Estudo de intera����es prote��na-carbohidrato em sistemas bacterianos

Bacillus subtilis tem a capacidade de utilizar arabinoligossac��ridos presentes na parede das c��lulas vegetais, atrav��s de um cons��rcios de enzimas envolvidas na hidr��lise enzim��tica dos mesmos. A capta����o destes carbohidratos por parte do Bacillus subtilis depende de dois transportadores membranares AraE e AraNPQ-MsmX. Ap��s a capta����o de L-arabinose e arabinoligossac��ridos, estes substratos ser��o metabolizados pelos produtos enzim��ticos codificados por genes pertencentes ao oper��o respons��vel pela metaboliza����o de arabinose araABDLMNPQabfA. Neste oper��o, para al��m dos genes respons��veis pelo transportador AraNPQ, o gene araL determina a s��ntese de uma enzima com atividade de fosfatase, AraL, que tem putativamente como principal fun����o a destoxifica����o de intermedi��rios metab��licos fosforilados, em situa����es particulares. O objectivo deste trabalho consistiu em descobrir quais os determinantes moleculares envolvidos em ambos os processos de reconhecimento molecular, ora o reconhecimento de arabinoligossac��ridos por parte da AraN, do ponto de vista dos carbohidratos ora de carbohidratos fosforilados do ponto de vista da AraL. Para elucidar estes processos de reconhecimento molecular prote��na-carbohidrato foi utilizada a t��cnica de RMN, modela����o computacional e mutag��nese dirigida. No primeiro capitulo s��o introduzidos conceitos fundamentais para a percep����o da a����o de ambas as prote��nas (AraN e AraL), no organismo Bacillus subtilis. O segundo capitulo, refere-se ao estudo dos mecanismos envolvidos no reconhecimento prote��na-carbohidrato atrav��s da t��cnica de STD-RMN para estudar intera����es do ponto de vista do carbohidrato, bem como abordagens bioinform��ticas tais como alinhamentos de sequencia prim��ria e dockings moleculares, para identificar res��duos do ponto de vista da prote��na passiveis de se encontrarem envolvidos no reconhecimento molecular, que numa ��ltima inst��ncia s��o mutados para se confirmar a sua relev��ncia no processo de reconhecimento molecular do ponto de vista da prote��na. Neste capitulo �� demonstrado que a AraN �� respons��vel pelo reconhecimento de arabinoligossac��ridos e celoligossac��ridos. No que diz respeito aos primeiros, a AraN reconhece preferencialmente arabinoligossac��ridos com tr��s subunidades, verificando-se a perda de satura����o na subunidade n��o redutora da arabinotetraose. O processo de reconhecimento molecular apresenta uma constante de dissocia����o com uma ordem de grandeza na ordem dos ��M. Do ponto de vista da prote��na s��o identificados res��duos (W253, Y254 e Y54) putativos de se encontrarem tamb��m eles envolvidos no reconhecimento molecular do ponto de vista da AraN atrav��s de intera����es CHstacking. Finalmente no terceiro capitulo, �� apresentada a optimiza����o da sobrexpress��o da AraL, dado que numa primeira fase a sua produ����o ocorria sob a forma de corpos de inclus��o. Para tal s��o descritas metodologias de solubiliza����o e renatura����o da mesmas, com recurso a agentes caotr��picos (ureia e GmdCl) e detergentes (SDS).

Prote��mica microbiana: an��lise comparativa da bact��ria redutora de sulfato Desulfovibrio desulfuricans ATCC 27774 e das actinobact��rias Salinispora arenicola e Salinispora pacifica

