Glial cells modulate the synaptic transmission of NTS neurons sending projections to ventral medulla of Wistar rats

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

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.

Screening, immobilization and utilization of whole cell biocatalysts to mediate the ethanolysis of babassu oil

The screening. biomass growth of lipase-producing fungus isolated from different sources and available at URM (University Recife Mycologia). as well as, the immobilization and utilization of the whole cells for the transesterification of babassu oil were investigated. Rhizopus oryzae (URM 3231, 4692), Mucor circinelloides (URM 4140, 4182) and Penicillium citrinum URM 4216 were considered to be good intracellular lipase producers whereas those from Mucor hiemalis URM 4144 and Mucor piriformis URM 4145 were weaker. Fungi biomass containing high lipase activities was immobilized on different biomass support particles (BSPs) and with the exception of Penicillium citrinum URM 4216 all the other fungi strains exhibited high lipase activity (20-50 Ug(-1)) when immobilized in situ using polyurethane foam particles. Transesterification activities of the immobilized whole cells were evaluated in the ethanolysis reaction with babassu oil and the highest performance was attained by M. circinelloides URM 4182 giving 83.22 +/- 3.68% ester yield in less than 96 h reaction. The biocatalyst operational stability was also assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 26 days and 2.6 x 10(-2)day(-1), for half-life and a deactivation coefficient, respectively. The purified product (biodiesel) exhibited viscosity (6.63 cSt) close to the value to attend specifications by the ASTM 06751 to be used as biofuel. Results are favorable compared with data already reported in the literature and demonstrated that M. circinelloides URM 4182 whole cells is a cheaper biocatalyst that can be used in the biodiesel synthesis. (C) 2012 Elsevier B.V. All rights reserved.

Effect of Nintendo Wii (TM)-based motor and cognitive training on activities of daily living in patients with Parkinson's disease: A randomised clinical trial

Objectives To investigate the effect of Nintendo Wii (TM)-based motor cognitive training versus balance exercise therapy on activities of daily living in patients with Parkinson's disease. Design Parallel, prospective, single-blind, randomised clinical trial. Setting Brazilian Parkinson Association. Participants Thirty-two patients with Parkinson's disease (Hoehn and Yahr stages 1 and 2). Interventions Fourteen training sessions consisting of 30 minutes of stretching, strengthening and axial mobility exercises, plus 30 minutes of balance training. The control group performed balance exercises without feedback or cognitive stimulation, and the experimental group performed 10 Wii Fit (TM) games. Main outcome measure Section II of the Unified Parkinson's Disease Rating Scale (UPDRS-II). Randomisation Participants were randomised into a control group (n = 16) and an experimental group (n = 16) through blinded drawing of names. Statistical analysis Repeated-measures analysis of variance (RM-ANOVA). Results Both groups showed improvement in the UPDRS-II with assessment effect (RM-ANOVA P < 0.001, observed power = 0.999). There was no difference between the control group and the experimental group before training {8.9 [standard deviation (SD) 2.9] vs 10.1 (SD 3.8)}, after training [7.6 (SD 2.9) vs 8.1 (SD 3.5)] or 60 days after training [8.1 (SD 3.2) vs 8.3 (SD 3.6)]. The mean difference of the whole group between before training and after training was -0.9 (SD 2.3, 95% confidence interval -1.7 to -0.6). Conclusion Patients with Parkinson's disease showed improved performance in activities of daily living after 14 sessions of balance training, with no additional advantages associated with the Wii-based motor and cognitive training. Registered on http://www.clinicaltrials.gov (identifier: NCT01580787). (C) 2012 Chartered Society of Physiotherapy. Published by Elsevier Ltd. All rights reserved.

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Segmental dataset and whole body expression data do not support the hypothesis that non-random movement is an intrinsic property of Drosophila retrogenes

