980 resultados para Cloning, Organism
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INTRODUCTION: his study evaluated the consumption of major classes of antibiotics, the colonization of the oropharynx of patients on mechanical ventilation, and the risk of ventilator-associated pneumonia (VAP) caused by Staphylococcus aureus in an intensive care unit for adults. METHODS: A case-control study was carried out using colonized patients (cases) by oxacillin-resistant S. aureus (ORSA) and (controls) oxacillin-sensitive S. aureus (OSSA) from May 2009 to August 2010. The occurrence of VAP by S. aureus was also evaluated in the same period. Antibiotic consumption was expressed as the number of defined daily doses (DDD)/1,000 patient-days for glycopeptides, carbapenems, and extended-spectrum cephalosporins. RESULTS: Three hundred forty-six (56.1%) patients underwent mechanical ventilation with a frequency of oropharyngeal colonization of 36.4%, corresponding to 63.5% for ORSA and 36.5% for OSSA. The risk of illness for this organism was significant (p<0.05), regardless of whether colonization/infection was by ORSA or OSSA. The consumption of antibiotics was high, mainly for broad-spectrum cephalosporins (551.26 DDDs/1,000 patient-days). The high density of use of glycopeptides (269.56 DDDs/1,000 patient-days) was related to colonization by ORSA (Pearson r=0.57/p=0.02). Additionally, age >60 years, previous antibiotic therapy, and previous use of carbapenems were statistically significant by multivariate analysis. CONCLUSIONS: There was a significant relationship between the colonization of the oropharyngeal mucosa and the risk of VAP by both phenotypes. The use of glycopeptides was related to colonization by ORSA.
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We report the case of a 36-year-old man who had acquired immune deficiency syndrome and developed suppurative mediastinitis extending over the left lung and anterior thoracic wall around the sternum, pericardial effusions, splenomegaly, and mesenteric and periaortic lymphadenomegaly due to Mycobacterium avium (genotype I). The organism was isolated from an axillary lymph node and the bone marrow. Mediastinitis associated with disseminated M. avium complex infection is uncommon and, to the best of our knowledge, this manifestation has not reported before.
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Although cryptococcal infections begin in the lungs, meningoencephalitis is the most frequently encountered manifestation of cryptococcosis among individuals with advanced immunosuppression. As the infection progresses along the Virchow-Robin spaces, these structures may become dilated with mucoid material produced by the capsule of the organism. We report a case of a 24-year-old man with cryptococcal meningoencephalitis in which magnetic resonance imaging showed clusters of gelatinous pseudocysts in the periventricular white matter, basal ganglia, mammillary bodies, midbrain peduncles and nucleus dentatus with a soap bubble appearance.
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Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections.
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Os resultados apresentados no captulo 2 foram includos no artigo Dantas JM, Campelo LM, Duke NEC, Salgueiro CA, Pokkuluri PR (2015) "The structure of PccH from Geobacter sulfurreducens a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode", FEBS Journal, 282, 2215-2231.
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Introduction This work presents the initial findings of a molecular epidemiological investigation of Trypanosoma cruzi in triatomine insects in State of Mato Grosso do Sul. Methods A total of 511 triatomines from different regions of the state were examined. Deoxyribonucleic acid (DNA) was extracted from the intestinal contents of the insects using phenol-chloroform-isoamyl alcohol (25:24:1). Polymerase chain reaction (PCR) using primers 121/122 targeting DNA kinetoplast (kDNA) was then performed to identify T. cruzi, and positive samples were subjected to PCR using the primer pair TcSC5D-F/R followed by restriction fragment length polymorphism (RFLP) with the restriction enzymes SphI and HpaI (1 U/reaction), cloning and sequencing. Results One hundred samples were positive for T. cruzi, and three discrete typing units (DTUs) were identified (TcI, TcII, and TcBat). Triatoma sordida had the highest T. cruzi occurrence (83.3%), and DTUs were found in three samples: 58.3% of the samples were TcI, 33.3% were TcII and 8.3% were TcBat. There was a clear geographical distribution of the DTUs throughout the state, with TcI, TcII and TcBat located in the center, TcI located in the east, and TcII located in the west. Conclusions This study showed the occurrence of overlapping DTUs in State of Mato Grosso do Sul. The distributions of the DTUs were different, with TcI, TcII and TcBat in the center of the state, TcI predominantly in the east, and TcII in the west. Further studies may reveal a more defined mosaic distribution of DTUs in MS.
