A sepse é uma disfunção orgânica causada por uma resposta do hospedeiro à infecção desregulada, com risco de morte. Quando o tratamento da sepse não é efetivo, o quadro pode progredir para hipotensão severa. O tecido adiposo perivascular (perivascular adipose tissue - PVAT) é reconhecido como um elemento regulador na biologia vascular, com implicações na fisiopatologia de doenças cardiovasculares. No entanto, em relação à sepse, pouco se sabe acerca dos efeitos desta sobre a ação modulatória que o PVAT exerce no tônus vascular. Dessa maneira a hipótese do presente trabalho foi a de que a sepse poderia aumentar o efeito anti-contrátil do PVAT. Portanto, o objetivo do trabalho foi avaliar o efeito da sepse experimental na ação modulatória que o PVAT exerce sobre tônus vascular e os possíveis mecanismos envolvidos nessa resposta. Para isso foram utilizados ratos Wistar Hannover com idade entre 60 e 70 dias (270 a 300g). Os ratos foram distribuídos aleatoriamente em 2 grupos: 1) Grupo Sham: foi realizada apenas uma laparotomia sem os procedimentos de ligadura e punção do ceco; 2) Grupo CLP (Cecal Ligation and Puncture) : a sepse letal foi induzida utilizando o modelo CLP no qual foi realizada uma laparotomia para exposição do ceco, onde foi feito uma ligadura e 20 punções intermediárias entre a ligadura e a ponta do ceco com agulha de calibre 18 gauge (G). Os animais foram anestesiados com quetamina/xilasina (80/10 mg/kg, i.p.) e mortos 6 h após a indução da sepse. A aorta torácica foi coletada para realização das análises bioquímicas e funcionais. A sepse letal reduziu a taxa de sobrevida, a pressão arterial média (PAM), não alterou os níveis de leucócitos e neutrófilos, mas aumentou a contagem de bactérias no sangue e no lavado peritoneal, aumentou os níveis plasmáticos de nitrato, uréia e CK-MB. A sepse diminuiu a contração induzida pela fenilefrina e serotonina nas aortas PVAT(-)/Endo(+) ou Endo (-) do grupo CLP, quando comparada ao Sham. Porém nas artérias PVAT(+)/Endo(+) ou Endo (-), o CLP induziu redução mais pronunciada da contração induzida tanto pela fenilefrina, quanto pela serotonina. O aumento do efeito anti-contrátil do PVAT na condição séptica não foi encontrado nas artérias após a incubação com L-NAME, 7-nitroindazol, 1400W, A779, carboxy-PTIO, ODQ, apamina, RO11384552 e indometacina. Tiron, catalase, 4-aminopiridina, glibenclamida e caribdotoxina não alteraram a contração induzida pela fenilefrina no grupo CLP. A sepse aumentou a concentração de H2O2 na aorta, mas não afetou a concentração no PVAT. Aumento dos níveis de ânion superóxido (O2-) e prostaglandina (PG)I2 foram detectados tanto na aorta, quanto no PVAT do grupo CLP. A sepse não alterou os níveis de PGE2 ou angiotensina (1-7) na aorta ou PVAT. Portanto, a sepse letal induzida por CLP aumenta a ação anti-contrátil do PVAT por um mecanismo que envolve a produção de NO pelas enzimas iNOS e nNOS, a participação de canais para KCa de baixa condutância e ativação da enzima guanilato ciclase solúvel. Além disso, sugere-se o envolvimento da PGI2 e angiotensina (1-7) na hiporresponsividade vascular mediada pelo PVAT durante a sepse / Sepsis is an organic dysfunction caused by an unregulated host response to lifethreatening infection. When treatment of sepsis is ineffective, the condition may progress to severe hypotension that is drug-irresponsive. Perivascular adipose tissue (PVAT) is recognized as a regulatory element in vascular biology that is implicated in the pathophysiology of cardiovascular diseases. However, little is known about the effects of sepsis in the modulatory action of PVAT. Thus, the hypothesis of the present study was that sepsis could increase the anti-contractile effect of PVAT. Therefore, the objective of the study was to evaluate the effect of experimental (lethal) sepsis in the modulatory action that PVAT exerts on vascular tone and the possible mechanisms underlying this response. With this purpose, male Wistar Hannover rats (250-300g) were divided in 2 groups: 1) Sham: the cecum was exteriorized without ligation and puncture; 2) CLP: lethal sepsis was induced using the cecal ligation and puncture (CLP) model. The thoracic aorta was isolated 6 h after sepsis for functional and biochemical assays. Lethal sepsis reduced survival rate, mean arterial pressure (MAP), did not alter leukocytes and neutrophils migration, but increased bacterial count in the blood and peritoneal cavity, increased plasma levels of nitrate, urea and CK-MB. We found that in aortas PVAT(-)/Endo(+) or Endo(-), sepsis decreased the contraction induced by phenylephrine or serotonin, when compared to sham. In PVAT(+)/Endo(+) or Endo (-) arteries sepsis induced a more pronounced reduction of phenylephrine-induced contraction. Sepsis-induced increase of anti-contractile action of PVAT was not found after incubation of arteries with L-NAME, 7-nitroindazole, 1400W, A779, carboxyPTIO , ODQ, apamin, indomethacin and RO1138452. Tiron, catalase, charybdotoxin, 4- aminopyridine and glibenclamide did not alter phenylephrine-induced contraction in the CLP group. Sepsis increased H2O2 concentration in the aorta, but did not affect H2O2 concentration in PVAT. Increased levels of superoxide anion (O2-) and prostaglandin (PG) I2 were detected in both aorta and PVAT. Sepsis did not alter the levels of PGE2 or angiotensin (1-7) in the aorta or PVAT. Conclusion: Lethal sepsis increases the anticontractile action of PVAT by a mechanism that involves the production of nitric oxide (NO) by iNOS and nNOS, the participation of calcium-dependent K+ channel of low conductance and activation of the enzyme soluble guanylate cyclase. Angiotensin (1-7) and PGI2 also contribute to the increased anti-contractile effect displayed by PVAT during sepsis. Financial Support: CAPES

Die ATP-abhängige Serinprotease ClpXP ist für die Kontrolle und Verfügbarkeit einer großen Anzahl von Enzymen und regulatorischer Proteine sowie für den Abbau fehlge-falteter Proteine verantwortlich. Sie besteht aus zwei Komponenten, der Protease ClpP und der ATPase ClpX, welche für die Substratspezifität verantwortlich ist und zusätzlich als Chaperon wirken kann. Durch den gezielten Abbau globaler Regulatoren wie dem alternativen Sigmafaktor RpoS kommt die regulatorische Funktion der Protease auf post-translationaler Ebene zum Tragen. Um den Einfluss der Protease ClpXP auf die Virulenz-eigenschaften uropathogener Escherichia coli zu studieren, wurde der uropathogene E. coli Stamm 536 als Modellorganismus verwendet. Uropathogene E. coli Stämme werden als häufigste Erreger von Harnwegsinfektionen des Menschen beschrieben. Der E. coli Stamm 536 (O6:K15:H31) unterscheidet sich von apathogenen Stämmen durch die Anwesenheit von bislang sechs charakterisierten Pathogenitätsinseln (PAIs), die für eine Reihe verschiedener Virulenzfaktoren wie Prf- und S-Fimbrien, alpha-Hämolysin, dem Kapselantigen K15 und Eisenaufnahmesystemen kodieren. Zudem exprimiert der Stamm Curli-Fimbrien, eine Flagelle vom Serotyp H31 und ist aufgrund seines glatten Lipopolysaccharid Phänotyps (O6 Antigen) serumresistent. Ziel dieser Arbeit war es, den Einfluss der Protease ClpXP auf die Regulation und Expression Virulenz-assoziierter Gene des E. coli Stammes 536 sowohl auf Protein-, als auch auf Transkriptebene näher zu charakterisieren. Um den Einfluss der clpX- und clpP-Deletion auf die Proteinexpression des E. coli Stammes 536 zu untersuchen, wurde die Methode der zweidimensionalen (2-D) Gelelektrophorese angewendet. Es wurden zytoplasmatische und extrazelluläre Proteine der logarithmischen und stationären Wachstumsphase untersucht