Aerosols of nitrate solutions were irradiated in the presence of radical scavengers in an attempt to measure the yield of hydroxyl radical in both the aqueous phase and the gas phase. Carbon monoxide, benzoic acid, benzene and cyclohexane were used as scavengers to trap hydroxyl radical. The products from the reaction of these scavengers with hydroxyl radical were analysed with High Performance Liquid Chromatography and mass spectrometry. The radiant flux in the chamber was measured via ferrioxalate actinometry, both with bulk liquid and aerosol droplets. Many quantitative results were obtained but several anomalies were found. This suggests that Mie theory is not capable of predicting rates of photochemical reactions within droplets.

Red blood cell hemolysis in sickle cell disease (SCD) releases free hemoglobin. Extracellular hemoglobin and its degradation products, free heme and iron, are highly toxic due to oxidative stress induction and decrease in nitric oxide availability. We propose an approach that helps to eliminate extracellular hemoglobin toxicity in SCD by employing a bacterial protein system that evolved to extract heme from extracellular hemoglobin. NEAr heme Transporter (NEAT) domains from iron-regulated surface determinant proteins from Staphylococcus aureus specifically bind free heme as well as facilitate its extraction from hemoglobin. We demonstrate that a purified NEAT domain fused with human haptoglobin beta-chain is able to remove heme from hemoglobin and reduce heme content and peroxidase activity of hemoglobin. We further use molecular dynamics (MD) simulations to resolve molecular pathway of heme transfer from hemoglobin to NEAT, and to elucidate molecular mechanism of such heme transferring process. Our study is the first of its kind, in which simulations are employed to characterize the process of heme leaving hemoglobin and subsequent rebinding with a NEAT domain. Our MD results highlight important amino acid residues that facilitate heme transfer and will guide further studies for the selection of best NEAT candidate to attenuate free hemoglobin toxicity.

O presente trabalho teve por objetivo avaliar alterações devido à exposição a Nanotubos de Carbono de Parede Simples (NTCPS) em duas linhagens celulares epiteliais (BBnt e HK2) e em uma linhagem celular monocítica (THP-1), enfocando os mecanismos de internalização e os efeitos sobre o ciclo celular. Foi avaliada a ação dos receptores scavenger na internalização dos NTCPS nas células HK2 e THP-1 e a interferência de duas concentrações de NTCPS sobre os elementos do citoesqueleto e no ciclo celular, nas células HK2 e BBnt. As concentrações utilizadas foram equivalentes as permitidas pelo The National Institute for Occupational Safety and Health: 2,4 e 24 mg/cm2. A análise de expressão de mRNA por RT-PCR para receptores scavenger, mostrou que a internalização do NTCPS pode ocorre por endocitose. Sendo que os receptores SCARA5 e SRA são os responsáveis pela internalização nas células THP-1, enquanto MARCO e SRA realizam o processo de internalização nas células HK2. Observou-se que em ambas as concentrações, as células BBnt apresentaram amplificação centrossômica, com a ocorrência de 25,38% e 28,46% de mitoses alteradas para cada concentração, respectivamente. Não houve interferência significativa na progressão do ciclo celular em ambas as linhagens. O estudo da interação dos NTCPS com vesículas lipídicas não apresentou evidencias de alterações ou danos na membrana das vesículas, porém as vesículas apresentaram-se associadas umas às outras após o tratamento com 24 mg/cm2. / This study aimed to assess changes due to exposure to of Single-wall Carbon Nanotubes (SWCNT) in two epithelial cell lines (BBnt and HK2) and a monocytic cell line (THP-1), focusing on the mechanisms of internalization and effects on the cell cycle. The action of scavenger receptors in the internalization of SWNTC in HK2 and THP-1 cells and the interference of two concentrations of SWNTC about elements of the cytoskeleton and the cell cycle, in BBnt and HK2 cells was evaluated. The concentrations used were equivalent to those allowed by The National Institute for Occupational Safety and Health: 2,4 to 24 mg/cm2. Analysis of mRNA expression by RT-PCR for scavenger receptors showed that the SWNTC internalization can occurs by endocytosis. Being that SCARA5 and SRA receptors are responsible for internalization in THP-1 cells, while MARCO and SRA perform the process of internalization in HK2 cells. It was observed that at both concentrations, the cells showed centrosome amplification in BBnt cells, with the occurrence of 25.38% and 28.46% of mitosis changed for each concentration, respectively. There was no significant interference with cell cycle progression in both strains. The study of the interaction of lipid vesicles with SWNTC showed no evidence of change or damage the membrane of the vesicles, but the vesicles were associated with each other after treatment with 24 mg/cm2.