As bact��rias redutoras de sulfato (BRS) s��o um grupo diversificado de micro-organismos anaer��bios que obt��m energia a partir da redu����o dissimilativa do sulfato. Algumas esp��cies de BRS possuem versatilidade a n��vel respirat��rio, como �� o caso de Desulfovibrio desulfuricans ATCC 27774, devido �� utiliza����o de aceitadores finais de eletr��es alternativos. Neste contexto, o objetivo da primeira parte desta disserta����o consistiu na identifica����o das ferramentas metab��licas (prote��nas) envolvidas na flexibilidade respirat��ria desta bacteria induzidas por diferentes aceitadores de eletr��es (nitrato vs sulfato). Assim, os extratos proteicos totais de c��lulas de D. desulfuricans crescidas em meios VMN com nitrato ou sulfato, foram analisados por eletroforese bidimensional (2DE), num gradiente de pH 4 - 7. Nas condi����es experimentais testadas, foram observados 604 e 519 spots de prote��nas nos g��is de c��lulas crescidas em meio contendo nitrato ou sulfato, respetivamente. Pela avalia����o estat��stica foi poss��vel observar aproximadamente 25 % de spots diferenciais. Os resultados obtidos sugerem que na presen��a de nitrato, a bact��ria n��o s�� cresce mais rapidamente e com maior rendimento, como tamb��m produz uma maior quantidade de prote��nas. Estes dados foram relacionados com a adapta����o de D. desulfuricans ao substrato respirat��rio alternativo. Tal como esperado, nos ensaios das atividades enzim��ticas das redutases do nitrito e do nitrato, foi poss��vel observar maior atividade nos extratos das c��lulas crescidas em nitrato do que em sulfato. As actinobact��rias marinhas do g��nero Salinispora, t��m vindo a ser exploradas como fontes de biof��rmacos naturais. Assim, na segunda parte deste trabalho, realizou-se um estudo preliminar, que pretendeu caracterizar as prote��nas envolvidas na bioss��ntese destes compostos bioactivos em S. arenicola e de S. pacifica. Para tal, foi utilizada uma abordagem prote��mica diferencial, baseada em 2DE. Surpreendentemente, os perfis proteicos das duas esp��cies mostraram-se bastante distintos, tendo-se identificado apenas 37 spots comuns entre os 650 observados no gel de S. arenicola e 510 no gel de S. pacifica.

Crystallographic studies of proteins involved in the mRNA localization mechanisms in Drosophila melanogaster and amidation of the peptidoglycan residues in Staphylococcus aureus

Part of the work described in this chapter, was the subject of the following publication: D. Vieira, T. a. Figueiredo, A. Verma, R. G. Sobral, A. M. Ludovice, H. de Lencastre, and J. Trincao, ���Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan,��� Acta Crystallogr. Sect. F Struct. Biol. Commun., vol. 70, no. 5, pp. 1���4, Apr. 2014.

Atypical lymphocytosis in leptospirosis: a cohort of hospitalized cases between 1996 and 2009 in State of Rio de Janeiro, Brazil

INTRODUCTION: Leptospirosis is a zoonotic disease found in tropical and temperate countries, and its clinical diagnostic confusion with arboviruses (dengue fever, oropouche fever and yellow fever), Brazilian spotted fever, viral hepatitis and hantaviruses has been an ongoing public health concern. The aim of this observational study was to demonstrate an association between findings of atypical lymphocytosis and the progression of endemic leptospirosis. METHODS: A retrospective analysis was performed on the demographic, epidemiological, clinical and laboratory aspects of 27 human leptospirosis cases that occurred over a period of 13 years (1996-2009) with no reported epidemic outbreaks in Rio de Janeiro, Brazil. RESULTS: The overall mortality rate was 11.1% in our cohort of hospitalized cases. However, there was no mortality among patients with atypical lymphocytosis (OR = 11.1; 95% CI = 1.12-110.9; p = 0.04). Two patients who were in the septicemic phase showed signs of expansion of γδ T cell responses in peripheral blood. CONCLUSIONS: Atypical lymphocytosis may be observed in patients with leptospirosis. Our observations suggest that these atypical leukocyte subsets are associated with partial protection during the disease course of leptospirosis.

Active surveillance of American tegumentary leishmaniasis in endemic areas in rural Bolivia

INTRODUCTION: American tegumentary leishmaniasis (ATL), including mucocutaneous leishmaniasis (MCL) and localized cutaneous leishmaniasis (LCL), is endemic in Bolivia. We describe the results of active surveillance of ATL from 2001 to 2006 and assess demographic data related to ATL epidemiology in the Yungas valleys. METHODS: Community-based active ATL surveillance was performed by the institutions SERVIR, C��RITAS, and the Health Services Department of La Paz, whose files were reviewed retrospectively. A cross-sectional survey was carried out to assess demographic data in two communities. RESULTS: Two thousand nine hundred nine cases of ATL were detected from 2001 to 2006: 2,488 (85.5%) corresponded to LCL and 421 (14.5%) to MCL. A reduction in the proportion of mucosal cases was observed between 2001 and 2006. The proportion of MCL cases increased with age and was higher among males (15.5% versus 12.1%, p=0.018). The rate of positivity via direct observation of the parasite in dermal scrapings and in parasite cultivation was significantly higher for LCL than for MCL (p<0.001 and p=0.009, respectively). The rate of reactivity in the leishmanin skin test was higher in the group with mucosal lesions (p=0.012). The cross-sectional survey showed that 40% of the families had emigrated from the Altiplano. CONCLUSIONS: It is necessary to undertake continuous case detection of ATL in the area, where the disease presents a high rate of mucosal cases. Increasing incidence seems to be associated with immigration and continuous deforestation to expand the crop-growing areas.

