Análise e expressão de genes de PKS II e de carboidrases provenientes de bibliotecas metagenômicas

Pós-graduação em Microbiologia Agropecuária - FCAV

Multilayer adsorption of Cu(II) and Cd(II) over Brazilian Orchid Tree (Pata-de-vaca) and its adsorptive properties

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear lucent after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded lucent fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the lucency of the fibrils indicated that they were composed of collagen I, whereas the lucency of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.

Correntes críticas e comportamento dinâmico dos vórtices em fitas supercondutoras do tipo II com arranjos conformes de centros de aprisionamento

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Correntes críticas e comportamento dinâmico dos vórtices em fitas supercondutoras do tipo II com arranjos conformes de centros de aprisionamento

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n = 58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.

Classifying A-field and B-field configurations in the presence of D-branes. Part II: Stacks of D-branes

In the paper of Bonora et al. (2008) [3] we have shown, in the context of type II superstring theory, the classification of the allowed B-field and A-field configurations in the presence of anomaly-free D-branes, the mathematical framework being provided by the geometry of gerbes. Here we complete the discussion considering in detail the case of a stack of D-branes, carrying a non-abelian gauge theory, which was just sketched in Bonora et al. (2008) [3]. In this case we have to mix the geometry of abelian gerbes, describing the B-field, with the one of higher-rank bundles, ordinary or twisted. We describe in detail the various cases that arise according to such a classification, as we did for a single D-brane, showing under which hypotheses the A-field turns out to be a connection on a canonical gauge bundle. We also generalize to the non-abelian setting the discussion about "gauge bundles with non-integral Chern classes", relating them to twisted bundles with connection. Finally, we analyze the geometrical nature of the Wilson loop for each kind of gauge theory on a D-brane or stack of D-branes.

A modified laparoscopic sleeve gastrectomy for the treatment of diabetes mellitus type 2 and metabolic syndrome in obesity

BACKGROUND: Ghrelin is a gastrointestinal peptide hormone (a 28-amino acid peptide) produced primarily by X/A cells in the oxyntic glands of the stomach fundus and cells lining the duodenum cavern. It suppresses insulin secretion and action and commands a significant role in regulating food intake. The aim of the present study was to show that modified laparoscopic sleeve gastrectomy (MLSG), in which a significant part of the gastric fundus and body of the stomach is removed up to 1 inch from the pylorus vein, may contribute to decreasing circulating ghrelin levels. METHODS: A study population consisting of 150 individuals was monitored after undergoing a MLSG, with individuals chosen based on a documented history of diabetes mellitus type 2 and metabolic syndrome, clinical results determining a body mass index (BMI) of 35 to 60 kg/m(2), peptide C level greater than 1, negative anti-glutamic acid decarboxylase, negative anti-insulin, and confirmed stability of drug/insulin treatment and glycosylated hemoglobin greater than 6.5% for at least 24 and 3 months, respectively, before enrollment. RESULTS: Twenty-four months after surgery, 150 patients (86.6%) presented with normal glycemic levels between 77 and 99 mg/dL. All patients improved average serum insulin levels by 9 mU/L and average glycosylated hemoglobin levels by 5.1% (normal range, 4%-6%). All patients tested negative for Helicobacter pylori and stopped using insulin, with 3 patients prescribed twice-daily use of an oral hypoglycemiant. In 14% of cases, patients experienced partial hair loss with low serum zinc levels and were prescribed oral zinc reposition and topical hair stimulants. The average weight loss recorded was 44.6% for patients with a BMI less than 45 kg/m(2) and 58% for patients with a BMI greater than 50 kg/m(2). CONCLUSIONS: The MLSG is a safe procedure with a low morbidity rate (2.7%) (4 cases of fistula and 2 of bleeding) and no surgical mortality in this study. This surgery can promote control of diabetes mellitus type 2 and aid the treatment of exogenous overweight and morbidly obese individuals. The results of this study show that only through resection of the ghrelin-producing gastric area can most obesity cases and diabetes type II conditions be reverted to nonobese and controlled diabetes. (c) 2012 Elsevier Inc. All rights reserved.

Human hemorrhagic pulmonary leptospirosis: pathological findings and pathophysiological correlations.

Abstract Background: Leptospirosis is a re-emerging zoonosis with protean clinical manifestations. Recently, the importance of pulmonary hemorrhage as a lethal complication of this disease has been recognized. In the present study, five human necropsies of leptospirosis (Weil‘s syndrome) with extensive pulmonary manifestations were analysed, and the antibodies expressed in blood vessels and cells involved in ion and water transport were used, seeking to better understand the pathophysiology of the lung injury associated with this disease. Principal Findings: Prominent vascular damage was present in the lung microcirculation, with decreased CD34 and preserved aquaporin 1 expression. At the periphery and even inside the extensive areas of edema and intraalveolar hemorrhage, enlarged, apparently hypertrophic type I pneumocytes (PI) were detected and interpreted as a non-specific attempt of clearence of the intraalveolar fluid, in which ionic transport, particularly of sodium, plays a predominant role, as suggested by the apparently increased ENaC and aquaporin 5 expression. Connexin 43 was present in most pneumocytes, and in the cytoplasm of the more preserved endothelial cells. The number of type II pneumocytes (PII) was slightly decreased when compared to normal lungs and those of patients with septicemia from other causes, a fact that may contribute to the progressively low PI count, resulting in deficient restoration after damage to the alveolar epithelial integrity and, consequently, a poor outcome of the pulmonary edema and hemorrhage. Conclusions: Pathogenesis of lung injury in human leptospirosis was discussed, and the possibility of primary noninflammatory vascular damage was considered, so far of undefinite etiopathogenesis, as the initial pathological manifestation of the disease.

