Inhibitory Action of Neem Aqueous Extract (Azadirachta indica A. Juss) on the Vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) Ticks

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

Inhibitory activity of compounds isolated from Polymnia sonchifolia on aflatoxin production by Aspergillus flavus

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

Follicle profile and plasma gonadotropin concentration in pubertal female ponies

Twelve female ponies were examined daily for 30 days and classified as ovulating (OV; N = 6; 197 ± 6 kg) or prepubertal (PP; N = 6; 196 ± 9 kg). Follicles were detected by ultrasound and gonadotropins quantified by radioimmunoassay. The mean diameter of the largest follicles was significantly larger in OV (38 ± 1 mm) than in PP (26 ± 2 mm) but there was no difference between groups in the size of the second largest follicle. There were more small follicles (<24 mm) in the PP than in the OV group, but PP fillies had a smaller number of follicles >29 mm than the OV fillies. Follicle-stimulating hormone (FSH) levels did not differ between groups but PP fillies had lower luteinizing hormone (LH) peak (8 ± 1 ng/ml) and basal (4 ± 0.5 ng/ml) levels, lower peak magnitude (2 ± 0.2 ng/ml) and period average (5 ± 0.6 ng/ml) than OV fillies (32 ± 4.5, 8 ± 1.2, 17.1 ± 6, and 15 ± 2.3 ng/ml, respectively). The PP group, in contrast to the OV group, showed no relationship between FSH surge and follicle wave emergence. We conclude that an LH concentration higher than 8 ng/ml is needed for follicle growth to a preovulatory size. Wave emergence and FSH secretion seem to be independent events, probably due to an inhibitory neural system in these PP animals. PP fillies may provide a physiological model for the study of follicle wave emergence which apparently does not depend on gonadotropin levels.

Inhibitory effects of jackbean (Canavalia ensiformis L.) leaf residues on germination and vigour of crops and weeds

The effects of jackbean leaf residues incorporated in the soil on germination and seedlings growth of cucumber, radish and some weeds was examined. Trials were carried out under greenhouse conditions to (a) determine the amount of incorporated residue that is inhibitory to two test plants, (b) to determine if decomposition time changes the inhibitory levels of jackbean residues on test plants and (c) to determine the amount of residue that is inhibitory to the weed species. Jackbean leaf residues incorporated in soil at concentration of 2% or higher and allowed to decompose for a period of 0 to 2 weeks before sowing, reduced the initial growth of cucumber and radish and at different concentrations, reduced germination and growth of three weed species. These results suggest the presence of allelopathic components in Jackbean leaves that could affect seed germination and seedling development.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: Reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Fluoride concentration in public water supply: 72 months of analysis

Known as one of the ten most important advances on Public Health in the 20th century, fluoridation of public water supply is a measure of wide population coverage, which is effective on caries control. The city of Araçatuba, in the Northwest region of the São Paulo state, Brazil, started public water supply fluoridation in 1972 and, based on the average annual highest temperature, has kept the fluoride concentration between 0.6 to 0.8 mgF/L. The purpose of this study was to analyze monthly the fluoride concentration in public water supply in the city of Araçatuba during 72 months. Water samples were collected monthly on weekdays, directly from the water distribution network, on pre-established locations and analyzed in duplicate between November 2004 and October 2010 at the Research Laboratory of the Nucleus for Public Health (NEPESCO) of the Public Health Graduate Program from Araçatuba Dental School/UNESP, Brazil, using an fluoride-specific electrode connected to an ion analyzer. From the total of samples (n=591), 67.2% (n=397) presented fluoride concentration between 0.6 and 0.8 mgF/L; 20.6% (n=122) below 0.6 mgF/L; 11.5% (n=68) between 0.8 and 1.2 mgF/L and 0.7% (n=4) above 1.2 mgF/L. Most samples showed fluoride levels within the recommended parameters. Minimal variation was observed among the analyzed collection locations, showing that the city has been able to control the fluoride levels in the public water supply and reinforcing the importance of surveillance and constant monitoring to assure the quality of the water delivered to the population.