und in diesem Zusammenhang jeweils ein internes Mastergel aller 93 identifizierten zytoplasmatischen und aller 127 identifizierten Proteinen des extrazellulären Proteoms angefertigt. In der zytoplasmatischen Fraktion konnte für 13 Proteine aus der logarithmischen und für 25 Proteine aus der stationären Wachstums-phase eine unterschiedliche Expression festgestellt werden. Die 2-D Analyse der Proteine aus dem Kulturüberstand ergab ein erhöhtes Sekretionsvermögen der clpX- und clpP-negativen Stämme sowohl in der logarithmischen als auch in der stationären Wachstums-phase. Darüber hinaus konnte eine reduzierte Expression von Hauptstrukturuntereinheiten der Prf-, S-, CS12-ähnlichen und Typ 1-Fimbrien sowie des Autotransporters Ag43 in den clpX- und clpP-Mutanten nachgewiesen werden. Zusätzlich wiesen diese Stämme eine verstärkte Expression des Flagellins FliC auf. Durch Western Blot-Analysen und weitere phänotypische Tests konnten die Ergebnisse aus den Proteomstudien für Prf-, S- und Typ 1-Fimbrien sowie für die Flagellenexpression bestätigt werden. Zudem war eine stark verlangsamte Expression von Curli und Cellulose bei Temperaturen unter 37 °C, eine erhöhte Motilität und ein „hyperflagellierter“ Phänotyp der clpX- und clpP-negativen Stämme zu beobachten. Demgegenüber hatte ClpXP keinen Einfluss auf die alpha-Hämolysinproduktion, die Expression des Lipopolysaccharides, die Serumresistenz und in vivo-Virulenz dieses Stammes. Bei anschließenden Transkriptionsanalysen (semiquantitative Real-Time Reverse Transkrip-tion (RT)-PCR) der Gene, welche für die Hauptstrukturuntereinheiten von Fimbrien kodieren, konnte eine reduzierte Transkription der Determinanten für Prf-, S-, und Curli-Fimbrien, aber eine erhöhte Transkription für Gencluster der Typ 1- und CS12-ähnlichen Fimbrien und keine Änderung der Transkriptmengen für Gene des Pix-Fimbrienoperons in den Mutantenstämmen nachgewiesen werden. Die Transkription der beiden Antigen 43 Varianten ORF52III und ORF47V war durch die Deletion von clpX und clpP gleichermaßen betroffen und deutlich reduziert. ClpXP hatte aber keinen Einfluss auf die Transkription des „Masterregulatorgens“ flhC der Flagellen-Regulationskaskade, beeinflusst jedoch die in der Hierarchie weiter unten liegender Operons positiv, da eine deutlich erhöhte Expression von fliA und fliC nachgewiesen werden konnte. Demzufolge hat ClpXP einen negativen regulatorischen Effekt auf die Flagellenexpression, der aber erst auf posttranskriptionaler oder posttranslationaler Ebene auftritt. Viele der beobachteten Einflüsse, v.a. auf die Flagellen- und Fimbrienexpression, konnten auf die fehlende regulatorische Wirkung von RpoS zurückgeführt werden, welches ausschließlich durch ClpXP degradiert wird. Zudem lassen die Ergebnisse vermuten, dass der globale Regulator Lrp, welcher eine wichtige Rolle bei der Regulation der Fimbrienexpression spielt, direkt oder indirekt durch ClpXP beeinflusst wird, wodurch die regulatorischen Netzwerke zusätzlich gestört werden. / ClpXP is an ATP-dependent serine protease that controls the level and availability of key metabolic enzymes or regulatory proteins and plays a role in breakdown of abnormal and misfolded proteins. ClpXP consists of two independent components. ClpP has proteolytic activity, whereas ClpX has ATPase activity, provides the substrate specificity and function as a molecular chaperone. The proteolysis of global regulators such as the alternative sigma factor RpoS contributes to its function as a regulator on posttranslational level. To analyse the impact of the protease ClpXP on the virulence factors of uropathogenic Escherichia coli, the uropathogenic E. coli strain 536 was chosen as a model organism. Uropathogenic E. coli are the most frequently isolated causative agents of infections of the urinary tract