Staphylococcus aureus thiaminase II: oligomerization warrants proteolytic protection against serine proteases

Staphylococcus aureus TenA (SaTenA) is a thiaminase type II enzyme that catalyzes the deamination of aminopyrimidine, as well as the cleavage of thiamine into 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 5-(2-hydroxyethyl)-4-methylthiazole (THZ), within thiamine (vitamin B1) metabolism. Further, by analogy with studies of Bacillus subtilis TenA, SaTenA may act as a regulator controlling the secretion of extracellular proteases such as the subtilisin type of enzymes in bacteria. Thiamine biosynthesis has been identified as a potential drug target of the multi-resistant pathogen S. aureus and therefore all enzymes involved in the S. aureus thiamine pathway are presently being investigated in detail. Here, the structure of SaTenA, determined by molecular replacement and refined at 2.7 A ° resolution to an R factor of 21.6% with one homotetramer in the asymmetric unit in the orthorhombic space group P212121, is presented. The tetrameric state of wild-type (WT) SaTenA was postulated to be the functional biological unit and was confirmed by small-angle X-ray scattering (SAXS) experiments in solution. To obtain insights into structural and functional features of the oligomeric SaTenA, comparative kinetic investigations as well as experiments analyzing the structural stability of the WT SaTenA tetramer versus a monomeric SaTenA mutant were performed.

Etablierung einer Ko-Kultur von Alveolarepithel und mikrovaskulärem Endothel als in-vitro-Modell einer humanen respiratorischen Einheit

Ziel der Promotionsarbeit war die Etablierung einer humanen respiratorischen Einheit in vitro zur Untersuchung von Wirkmechanismen einer akuten Lungenschädigung. Als Ko-Kulturmodell wurde eine Kultur humaner Alveoloarepithelzellen vom Typ II (A549, NCI H441, primäre hATII Zellen) mit mikrovaskulären Endothelzellen (ISO-HAS-1, primäre HPMEC) auf den beiden Seiten einer mikroporösen Filtermembran (Bilayer) gewählt. Ein differenzierter Monolayer von NCI H441 konnte in Bilayer-Ko-Kultur mit ISO-HAS-1 oder HPMEC durch die Zugabe von Dexamethason (1 µM) unter Verwendung eines serumhaltigen Mediums induziert werden. Dabei wurde eine von Tag 10 bis Tag 12 phänotypisch stabile Ko-Kultur mit TER-Werten um 500 Ohm x cm2 erhalten. Im Hinblick auf die Freisetzung von IL-8 und MCP-1 und die fehlende Freisetzung von RANTES nach Stimulation waren NCI H441 den hATII Zellen ähnlicher als die häufig als hATII-analog eingesetzte Zell-Linie A549, die RANTES freisetzte. Außerdem bildeten A549 trotz zahlreicher Variatione

Spin-crossover in metallomesogens of iron(II)

Covalent grafting mesogenic groups to the coordination cores of the parent mononuclear low-spin and spin-crossover compounds afforded metallomesogenic complexes of iron(II). In comparison with the parent complexes the spin-crossover properties of the alkylated derivatives are substantially modified. The type of the modification was found to be dependent on the properties of the parent system and the nature of the used anion, however, the general tendency is the destabilization of the low-spin state at the favor of spin-crossover or high-spin behavior below 400 K. The structural insight revealed the micro-segregated layered organization. The effect of the alkylation of the parent compounds consists first of all in the change of the lattice to a two-dimensional lamellar one retaining significant intermolecular contacts only within the ionic bilayers. The comprehensive analysis of the structural and thermodynamic data in the homologous series pointed at the mechanism of the interplay between the structural modification on melting and the induced anomalous change of the magnetic properties. A family of one-dimensional spin-crossover polymers was synthesized and characterized using a series of spectroscopic methods, X-ray powder diffraction, magnetic susceptibility measurements and differential scanning calorimetry. The copper analogue of was also synthesized and its crystal structure solved. In comparison with the mononuclear systems, the polymeric mesogens of iron(II) are less sensitive to the glass transition, which was attributed to the moderate concomitant variation of the structure. Nevertheless, the observed increase of the magnetic hysteresis with lengthening of the alkyl substituents was ascribed to the interplay of the structural reorganization of the coordination core due to spin-crossover with the structural delay in the spatial reorganization of the mesogenic substituents. The classification of mononuclear and polymeric metallomesogens according to the interactions between the structural- and the spin-transition and analysis of the data on the reported spin-crossover metallomesogens led to the separation of three types, namely: Type i: systems with coupling between the electronic structure of the iron(II) ions and the mesomorphic behavior of the substance; Type ii: systems where both transitions coexist in the same temperature region but are not coupled due to competition with the dehydration or due to negligible structural transformation; Type iii: systems where both transitions occur in different temperature regions and therefore are uncoupled. Fine-tuning, in particular regarding the temperature at which the spin-transition occurs with hysteresis properties responsible for the memory effect, are still a major challenge towards practical implementation of spin-crossover materials. A possible answer to the problem could be materials in which the spin-crossover transition is coupled with another transition easily controllable by external stimuli. In the present thesis we have shown the viability of the approach realized in the mesogenic systems with coupled phase- and spin-transitions.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules – dexamethasone, triiodothyronine (T3) and insulin – on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.