Biochemical and nutritional evaluation of patients with visceral leishmaniasis before and after treatment with leishmanicidal drugs

Introduction Visceral leishmaniasis (VL) is caused by the intracellular protozoan Leishmania donovani complex. VL may be asymptomatic or progressive and is characterized by fever, anemia, weight loss and the enlargement of the spleen and liver. The nutritional status of the patients with VL is a major determinant of the progression, severity and mortality of the disease, as it affects the clinical progression of the disease. Changes in lipoproteins and plasma proteins may have major impacts in the host during infection. Thus, our goal was evaluate the serum total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, glucose, albumin, globulin and total protein levels, as well as the body composition, of VL patients before and after treatment. Methods Nutritional evaluation was performed using the bioelectrical impedance analysis (BIA) to assess body composition. Biochemical data on the serum total cholesterol, HDL, LDL, triglycerides, glucose, albumin, globulin and total protein were collected from the medical charts of the patients. Results BIA indicated that both pre-treatment and post-treatment patients exhibited decreased phase angles compared to the controls, which is indicative of disease. Prior to treatment, the patients exhibited lower levels of total body water compared to the controls. Regarding the biochemical evaluation, patients with active VL exhibited lower levels of total cholesterol, HDL, LDL and albumin and higher triglyceride levels compared to patients after treatment and the controls. Treatment increased the levels of albumin and lipoproteins and decreased the triglyceride levels. Conclusions Our results suggest that patients with active VL present biochemical and nutritional changes that are reversed by treatment.

Clinical conditions associated withintestinal strongyloidiasis in Rio de Janeiro, Brazil

INTRODUCTION: Strongyloides stercoralis is a soil-transmitted helminth that produces an infection that can persist for decades. The relationships between certain clinical conditions and strongyloidiasis remains controversial. This study aims to identify the clinical conditions associated with intestinal strongyloidiasis at a reference center for infectious diseases in Rio de Janeiro, Brazil. METHODS: The clinical conditions that were assessed included HIV/AIDS, HTLV infection, cardiovascular diseases, diabetes, obstructive respiratory diseases, viral hepatitis, tuberculosis, cancer, chronic renal disease, nutritional/metabolic disorders, psychiatric conditions, rheumatic diseases and dermatologic diseases. We compared 167 S. stercoralis-positive and 133 S. stercoralis-negative patients. RESULTS: After controlling for sex (male/female OR = 2.29; 95% (CI): (1.42 - 3.70), rheumatic diseases remained significantly associated with intestinal strongyloidiasis (OR: 4.96; 95% CI: 1.34-18.37) in a multiple logistic regression model. With respect to leukocyte counts, patients with strongyloidiasis presented with significantly higher relative eosinophil (10.32% &#177; 7.2 vs. 4.23% &#177; 2.92) and monocyte (8.49% &#177; 7.25 vs. 5.39% &#177; 4.31) counts and lower segmented neutrophil (52.85% &#177; 15.31 vs. 61.32% &#177; 11.4) and lymphocyte counts (28.11% &#177; 9.72 vs. 30.90% &#177; 9.51) than S. stercoralis-negative patients. CONCLUSIONS: Strongyloidiasis should be routinely investigated in hospitalized patients with complex conditions facilitate the treatment of patients who will undergo immunosuppressive therapy. Diagnoses should be determined through the use of appropriate parasitological methods, such as the Baermann-Moraes technique.

Prevalence of intestinal parasites and risk factors forspecific and multiple helminth infections in a remote city of the Brazilian Amazon

Abstract: INTRODUCTION: Few studies have described the risk factors of intestinal parasitic infections in the Amazon. METHODS: A cross-sectional survey was performed in a City of the State of Amazonas (Brazil) to estimate the prevalence of intestinal parasites and determine the risk factors for helminth infections. RESULTS: Ascaris lumbricoides was the most prevalent parasite. The main risk factors determined were: not having a latrine for A. lumbricoides infection; being male and having earth or wood floors for hookworm infection; and being male for multiple helminth infections. CONCLUSIONS: We reported a high prevalence of intestinal parasites and determined some poverty-related risk factors.