in humans. The E. coli isolate 536 (O6:K15:H31) carries six pathogenicity islands (PAIs) that encode its key virulence factors such as alpha-hemolysins, P-related and S-fimbriae, capsule and iron-uptake systems, that are absent from non-pathogenic isolates. In addition, the strain expresses curli, flagella of serotype H31 and is due to its smooth lipopolysaccharide resistant to human serum. The aim of this study was to characterize the impact of the ATP-dependent protease ClpXP on the expression and regulation of virulence-associated genes of strain E. coli 536 and its clpX- and clpP-deficient mutants on the protein- as well as on the transcriptional level. In order to study the impact of ClpXP on global protein expression of E. coli strain 536, the proteome pattern of total cell extracts as well as of secreted proteins (“secretome”) from the exponential and stationary phase of strain 536 and its isogenic clpX and clpP mutants were compared by two-dimensional (2-D) gel electrophoresis. Internal master gels were prepared for all identified 93 proteins from the cytoplasmic and 127 proteins from the extracellular fraction. Analysis of the cytoplasmic fraction revealed an altered expression of 13 proteins from the exponential and 25 proteins from the stationary phase. 2-D analysis of the secretome exhibited a highly increased secretion ability of the clpX- and clpP-deficient strains in both, exponential and stationary phase, compared to wild type strain 536. In contrast, both mutant strains showed a reduced abundance of the major subunits of S-, P-related, putative CS12-like and type 1-fimbriae as well as the autotransporter protein antigen 43. Additionally, the mutant strains showed a higher expression of flagellin FliC compared to the wild type strain. Results from proteome studies concerning P-related, S- and type 1-fimbriae as well as flagella expression were confirmed by Western blot-analysis and further phenotypic assays. Additionally, a retarded expression of curli and cellulose at temperatures of 30 °C and below, as well as a “hyperflagellated” phenotype with higher motility could be detected upon clpX- and clpP deletion. In contrast, ClpXP had no influence on alpha-hemolysin- and lipopolysaccharide expression, serum resistance and in vivo virulence of strain 536. Following transcriptional analysis (semi-quantitative real-time reverse transcription (RT)-PCR) of genes coding for major fimbrial subunits, a reduced transcription of the determinants coding for S- and P-related fimbriae and curli was observed in the clpX- and clpP-deficient variants. Besides, enhanced transcription of CS12-like and type 1-fimbrial determinants and no differences with respect to transcription of the pix fimbrial gene cluster were detected in the clpX- and clpP mutants relative to the wild type. Transcription of both antigen 43 gene variants, ORF52III and ORF47V, was negatively influenced upon clpX- and clpP deletion and the relative expression was reduced compared to the wild type. Furthermore, it has been shown, that ClpXP had no influence on the “master regulator gene” flhC of flagellar regulation cascade, but enhanced transcription of genes from operons downstream of flhC, like fliA and fliC. These results suggest that ClpXP plays a role as a negative regulator of flagella regulation at the post-transcriptional or post-translational level. Many of the observed effects, particularly on flagellar- and fimbrial gene expression, are due to malfunction of RpoS, which is degraded especially by ClpXP. Additionally, the obtained results indicate that Lrp, a global regulator that plays a dominant role in fimbrial gene expression, is influenced, directly or indirectly by ClpXP and that therefore the corresponding regulatory networks are disordered in clpX and clpP mutants.