Sugarcane bagasse ozonolysis pretreatment: Effect on enzymatic digestibility and inhibitory compound formation

Sugarcane bagasse was pretreated with ozone to increase lignocellulosic material digestibility. Bagasse was ozonated in a fixed bed reactor at room temperature, and the effect of the two major parameters, ozone concentration and sample moisture, was studied. Acid insoluble and total lignin decreased whereas acid soluble lignin increased in all experiments. Pretreatment barely attacked carbohydrates, with cellulose and xylan recovery rates being >92%. Ozonolysis increased fermentable carbohydrate release considerably during enzymatic hydrolysis. Glucose and xylose yields increased from 6.64% and 2.05%, for raw bagasse, to 41.79% and 52.44% under the best experimental conditions. Only xylitol, lactic, formic and acetic acid degradation compounds were found, with neither furfural nor HMF (5-hydroxymethylfurfural) being detected. Washing detoxification provided inhibitor removal percentages above 85%, increasing glucose hydrolysis, but decreasing xylose yield by xylan solubilization. SEM analysis showed structural changes after ozonization and washing. © 2013 Elsevier Ltd.

Inhibitory mechanism of the nucleus of the solitary tract involved in the control of cardiovascular, dipsogenic, hormonal, and renal responses to hyperosmolality

The nucleus of the solitary tract (NTS) is the primary site of visceral afferents to the central nervous system. In the present study, we investigated the effects of lesions in the commissural portion of the NTS (commNTS) on the activity of vasopressinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, plasma vasopressin, arterial pressure, water intake, and sodium excretion in rats with plasma hyperosmolality produced by intragastric 2 M NaCl (2 ml/rat). Male Holtzman rats with 15-20 days of sham or electrolytic lesion (1 mA; 10 s) of the commNTS were used. CommNTS lesions enhanced a 2 M NaCl intragastrically induced increase in the number of vasopressinergic neurons expressing c-Fos in the PVN (28 ± 1, vs. sham: 22 ± 2 c-Fos/AVP cells) and SON (26 ± 4, vs. sham: 11 ± 1 c-Fos/AVP cells), plasma vasopressin levels (21 ± 8, vs. sham: 6.6 ± 1.3 pg/ml), pressor responses (25 ± 7 mmHg, vs. sham: 7 ± 2 mmHg), water intake (17.5 ± 0.8, vs. sham: 11.2 ± 1.8 ml/2 h), and natriuresis (4.9 ± 0.8, vs. sham: 1.4 ± 0.3 meq/1 h). The pretreatment with vasopressin antagonist abolished the pressor response to intragastric 2 M NaCl in commNTS-lesioned rats (8 ± 2.4 mmHg at 10 min), suggesting that this response is dependent on vasopressin secretion. The results suggest that inhibitory mechanisms dependent on commNTS act to limit or counterbalance behavioral, hormonal, cardiovascular, and renal responses to an acute increase in plasma osmolality. © 2013 the American Physiological Society.

Virulência de linhagens de Rhodococcus equi isoladas de linfonodo de suínos e javalis (Sus scrofa) de abatedouros

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

Atividade antimicrobiana da carbodiimida (EDC) sobre microorganismos presentes em lesões cariosas

Pós-graduação em Ciências Odontológicas - FOAR

Antimicrobial Properties and Cytotoxicity of an Antimicrobial Monomer for Application in Prosthodontics