Changes in body fluid and energy compartments during prolonged hunger strike

Prolonged total food deprivation in non-obese adults is rare, and few studies have documented body composition changes in this setting. In a group of eight hunger strikers who refused alimentation for 43 days, water and energy compartments were estimated, aiming to assess the impact of progressive starvation. Measurements included body mass index (BMI), triceps skinfold (TSF), arm muscle circumference (AMC), and bioimpedance (BIA) determinations of water, fat, lean body mass (LBM), and total resistance. Indirect calorimetry was also performed in one occasion. The age of the group was 43.3��6.2 years (seven males, one female). Only water, intermittent vitamins and electrolytes were ingested, and average weight loss reached 17.9%. On the last two days of the fast (43rd-44th day) rapid intravenous fluid, electrolyte, and vitamin replenishment were provided before proceeding with realimentation. Body fat decreased approximately 60% (BIA and TSF), whereas BMI reduced only 18%. Initial fat was estimated by BIA as 52.2��5.4% of body weight, and even on the 43rd day it was still measured as 19.7��3.8% of weight. TSF findings were much lower and commensurate with other anthropometric results. Water was comparatively low with high total resistance, and these findings rapidly reversed upon the intravenous rapid hydration. At the end of the starvation period, BMI (21.5��2.6 kg/m��) and most anthropometric determinations were still acceptable, suggesting efficient energy and muscle conservation. Conclusions: 1) All compartments diminished during fasting, but body fat was by far the most affected; 2) Total water was low and total body resistance comparatively elevated, but these findings rapidly reversed upon rehydration; 3) Exaggerated fat percentage estimates from BIA tests and simultaneous increase in lean body mass estimates suggested that this method was inappropriate for assessing energy compartments in the studied population; 4) Patients were not morphologically malnourished after 43 days of fasting; however, the prognostic impact of other impairments was not considered in this analysis.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor ��-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to ���in-cell��� Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

On the function of the floccular complex of the vertebrate cerebellum: implications in paleoneuroanatomy

The cerebellum floccular complex lobes (FCLs) are housed in the FCL fossa of the periotic complex. There is experimental evidence indicating that the FCLs integrate visual and vestibular information, responsible for the vestibulo-ocular reflex, vestibulo-collic reflex, smooth pursuit and gaze holding. Thus, the behavior of extinct animals has been correlated with FCLs dimension in multiple paleoneuroanatomy studies. Here I analyzed braincase endocasts of a representative sample of Mammalia (48 species) and Aves (59 species) rendered using tomography and image segmentation and tested statistical correlations between the floccular complex volume, ecological and behavioral traits to assess various previously formulated paleobiological speculations. My results demonstrate: 1) there is no significant correlation between relative FCL volume and body mass; 2) there is no significant correlation between relative FCL and optic lobes size in birds; 3) average relative FCL size is larger in diurnal than in nocturnal birds but there is no statistically significant difference in mammals; 4) feeding strategies are related with different FCL size patterns in birds, but not in mammals; 5) locomotion type is not related with relative FCL size in mammals; 6) agility is not significantly correlated with FCL size in mammals. I conclude that, despite the apparent relation between FCL size and ecology in birds, the cerebellum of tetrapods is a highly plastic structure and may be adapted to control different functions across different taxonomic levels. For example, the european mole (Talpa europaea) which is fossorial and practically blind, has a FCL fossae relative size larger than those of bats, which are highly maneuverable. Therefore, variation in FCL size may be better explained by a combination of multiple factors with relation to anatomical and phylogenetic evolutionary constraints.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ci��ncias - Especialidade em Biologia

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - �� + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections

Assessment of liposome disruption to quantify drug delivery in vitro

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

Hair coloration by gene regulation: fact or fiction?

The unravelling of hair pigmentation genetics and robust delivery systems to the hair follicle (HF) will allow the development of a new class of colouring products. The challenge will be changing hair colour from inside out by safely regulating the activity of target genes through the specific delivery of synthetic/natural compounds, proteins, genes, or small RNAs.