O Irradiador Multipropósito de Cobalto-60 é uma unidade construída no Instituto de Pesquisas Energéticas e Nucleares, órgão da Comissão Nacional de Energia Nuclear. Esta instalação utiliza dos efeitos que a radiação gama produz ao interagir com um meio material e suas consequências. A radiação gama emitida pelo radioisótopo cobalto-60 é ionizante, com elevado poder de penetração, que ao interagir com os produtos, inclusive no interior de suas embalagens, transfere sua energia por meio de colisões aos elétrons dos átomos que constituem os produtos. Ao mesmo tempo que este processo de ionização é desejável em função de suas características deletérias aos microrganismos, a exposição indiscriminada às radiações ionizantes pelo ser humano ou animais apresentará danos e em doses elevadas poderão levar a morte. Diante destas circunstâncias, para garantir a segurança, os equipamentos utilizados para irradiação são construídos e operados sob normas rígidas de construção e operação. O sistema de segurança do Irradiador, constituído por um gerenciamento eletrônico de intertravamento de portas e exposição das fontes radioativas, é feito simultaneamente por um controlador lógico programável (CLP) e uma lógica de relés. Todas as informações, obtidas através das entrada e saídas do Irradiador, são monitoradas pelo sistema de segurança e enviadas para um computador com um programa supervisório. O propósito do trabalho foi a construção de uma Unidade Autônoma de Monitoramento, Sinalização e Registro para o Sistema de Segurança do Irradiador Multipropósito de Cobalto-60. O desenvolvimento deste equipamento permitiu o monitoramento e registro de eventos, até mesmo de tempos muito curtos, não detectáveis pelo CLP. Cada evento foi registrado em um cartão de memória, de forma a permitir que estes eventos possam ser posteriormente analisados em qualquer computador, mantendo todo e qualquer histórico de ocorrências. Este é um equipamento de monitoramento totalmente independente, não interferindo no funcionamento do sistema atual já homologado. / The Cobalto-60 Multipurpose Irradiator is a unit built at the Nuclear and Energy Research Institute, an agency of the National Nuclear Energy Commission. This installation uses the effects that gamma radiation produces when interacting with a material medium and its consequences. The gamma radiation emitted by the cobalt-60 radioisotope is ionizing, with high penetration power, which when interacting with the products, even inside its packages, transfers its energy by means of collisions to the electrons of the atoms that make up the products. At the same time as this ionization process is desirable because of its deleterious characteristics to the microorganisms, indiscriminate exposure to ionizing radiation by humans or animals will present damages and in high doses could lead to death. In view of these circumstances, to ensure safety, the equipment used for irradiation is constructed and operated under strict construction and operation standards. The safety system of the Irradiator, consisting of an electronic management of door interlocking and exposure of the radioactive sources, is made simultaneously by a programmable logic controller (PLC) and a relay logic. All information, obtained through the inputs and outputs of the Irradiator, is monitored by the security system and sent to a computer with a supervisory program. The purpose of the work was the construction of an Autonomous Monitoring, Signaling and Registration Unit for the Cobalto-60 Multipurpose Irradiance Safety System. The development of this equipment allowed the monitoring and recording of events, even of very short times, not detectable by the CLP. Each event has been recorded on a memory card, so that these events can be later analyzed on any computer, maintaining any history of occurrences. This is a fully independent monitoring equipment, not interfering with the operation of the current system already approved.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic Mtb:ΔRv2745c mutant is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb, implicating a role for ClgR in the management of intraphagosomal redox stress. Our data indicates that ClgR plays a role in multiple regulatory networks in response to different stress conditions. Thus, redox stress leads to dysregulation of the σH/σE regulon in Mtb:ΔRv2745c. Induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions. Disruption of genes involved in sulfate assimilation also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. On the other hand, the expression of clgR during hypoxia is known to result in Clp protease induction. As such, the isogenic mutant has a significantly different growth profile upon hypoxia and reaeration. Transcriptomics reveal disruption of the dosR regulon, σH/σE regulon, and mycolic acid synthesis genes. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in vivo. Our in vivo findings in a low dose aerosolized model reveal deficiencies of the isogenic mutant when establishing an infection, leading to skewed immune responses throughout the course of infection. Thus, clgR plays a critical role in both establishing an infection that influence the immunogenic outcome. Additional studies investigating the role of clgR in a nonhuman primate model will further elucidate the contributions of clgR to the pathogenesis of Mtb in an animal model that is more representative of human TB disease. / acase@tulane.edu