O presente trabalho teve por objetivo avaliar alterações devido à exposição a Nanotubos de Carbono de Parede Simples (NTCPS) em duas linhagens celulares epiteliais (BBnt e HK2) e em uma linhagem celular monocítica (THP-1), enfocando os mecanismos de internalização e os efeitos sobre o ciclo celular. Foi avaliada a ação dos receptores scavenger na internalização dos NTCPS nas células HK2 e THP-1 e a interferência de duas concentrações de NTCPS sobre os elementos do citoesqueleto e no ciclo celular, nas células HK2 e BBnt. As concentrações utilizadas foram equivalentes as permitidas pelo The National Institute for Occupational Safety and Health: 2,4 e 24 mg/cm2. A análise de expressão de mRNA por RT-PCR para receptores scavenger, mostrou que a internalização do NTCPS pode ocorre por endocitose. Sendo que os receptores SCARA5 e SRA são os responsáveis pela internalização nas células THP-1, enquanto MARCO e SRA realizam o processo de internalização nas células HK2. Observou-se que em ambas as concentrações, as células BBnt apresentaram amplificação centrossômica, com a ocorrência de 25,38% e 28,46% de mitoses alteradas para cada concentração, respectivamente. Não houve interferência significativa na progressão do ciclo celular em ambas as linhagens. O estudo da interação dos NTCPS com vesículas lipídicas não apresentou evidencias de alterações ou danos na membrana das vesículas, porém as vesículas apresentaram-se associadas umas às outras após o tratamento com 24 mg/cm2. / This study aimed to assess changes due to exposure to of Single-wall Carbon Nanotubes (SWCNT) in two epithelial cell lines (BBnt and HK2) and a monocytic cell line (THP-1), focusing on the mechanisms of internalization and effects on the cell cycle. The action of scavenger receptors in the internalization of SWNTC in HK2 and THP-1 cells and the interference of two concentrations of SWNTC about elements of the cytoskeleton and the cell cycle, in BBnt and HK2 cells was evaluated. The concentrations used were equivalent to those allowed by The National Institute for Occupational Safety and Health: 2,4 to 24 mg/cm2. Analysis of mRNA expression by RT-PCR for scavenger receptors showed that the SWNTC internalization can occurs by endocytosis. Being that SCARA5 and SRA receptors are responsible for internalization in THP-1 cells, while MARCO and SRA perform the process of internalization in HK2 cells. It was observed that at both concentrations, the cells showed centrosome amplification in BBnt cells, with the occurrence of 25.38% and 28.46% of mitosis changed for each concentration, respectively. There was no significant interference with cell cycle progression in both strains. The study of the interaction of lipid vesicles with SWNTC showed no evidence of change or damage the membrane of the vesicles, but the vesicles were associated with each other after treatment with 24 mg/cm2.

Cob, as an arts-based research process, creates movements and moments of survival. Survival is an ideological construction and an actual, local practice. Survival is also about desiring and fulfilling arts-based desires to work with the land through academic and material scavenging. Cob creates strategies for surviving, for working with our respective environments wisely. Cob building teaches people how to negotiate the natural economy and their relationships to labor and each other through an artistic and intimate practice. From a feminist poststructural lens, survival happens on the local level, between and with people. Cob building creates knowledge through creative, kinesthetic, and collaborative engagement. As a feminist poststructuralist, arts-based research allows me to examine local action and interaction among people, positionalities, and competing differences. Rather than appeasing the modern impulse to objectify and rationalize an end-point or an object- oriented view of the production of art, feminist poststructural theory works to problematize the end-point. Through cob building, a rich, arts-based process, I call into question the modern impulse to find Truth and ask that we be aware of developing new oppressions when working toward equity and justice. Cob building teaches people how to engage together within the form of artistic creation. Cob is an arts-based research process that includes the land as an integral part of its canvas. In order to articulate, uncover, and engage the claim that, as an arts-based process, cob creates movements and moments of survival, I use the arts-based process, a/r/tography. This a/r/tographical text does not offer an end point but works to recreate moments and movements of cob building as an arts-based research project. A/r/tography helps to layer the movements of arts-based survival within cob building and this text. Throughout this work, the arts-based process of cob building is the overlying metaphor for the construction of the structure of this text. As the chapters move forward, the structure builds up.

Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted. Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles. / At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper nr 4: Submitted

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.

Aerosols of nitrate solutions were irradiated in the presence of radical scavengers in an attempt to measure the yield of hydroxyl radical in both the aqueous phase and the gas phase. Carbon monoxide, benzoic acid, benzene and cyclohexane were used as scavengers to trap hydroxyl radical. The products from the reaction of these scavengers with hydroxyl radical were analysed with High Performance Liquid Chromatography and mass spectrometry. The radiant flux in the chamber was measured via ferrioxalate actinometry, both with bulk liquid and aerosol droplets. Many quantitative results were obtained but several anomalies were found. This suggests that Mie theory is not capable of predicting rates of photochemical reactions within droplets.

Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-02-13T17:19:32Z No. of bitstreams: 1 Dissertação Ivonete Queiroz.pdf: 1073414 bytes, checksum: af4071808ba3af365237d21558158bf4 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-28T12:19:20Z (GMT) No. of bitstreams: 1 Dissertação Ivonete Queiroz.pdf: 1073414 bytes, checksum: af4071808ba3af365237d21558158bf4 (MD5) / Made available in DSpace on 2017-06-28T12:19:20Z (GMT). No. of bitstreams: 1 Dissertação Ivonete Queiroz.pdf: 1073414 bytes, checksum: af4071808ba3af365237d21558158bf4 (MD5) / CAPES / A hanseníase é uma doença infectocontagiosa crônica, causada pelo Mycobacterium leprae, caracterizada por lesões na pele e ampla apresentação clínica, a qual depende principalmente da resposta imunológica do hospedeiro frente à infecção pelo Mycobacterium leprae. Esta infecção está distribuída mundialmente persistindo como grave problema de saúde pública no Brasil. As incapacidades e deformidades duradouras decorrentes dos quadros reacionais podem ocorrer antes, durante ou após a instituição do tratamento específico. Os episódios reacionais inflamatórios são decorrentes da exacerbação abrupta da resposta imune mediada por célula contra antígenos do M. leprae. O episódio reacional tipo 1 ou reação reversa é uma ocorrência inflamatória aguda que acomete a pele e nervos periféricos, sendo causa comum de incapacidade física. A reação tipo 2 ou eritema nodoso hansênico acomete somente pacientes virchowianos (LL) e borderline-virchowianos (BL) com presença de muitos bacilos e pouca ou nenhuma imunidade celular ao M. leprae. O objetivo principal deste estudo foi avaliar a moléculas MARCO Scavenger, Receptor peptídeo formil 1(FPR1) e Receptor peptídeo N-formil 2 (FPR2) em monócitos de pacientes com hanseníase em associação ao desenvolvimento de reações hansênicas, para alcançar este objetivo células mononucleares do sangue periférico foram obtidas de pacientes com diferentes formas clínicas da hanseníase. A marcação de superfície celular foi realizada por citometria de fluxo e os dados analisados com FlowJo em 35 pacientes com hanseníase, divididos em 19 paucibacilar e 16 multibacilar. Os nossos resultados mostraram que os pacientes com a forma paucibacilar apresentam a frequência de monócitos intermediários aumentada. Não houve diferença estatística na média de intensidade de fluorescência (MFI) de CD80, CD86 e MHC II entre monócitos de pacientes com a forma paucibacilar e multibacilar da hanseníase. No entanto, monócitos intermediários de pacientes com a forma multibacilar apresentaram média de intensidade de fluorescência maior em CD86 e MCHII. Os nossos dados sugerem que o receptor FPR1 pode ter um papel relevante para detecção na amplificação da resposta inflamatória de indivíduos com hanseníase quando comparado com as outras moléculas aplicadas neste estudo. / Leprosy is a chronic infectious disease caused by Mycobacterium leprae, characterized by skin lesions and extensive clinical presentation which depends mainly on the immune front of the host response to infection by Mycobacterium leprae. This infection is distributed worldwide persist as a serious public health problem in Brazil. Disability and lasting deformities resulting from reaction symptoms can occur before, during or after the institution of specific treatment. Reactional inflammatory episodes are due to the abrupt exacerbation of immune cell-mediated response against M. leprae antigens. The episode reaction type 1 or reverse reaction is an acute inflammatory occurrence that affects the skin and peripheral nerves, and common cause of disability. Reaction type 2 or ENL only affects lepromatous patients (LL) and borderline-lepromatous (BL) with the presence of many bacilli and little or no cellular immunity to M. leprae. To assess the association of biomarkers MARCO Scavenger, Formyl peptide receptor (FPR1) and N-formyl peptide receptor 2 (FPR2) in monocytes of patients with leprosy associated with the development of leprosy reactions, mononuclear cells from peripheral blood were obtained from patients with different clinical forms of leprosy. To achieve this goal peripheral blood mononuclear cells were obtained from patients with different clinical forms of leprosy. The cell surface stain was performed by flow cytometry and the data analyzed with FlowJo in 35 patients with leprosy, divided into 19 paucibacillary and 16 multibacillary patients. Our results showed that patients with the paucibacillary form exhibit increased monocyte frequency. There was no statistical difference in the mean fluorescence intensity (MFI) of CD80, CD86 and MHC II between monocytes of paucibacillary and multibacillary leprosy patients. However, intermediate monocytes from patients with the multibacillary form had higher mean fluorescence intensity in CD86 and MCHII. Our data suggest that the FPR1 receptor may play a relevant role in detecting the amplification of the inflammatory response of individuals with leprosy when compared with the other molecules applied in this study.