Purpose: This study aimed to investigate the antimicrobial properties and cytotoxicity of the monomer methacryloyloxyundecylpyridinium bromide (MUPB), an antiseptic agent capable of copolymerizing with denture base acrylic resins. Materials and Methods: The antimicrobial activity of MUPB was tested against the species Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus, and Streptococcus mutans. The minimum inhibitory and fungicidal/bactericidal concentrations (MIC, MFC/MBC) of MUPB were determined by serial dilutions in comparison with cetylpyridinium chloride (CPC). The cytotoxic effects of MUPB at concentrations ranging from 0.01 to 1 g/L were assessed by MTT test on L929 cells and compared with methyl methacrylate (MMA). The antimicrobial activity of copolymerized MUPB was tested by means of acrylic resin specimens containing three concentrations of the monomer (0, 0.3, 0.6% w/w). Activity was quantified by means of a disc diffusion test and a quantification of adhered planktonic cells. Statistical analysis employed the Mann-Whitney test for MIC and MFC/MBC, and ANOVA for the microbial adherence test (a= 0.05). Results: MUBP presented lower MIC values when compared with CPC, although differences were significant for C. dubliniensis and S. mutans only (p= 0.046 and 0.043, respectively). MFC/MBC values were similar for all species except C. albicans; in that case, MUPB presented significantly higher values (p= 0.046). MUPB presented higher cytotoxicity than MMA for all tested concentrations (p < 0.001) except at 0.01 g/L. Irrespective of the concentration incorporated and species, there was no inhibition halo around the specimens. The incorporation of MUPB influenced the adhesion of C. albicans only (p= 0.003), with lower CFU counts for the 0.6% group. Conclusions: It was concluded that non-polymerized MUPB has an antimicrobial capacity close to that of CPC and high cytotoxicity when compared with MMA. The antimicrobial activity of MUPB after incorporation within a denture base acrylic resin did not depend on its elution, but was shown to be restricted to C. albicans.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Transport of nanoparticles into polymersomes : a minimal model system of particles passage through biological membranes

This thesis focuses on the design and characterization of a novel, artificial minimal model membrane system with chosen physical parameters to mimic a nanoparticle uptake process driven exclusively by adhesion and softness of the bilayer. The realization is based on polymersomes composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMDS-b-PMOXA) and nanoscopic colloidal particles (polystyrene, silica), and the utilization of powerful characterization techniques. rnPDMS-b-PMOXA polymersomes with a radius, Rh ~100 nm, a size polydispersity, PD = 1.1 and a membrane thickness, h = 16 nm, were prepared using the film rehydratation method. Due to the suitable mechanical properties (Young’s modulus of ~17 MPa and a bending modulus of ~7⋅10-8 J) along with the long-term stability and the modifiability, these kind of polymersomes can be used as model membranes to study physical and physicochemical aspects of transmembrane transport of nanoparticles. A combination of photon (PCS) and fluorescence (FCS) correlation spectroscopies optimizes species selectivity, necessary for a unique internalization study encompassing two main efforts. rnFor the proof of concepts, the first effort focused on the interaction of nanoparticles (Rh NP SiO2 = 14 nm, Rh NP PS = 16 nm; cNP = 0.1 gL-1) and polymersomes (Rh P = 112 nm; cP = 0.045 gL-1) with fixed size and concentration. Identification of a modified form factor of the polymersome entities, selectively seen in the PCS experiment, enabled a precise monitor and quantitative description of the incorporation process. Combining PCS and FCS led to the estimation of the incorporated particles per polymersome (about 8 in the examined system) and the development of an appropriate methodology for the kinetics and dynamics of the internalization process. rnThe second effort aimed at the establishment of the necessary phenomenology to facilitate comparison with theories. The size and concentration of the nanoparticles were chosen as the most important system variables (Rh NP = 14 - 57 nm; cNP = 0.05 - 0.2 gL-1). It was revealed that the incorporation process could be controlled to a significant extent by changing the nanoparticles size and concentration. Average number of 7 up to 11 NPs with Rh NP = 14 nm and 3 up to 6 NPs with Rh NP = 25 nm can be internalized into the present polymersomes by changing initial nanoparticles concentration in the range 0.1- 0.2 gL-1. Rapid internalization of the particles by polymersomes is observed only above a critical threshold particles concentration, dependent on the nanoparticle size. rnWith regard possible pathways for the particle uptake, cryogenic transmission electron microscopy (cryo-TEM) has revealed two different incorporation mechanisms depending on the size of the involved nanoparticles: cooperative incorporation of nanoparticles groups or single nanoparticles incorporation. Conditions for nanoparticle uptake and controlled filling of polymersomes were presented. rnIn the framework of this thesis, the experimental observation of transmembrane transport of spherical PS and SiO2 NPs into polymersomes via an internalization process was reported and examined quantitatively for the first time. rnIn a summary the work performed in frames of this thesis might have significant impact on cell model systems’ development and thus improved understanding of transmembrane transport processes. The present experimental findings help create the missing phenomenology necessary for a detailed understanding of a phenomenon with great relevance in transmembrane transport. The fact that transmembrane transport of nanoparticles can be performed by artificial model system without any additional stimuli has a fundamental impact on the understanding, not only of the nanoparticle invagination process but also of the interaction of nanoparticles with biological as well as polymeric membranes. rn