Microbiologists have long focused on transcription as a main source of physiological regulation in bacterial adaptation. However, the time scale on which certain cellular responses must be coordinated dictates that a more rapid system be in place to deal with sudden environmental stresses. In eukaryotes, understanding the proteasome and ubiquitin-tagging has led to an appreciation for protein turnover as a mechanism for rapid adaptation. Like eukaryotes, bacteria possess several proteolytic complexes that degrade proteins into smaller polypeptides and amino acids. These enzymes were discovered as maintainers of protein quality control, through recognition of aberrant protein products, but recent studies have suggested that they play an active role in regulation of cell processes through degradation of endogenous proteins. Surprisingly, a genome wide screen for essential genes in Mycobacterium tuberculosis (Mtb) found numerous proteases to be essential for growth, providing evidence that degradative regulation may be critical for survival. One essential complex, Clp protease, was intriguing as it appeared to have a divergent structure in Mtb, and was largely dispensable for growth in most other organisms. In order to study the importance of protein turnover and degradative regulation in Mtb, I chose to study Clp as a model. I confirmed that Clp was required for normal growth in mycobacteria through targeted genetic engineering, and demonstrated that depletion of Clp was bactericidal. We hypothesized that a protease would be essential because it might prevent accumulation of toxic proteins or repressors of vital processes. To understand why Clp protease was so critical, I conducted proteomic analysis comparing wildtype and Clp-depleted cells to identify substrates of the protease. In line with our hypothesis, I identified WhiB1, a redox-sensitive transcriptional repressor. Blocking degradation of WhiB1 by Clp resulted in death, suggesting that the importance of Clp can be partially explained by its action on the repressor. Finally, taking advantage of known Clp-specific inhibitors in S. aureus, we showed that Clp could be targeted with small molecules in Mtb. The elucidation of novel drug targets and small molecules active against Mtb is crucial due to the overwhelming prevalence of the disease and rises in drug resistant forms.

The Clp chaperones and proteases play a pivotal role in maintaining cellular homeostasis. They are highly conserved across prokaryotes and can also be found in the mitochondria of eukaryotes and chloroplast of plants. For my thesis, I provide an analysis of the Clp chaperones and protease in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which I term PfClpB1, PfClpB2, PfClpC, and PfClpM. One PfClpP, the proteolytic protomer, and one PfClpR, an inactive isoform, were also identified. All proteins, with the exception of PfClpB2, were found to be localized to the apicoplast, a non-photosynthetic relic plastid in P. falciparum. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by various techniques. Through X-ray crystallography, PfClpP assumed a compacted tetradecamer structure similar to that observed for other ClpPs. My data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

The Clp chaperones and proteases play a pivotal role in maintaining cellular homeostasis. They are highly conserved across prokaryotes and can also be found in the mitochondria of eukaryotes and chloroplast of plants. For my thesis, I provide an analysis of the Clp chaperones and protease in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which I term PfClpB1, PfClpB2, PfClpC, and PfClpM. One PfClpP, the proteolytic protomer, and one PfClpR, an inactive isoform, were also identified. All proteins, with the exception of PfClpB2, were found to be localized to the apicoplast, a non-photosynthetic relic plastid in P. falciparum. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by various techniques. Through X-ray crystallography, PfClpP assumed a compacted tetradecamer structure similar to that observed for other ClpPs. My data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Mecânica, 2014. