C3H/HePas foram primados com IFN-γ e desafiados com LPS para a dosagem de citocinas, simulando quadro agudo e crônico de infecção. Neste trabalho, determinamos que o IFN-γ modula positivamente a produção de diversas citocinas in vitro por células HL-1 (IP-10, MCP-1, G-CSF, RANTES, MIG, IL-6, MIF) e também in vivo no coração (IP-10, KC, G-CSF, LIF e IL-6). Além disso, in vitro, o IFN-γ foi capaz de diminuir a produção de VEGF e GM-CSF em relação aos grupos tratados apenas com LPS. Os dados corroboram a literatura e permitem concluir que os cardiomiócitos são capazes de participar ativamente da resposta inflamatória no coração e que são sensíveis aos produtos da mesma. O trabalho serve ainda de base para novos estudos sobre o perfil de citocinas expressas no coração no curso da infecção por T. cruzi e como os cardiomiócitos participam da resposta inflamatória em questão. / Chronic Chagasic Cardiomyopathy (CCC) is the most morbid element of Chagas Disease, the elucidation of its physiopathology being fundamental. However, little is known about the role of structural cells, such as cardiomyocytes, in the course of the disease. In other cardiac pathologies, it has been shown that IFN-γ determines the priming of several resident populations, positively modulating their response. In this work, HL-1 cardiomyocytes and C3H/HePas mice were primed with IFN-γ (in brief or extended protocols) and challenged with LPS, the cytokines produced being measured in the supernatants. We observed that IFN-γ positively modulates the in vitro production of many cytokines by HL-1 cells (IP-10, MCP-1, G- CSF, RANTES, MIG, IL-6, MIF) and also their in vivo production at the heart (IP-10, KC, G-CSF, LIF and IL-6). Besides, IFN-γ was able to decrease the LPS-induced production of VEGF and GM-CSF by HL-1 cells. Our data allow us to conclude that cardiomyocytes actively participate in the inflammatory response of the heart, being sensitive to products released by professional immune cells. This work may serve as a basis for further studies on the profile of the cytokines secreted in the heart tissue along the course of cardiac inflammation.