Effect of Acute Psychological Stress on Prefrontal GABA Concentration Determined by Proton Magnetic Resonance Spectroscopy

Objective Impaired function of the central gamma-aminobutyric acid (GABA) system, which provides the brain’s major inhibitory pathways, is thought to play an important role in the pathophysiology of anxiety disorders. The effect of acute psychological stress on the human GABA-ergic system is still unknown, however. The purpose of this study was to determine the effect of acute stress on prefrontal GABA levels. Method A recently developed noninvasive magnetic resonance spectroscopy method was used to measure changes in the GABA concentration of the prefrontal cortex in 10 healthy human subjects during a threat-of-shock condition and during a safe condition (two sessions on different days). The main outcome measure was the mean GABA concentration within a 3×3×2-cm3 voxel selected from the medial prefrontal cortex. Results Prefrontal GABA decreased by approximately 18% in the threat-of-shock condition relative to the safe condition. This reduction was specific to GABA, since the concentrations of N-acetyl-aspartate, choline-containing compounds, and glutamate/glutamine levels obtained in the same spectra did not change significantly. Conclusions This result appeared compatible with evidence from preclinical studies in rodents, which showed rapid presynaptic down-regulation of GABA-ergic neurotransmission in response to acute psychological stress. The molecular mechanism and functional significance of this reduced inhibitory effect of acute psychological stress in relation to impaired GABA-ergic function in anxiety disorders merit further investigation.

Relationship between antibiotic concentration in bone and efficacy of treatment of staphylococcal osteomyelitis in rats: azithromycin compared with clindamycin and rifampin

We examined the effect of azithromycin (CP-62,993), a new oral macrolide-like antibiotic, alone and in combination with rifampin, as treatment for experimental staphylococcal osteomyelitis. Clindamycin was used as a comparison drug. Rats (n = 10 to 15 per group) were infected by direct instillation of Staphylococcus aureus into the tibial medullary cavity. After 10 days, 21-day treatments with azithromycin (50 mg/kg of body weight, once daily, by the oral route), rifampin (20 mg/kg, once daily, subcutaneously), or clindamycin (90 mg/kg, three times daily, by the oral route) were started. The drugs were used singly or in combination (azithromycin plus rifampin or clindamycin plus rifampin). Peak azithromycin concentrations in bone were > 30 times higher than levels in serum, but the drug had little effect on final bacterial titers (5.13 +/- 0.46 log10 CFU/g of bone; for controls, 6.54 +/- 0.28 log10 CFU/g). Clindamycin was more active than azithromycin (3.26 +/- 2.14 log10 CFU/g of bone; 20% of sterilized bones), but rifampin was the most active single drug (1.5 +/- 1.92 log10 CFU/g; 53% of sterilized bones). Therapy with rifampin or clindamycin alone was associated with the emergence of resistance. Rifampin plus azithromycin (0.51 +/- 1.08 log10 CFU/g of bone; 80% of sterilized bones) and rifampin plus clindamycin (0.87 +/- 1.34 log10 CFU/g of bone; 66% of sterilized bones) were the most active regimens. Thus, azithromycin is ineffective as a single drug for the treatment of experimental staphylococcal osteomyelitis, despite high levels in bone that markedly exceeded the MIC, but it may be an attractive partner drug for rifampin.