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2015-11-25T15:13:31Z No. of bitstreams: 1 2015_IltonPereiradeOliveira.pdf: 8452704 bytes, checksum: 07e1001d9ac0c50320df03b95c713d97 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-05-17T20:23:45Z (GMT) No. of bitstreams: 1 2015_IltonPereiradeOliveira.pdf: 8452704 bytes, checksum: 07e1001d9ac0c50320df03b95c713d97 (MD5) / Made available in DSpace on 2016-05-17T20:23:46Z (GMT). No. of bitstreams: 1 2015_IltonPereiradeOliveira.pdf: 8452704 bytes, checksum: 07e1001d9ac0c50320df03b95c713d97 (MD5) / O objetivo deste trabalho é projetar um sistema embarcado baseado em um microcontrolador da família ARM, com o objetivo de simular um CLP (Controlador Lógico Programável) para aplicação em automação residencial, levando em conta vários requisitos, seguindo normas de segurança brasileiras e internacionais, programação simples, usando uma ferramenta gratuita de desenvolvimento. Neste contexto, há uma grande variedade aplicações que podem ser desenvolvidas usando um CLP residencial, tais como, controle de iluminação, controle básico de equipamentos eletrônicos, climatização de ambientes, acionamento do movimentador de portão, irrigação de jardins, abertura de persianas, alarme de segurança por meio da leitura de sensores na casa, entre outros. A proposta deste trabalho é que o CLP residencial torne-se um projeto de código aberto, incluindo todos os códigos fontes, esquemáticos e manuais de usuário, disponibilizando-os em uma página web disponível para receber modificações para novas aplicações. O sistema embarcado desenvolvido é baseado em um ARM CORTEX M3 LPC1768, incluindo também uma porta Ethernet e interfaces CAN, RS232, RS485, USBe e XBee. O sistema também conta com um RTC (relógio em tempo real), memoria interna de 512k, assim como memórias externas do tipo SD-card e EPROM. Além disso, foi desenvolvido um módulo de expansão para controle de cargas, que pode ser controlado a partir da placa-mãe usando tanto conexão XBee quanto via cabo serial. Este módulo é composto de 8 relés que oferecem conexões opto-isoladas que são dirigidas para as tarefas de automação residencial. Adicionalmente, o sistema desenvolvido foi projetado para ser aplicado em cursos de graduação com foco em disciplinas voltadas para microcontroladores e sistemas embarcados para automação e controle. Neste trabalho o quesito de interface, implícito nos CLPs comerciais, envolvendo linguagens de programação tais como Ladder (Logic Diagram Programming), FBD (Function Block Diagram), LD (Ladder Diagram), ST (Structured Text), SFC (Sequential Function Chart), entre outros, é deixado como trabalho futuro. _______________________________________________________________________________________________ ABSTRACT / The purpose of this work is to design an ARM-based embedded system, addressed to achieve the simulation of a PLC (Programmable Logic Controller) for home automation; taking into account several requirements such as low-cost, international safety standards, following the Brazilian rules, and offering simple programming by using a free tool development. In this context, there are a wide variety of applications that can be developed using a residential PLC, such as lighting control, basic control electronic equipment, air conditioning environments, gate mover driver, irrigation of gardens, opening blinds, security alarm by reading sensors in the home, among others. The purpose of this work is that the proposed residential CLP becomes an open source design, including all software files, data-sheets and user manuals, making available them on an online site, and thus available for receiving modifications for new applications. The developed embedded system is based on an ARM-3 (16-68C), and includes also an Ethernet port, as well as CAN, RS232, RS485, USB, and XBee interfaces. Additionally, the system also has a real time clock, as well as internal 512K memory and SD-card and EPROM external memories. Additionally, a daughter board has been also developed which can be accessed from the mother board using both XBee connection and via parallel cable. This daughter board is composed of 8 relays offering opto-isolated connections which are addressed for home automation tasks. Finally, the developed system has been designed to be applied in undergraduate courses focused in microcontroller application, and automation and control lectures. In this work the interface issue, implicit in commercial PLCs, involving programming languages such as Ladder (Logic Diagram Programming), FBD (Function Block Diagram), LD (Ladder Diagram), ST (Structured Text), SFC (Sequential Function Chart) among others, it is proposed as a future work.