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Primary inoculation tuberculosis is an exogenous infection resulting from direct inoculation of bacteria into individuals with no acquired immunity to the organism. We report a 63-year-old male patient who was diagnosed with primary inoculation tuberculosis on the basis of clinical appearance and histopathological examination. The findings from this case emphasize the importance of clinical and histopathological findings in this rarely seen form of skin tuberculosis if the organism cannot be shown to grow in culture.
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Abstract:INTRODUCTION:The frequency of methicillin-resistant Staphylococcus aureus (MRSA) has increased in the community. This study evaluated the prevalence of MRSA and community-acquired (CA)-MRSA in 120 healthy elderly.METHODS:The MRSA were evaluated for the presence of the IS256, mecA, agr, icaA, icaD, fnbB , and pvl genes with PCR. Results: Frequency of S. aureus and MRSA colonization was 17.8% and 19%, respectively. CA-MRSA isolate showed SCC mec IV, fnbB+ , and icaD+ .CONCLUSIONS:CA-MRSA was detected, with genotype determined as SCC mec type IV/IS256/ fnbB+ / icaA / icaD+ / bbp-/agr2 / bap / pvl, characterizing this population as a possible reservoir of this organism in the community.
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RESUMO: Clostridium difficile presentemente a principal causa de doena gastrointestinal associada utilizao de antibiticos em adultos. C. difficile uma bactria Gram-positiva, obrigatoriamente anaerbica, capaz de formar endsporos. Tem-se verificado um aumento dos casos de doena associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrncia, em parte, devido emergncia de estirpes mais virulentas, mas tambm devido m gesto do uso de antibiticos. C. difficile produz duas toxinas, TcdA e TcdB, que so os principais fatores de virulncia e responsveis pelos sintomas da doena. Estas so codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxignio so essenciais para a transmisso do organismo e recorrncia da doena. A expresso dos genes do PaLoc ocorre em clulas vegetativas, no final da fase de crescimento exponencial, e em clulas em esporulao. Neste trabalho construmos dois mutantes de eliminao em fase dos genes tcdR e tcdE. Mostrmos que a auto-regulao do gene tcdR no significativa. No entanto, tcdR sempre necessrio para a expresso dos genes presentes no PaLoc. Trabalho anterior mostrou que, com a exceo de tcdC, os demais genes do PaLoc so expressos no pr-esporo. Mostrmos aqui que TcdA detectada superfcie do esporo maduro e que a eliminao do tcdE no influencia a acumulao de TcdA no meio de cultura ou em associao s clulas ou ao esporo. Estas observaes tm consequncias para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser tambm um veculo para a entrega da toxina nos estgios iniciais da infeco, que TcdA possa ser libertada durante a germinao do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligao mucosa do clon.---------------------------ABSTRACT: Clostridium difficile is currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract. Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of hypervirulent strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence. The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation of tcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes. A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.
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RESUMO: As clulas eucariticas evoluram um sistema de sinalizao complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinalizao so essenciais para a sobrevivncia da clula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transduo de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a protena Smoothened (Smo) na ausncia dos seus ligandos, as protenas Sonic hedgehog (Shh). Estas protenas so reguladores fundamentais do desenvolvimento embrionrio, como ilustrado pelas malformaes drsticas observadas em embries humanos e de murganho com perturbaes da transduo de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malformaes sseas. Igualmente importantes so as consequncias da ativao inapropriada da via de sinalizao Hh na formao de tumores. Curiosamente, os componentes desta via localizam-se nos clios primrios. Alm disso, demonstrou-se que esta localizao crucial para a sinalizao atravs da via Hh. Na presena dos ligandos, Ptch1 internalizado e destinado a degradao ou sequestrado num compartimento da clula de onde no pode desempenhar o seu papel inibitrio. A protena Arl13b uma pequena GTPase pertencente famlia Arf/Arl da superfamlia Ras de pequenas GTPases e foi implicada no sndrome de Joubert, uma ciliopatia caracterizada por ataxia congnita cerebelar, hipotonia, atrso mental e cardiopatia congnita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gestao (E13,5) e exibem anomalias morfolgicas nos clios que levam interrupo da sinalizao Hh. Alm disso, a Arl13b est diretamente envolvida na regulao da via Hh, controlando a localizao de vrios componentes desta via nos clios primrios. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que so estruturas de actina envolvidas em macropinocitose e internalizao de recetores, e que regula a sua formao. Alm disso, aprofundmos o conhecimento do processo de ativao da via de sinalizao Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos aps ativao da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir da, sugerindo uma induo contnua da reorganizao do citoesqueleto de actina quando a via est ativada. Observmos ainda que a inibio da formao de CDRs atravs do silenciamento de WAVE1, uma protena necessria para a formao destas estruturas, resulta na diminuio da ativao da via de sinalizao Hh. Alm disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, atravs do silenciamento de uma protena necessria para a ciso de macropinossomas, nomeadamente a protena BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose so necessrias para a ativao da via de sinalizao Hh e indicam que esta via de internalizao controla os nveis de sinal Hh. Durante o desenvolvimento, as clulas proliferativas dependem do clio primrio para a transduo de vrias vias de sinalizao. A via Hh induz a diferenciao do msculo cardaco. Por conseguinte, os murganhos deficientes na via de sinalizao Hh exibem uma variedade de defeitos de lateralidade, incluindo alterao do looping do corao, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investigmos o papel da Arl13b no desenvolvimento do corao. Mostramos que a Arl13b altamente expressa no corao de embries de murganho e de murganhos adultos ao nvel do mRNA e da protena. Alm disso, o perfil de distribuio da Arl13b no corao segue o dos clios primrios, que so essenciais para o desenvolvimento cardaco. Coraes de murganhos hnn no estadio E12,5 mostram um canal trio-ventricular aberto, espessamento da camada compacta ventricular e aumento do ndice mittico no ventrculo esquerdo. Alm disso, um atraso de 1 a 2 dias no desenvolvimento observado em coraes de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b necessria para o desenvolvimento embrionrio do corao e que defeitos cardacos podem contribuir para a letalidade embrionria de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regulao dos nveis de superfcie do recetor Ptch1, que envolve a remodelao do citoesqueleto de actina e a formao de CDRs aps a ativao da via de sinalizao Hh. Este mecanismo permite um feedback negativo que evita a represso excessiva da via atravs da remoo de Ptch1 da superfcie da clula. Alm disso, determinou-se que uma mutao de perda de funo na Arl13b causa defeitos cardacos durante o desenvolvimento, possivelmente relacionados com a associao dos defeitos em clios primrios e na sinalizao Hh, existentes em murganhos deficientes para Arl13b. A via de sinalizao Hh tem tido um papel central entre as vias de sinalizao, uma vez que a sua regulao crucial para o funcionamento apropriada da clula. Assim, a descoberta de um novo mecanismo de trfego atravs de macropinocitose e CDRs que controla a ativao e represso da via de sinalizao Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir identificao de novos alvos e estratgias teraputicas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.
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Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters.
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The immune system comprises of different cell types whose role is to protect us against pathogens. This thesis investigates a very important mechanism for our organism protection in a specific disorder: cross-presentation in Wiskott-Aldrich Syndrome (WAS). WAS is caused by loss-of-function mutations in the cytoskeletal regulator WASp and WAS patients suffer from eczema, thrombocytopenia, and immunodeficiency. X-linked neutropenia (XLN) is caused by gain-of-function mutations in WASp and XLN patients suffer from severe congenital neutropenia and immunodeficiency. This thesis was focused on the role of B and T lymphocytes and dendritic cells (DCs). This work will be divided into two main topics: 1) In the first part I studied the capacity of B cells to take up, degrade and present antigen. Moreover I studied the capacity of B cells to induce T cell proliferation. 2) In the second part, I studied T cell proliferation induced by dendritic cells. To increase our understanding about this mechanism, additional experiments were performed, including acidification capacity of CD8+ and CD8- DCs, reactive oxygen species (ROS) production since it is directly connected to acidification. These assays were measured by flow cytometry. Localization of Rac1 and Rac2 GTPases was assessed by confocal microscopy. Proliferation, acidification and ROS production assays were performed also with cells from X-linked neutropenia (XLN) mice. From this study we concluded that B cells cannot induce CD8+ T cell proliferation however they take up and present antigen. Moreover I have shown that increased cross-presentation by WASp KO DCs with ovalbumin is associated with decreased capacity to acidify endosomal compartment; and WASp KO CD8- DCs have increased Rac2 localization to the phagosome. XLN dendritic cells have similar acidification and ROS production capacity than wildtype cells. In conclusion, our data suggests that WASp regulates antigen processing and presentation in DCs.
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The Gallus gallus (chicken) embryo is a central model organism in evolutionary developmental biology. Its anatomy and developmental genetics have been extensively studied and many relevant evolutionary implications have been made so far. However, important questions regarding the developmental origin of the chicken skull bones are still unresolved such that no solid homology can be established across organisms. This precludes evolutionary comparisons between this and other avian model systems in which skull anatomy has evolved significantly over the last millions of years.(...)
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PhD Thesis in Bioengineering
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PhD thesis in Bioengineering