O Irradiador Multipropósito de Cobalto-60 é uma unidade construída no Instituto de Pesquisas Energéticas e Nucleares, órgão da Comissão Nacional de Energia Nuclear. Esta instalação utiliza dos efeitos que a radiação gama produz ao interagir com um meio material e suas consequências. A radiação gama emitida pelo radioisótopo cobalto-60 é ionizante, com elevado poder de penetração, que ao interagir com os produtos, inclusive no interior de suas embalagens, transfere sua energia por meio de colisões aos elétrons dos átomos que constituem os produtos. Ao mesmo tempo que este processo de ionização é desejável em função de suas características deletérias aos microrganismos, a exposição indiscriminada às radiações ionizantes pelo ser humano ou animais apresentará danos e em doses elevadas poderão levar a morte. Diante destas circunstâncias, para garantir a segurança, os equipamentos utilizados para irradiação são construídos e operados sob normas rígidas de construção e operação. O sistema de segurança do Irradiador, constituído por um gerenciamento eletrônico de intertravamento de portas e exposição das fontes radioativas, é feito simultaneamente por um controlador lógico programável (CLP) e uma lógica de relés. Todas as informações, obtidas através das entrada e saídas do Irradiador, são monitoradas pelo sistema de segurança e enviadas para um computador com um programa supervisório. O propósito do trabalho foi a construção de uma Unidade Autônoma de Monitoramento, Sinalização e Registro para o Sistema de Segurança do Irradiador Multipropósito de Cobalto-60. O desenvolvimento deste equipamento permitiu o monitoramento e registro de eventos, até mesmo de tempos muito curtos, não detectáveis pelo CLP. Cada evento foi registrado em um cartão de memória, de forma a permitir que estes eventos possam ser posteriormente analisados em qualquer computador, mantendo todo e qualquer histórico de ocorrências. Este é um equipamento de monitoramento totalmente independente, não interferindo no funcionamento do sistema atual já homologado. / The Cobalto-60 Multipurpose Irradiator is a unit built at the Nuclear and Energy Research Institute, an agency of the National Nuclear Energy Commission. This installation uses the effects that gamma radiation produces when interacting with a material medium and its consequences. The gamma radiation emitted by the cobalt-60 radioisotope is ionizing, with high penetration power, which when interacting with the products, even inside its packages, transfers its energy by means of collisions to the electrons of the atoms that make up the products. At the same time as this ionization process is desirable because of its deleterious characteristics to the microorganisms, indiscriminate exposure to ionizing radiation by humans or animals will present damages and in high doses could lead to death. In view of these circumstances, to ensure safety, the equipment used for irradiation is constructed and operated under strict construction and operation standards. The safety system of the Irradiator, consisting of an electronic management of door interlocking and exposure of the radioactive sources, is made simultaneously by a programmable logic controller (PLC) and a relay logic. All information, obtained through the inputs and outputs of the Irradiator, is monitored by the security system and sent to a computer with a supervisory program. The purpose of the work was the construction of an Autonomous Monitoring, Signaling and Registration Unit for the Cobalto-60 Multipurpose Irradiance Safety System. The development of this equipment allowed the monitoring and recording of events, even of very short times, not detectable by the CLP. Each event has been recorded on a memory card, so that these events can be later analyzed on any computer, maintaining any history of occurrences. This is a fully independent monitoring equipment, not interfering with the operation of the current system already approved.

This study was an examination of responses to a standardized assessment of social-emotional behaviors and correlation with speech and language development in young children with cleft palate and/or lip. Twenty-eight participants aged 14-35 months with nonsyndromic cleft palate and or lip were included in this study. The Infant-Toddler Social and Emotional Assessment (ITSEA) was used to identify emerging social and emotional behaviors. Descriptive analysis of ITSEA results was completed. Pearson correlation coefficient and effect size estimates were calculated between ITSEA domain raw scores and measures of speech and language development. A small proportion of participants (14%) showed ITSEA scores beyond the test cut-off scores across all domains ; 43% demonstrated concerns at the subdomain level. Correlational analysis indicated significant relationships between Externalizing, Dysregulation, and Competence Domains and speech accuracy and language measures. Interpretation of the outcomes suggests that early social emotional behaviors are emerging simultaneously with speech and language skills during early communicative development.