Background: Several studies in Drosophila have shown excessive movement of retrogenes from the X chromosome to autosomes, and that these genes are frequently expressed in the testis. This phenomenon has led to several hypotheses invoking natural selection as the process driving male-biased genes to the autosomes. Metta and Schlotterer (BMC Evol Biol 2010, 10:114) analyzed a set of retrogenes where the parental gene has been subsequently lost. They assumed that this class of retrogenes replaced the ancestral functions of the parental gene, and reported that these retrogenes, although mostly originating from movement out of the X chromosome, showed female-biased or unbiased expression. These observations led the authors to suggest that selective forces (such as meiotic sex chromosome inactivation and sexual antagonism) were not responsible for the observed pattern of retrogene movement out of the X chromosome. Results: We reanalyzed the dataset published by Metta and Schlotterer and found several issues that led us to a different conclusion. In particular, Metta and Schlotterer used a dataset combined with expression data in which significant sex-biased expression is not detectable. First, the authors used a segmental dataset where the genes selected for analysis were less testis-biased in expression than those that were excluded from the study. Second, sex-biased expression was defined by comparing male and female whole-body data and not the expression of these genes in gonadal tissues. This approach significantly reduces the probability of detecting sex-biased expressed genes, which explains why the vast majority of the genes analyzed (parental and retrogenes) were equally expressed in both males and females. Third, the female-biased expression observed by Metta and Schltterer is mostly found for parental genes located on the X chromosome, which is known to be enriched with genes with female-biased expression. Fourth, using additional gonad expression data, we found that autosomal genes analyzed by Metta and Schlotterer are less up regulated in ovaries and have higher chance to be expressed in meiotic cells of spermatogenesis when compared to X-linked genes. Conclusions: The criteria used to select retrogenes and the sex-biased expression data based on whole adult flies generated a segmental dataset of female-biased and unbiased expressed genes that was unable to detect the higher propensity of autosomal retrogenes to be expressed in males. Thus, there is no support for the authors' view that the movement of new retrogenes, which originated from X-linked parental genes, was not driven by selection. Therefore, selection-based genetic models remain the most parsimonious explanations for the observed chromosomal distribution of retrogenes.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Brossi P.M., Baccarin R.Y.A. & Massoco C.O. 2012 Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro? Pesquisa Veterinaria Brasileira 32(12):1355-1360. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Butanta, Sao Paulo, SP 5508-210, Brazil. E-mail: baccarin@ usp.br Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)(4) - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Abstract Background The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. Methods Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. Results The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. Conclusion This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Biomechanical modelling of human knee during living activities

The knee joint is a key structure of the human locomotor system. The knowledge of how each single anatomical structure of the knee contributes to determine the physiological function of the knee, is of fundamental importance for the development of new prostheses and novel clinical, surgical, and rehabilitative procedures. In this context, a modelling approach is necessary to estimate the biomechanic function of each anatomical structure during daily living activities. The main aim of this study was to obtain a subject-specific model of the knee joint of a selected healthy subject. In particular, 3D models of the cruciate ligaments and of the tibio-femoral articular contact were proposed and developed using accurate bony geometries and kinematics reliably recorded by means of nuclear magnetic resonance and 3D video-fluoroscopy from the selected subject. Regarding the model of the cruciate ligaments, each ligament was modelled with 25 linear-elastic elements paying particular attention to the anatomical twisting of the fibres. The devised model was as subject-specific as possible. The geometrical parameters were directly estimated from the experimental measurements, whereas the only mechanical parameter of the model, the elastic modulus, had to be considered from the literature because of the invasiveness of the needed measurements. Thus, the developed model was employed for simulations of stability tests and during living activities. Physiologically meaningful results were always obtained. Nevertheless, the lack of subject-specific mechanical characterization induced to design and partially develop a novel experimental method to characterize the mechanics of the human cruciate ligaments in living healthy subjects. Moreover, using the same subject-specific data, the tibio-femoral articular interaction was modelled investigating the location of the contact point during the execution of daily motor tasks and the contact area at the full extension with and without the whole body weight of the subject. Two different approaches were implemented and their efficiency was evaluated. Thus, pros and cons of each approach were discussed in order to suggest future improvements of this methodologies. The final results of this study will contribute to produce useful methodologies for the investigation of the in-vivo function and pathology of the knee joint during the execution of daily living activities. Thus, the developed methodologies will be useful tools for the development of new prostheses, tools and procedures both in research field and in diagnostic, surgical and rehabilitative fields.

Innovative treatments for shelf-life extension of whole and fresh-cut fruit

Maintaining the postharvest quality of whole and fresh-cut fruit during storage and distribution is the major challenge facing fruit industry. For this purpose, industry adopt a wide range of technologies to enable extended shelf-life. Many factors can lead to loss of quality in fresh product, hence the common description of these products as ‘perishable’. As a consequence normal factors such as transpiration and respiration lead ultimately to water loss and senescence of the product. Fruits and vegetables are living commodities and their rate of respiration is of key importance to maintenance of quality. It has been commonly observed that the greater the respiration rate of a product, the shorter the shelf-life. The principal problem for fresh-cut fruit industries is the relative shorter shelf-life of minimally processed fruit (MPF) compared to intact product. This fact is strictly connected with the higher ethylene production of fruit tissue stimulated during fresh-cut processing (peeling, cutting, dipping). 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene action and several researches have shown its effectiveness on the inhibition of ripening and senescence incidence for intact fruit and consequently on their shelf-life extension. More recently 1-MCP treatment has been tested also for shelf-life extension of MPF but discordant results have been obtained. Considering that in some countries 1-MCP is already a commercial product registered for the use on a number of horticultural products, the main aim of this actual study was to enhance our understanding on the effects of 1-MCP treatment on the quality maintenance of whole and fresh-cut climacteric and non-climacteric fruit (apple, kiwifruit and pineapple). Concerning the effects of 1-MCP on whole fruit, was investigated the effects of a semi-commercial postharvest treatment with 1-MCP on the quality of Pink Lady apples as functions of fruit ripening stage, 1-MCP dose, storage time and also in combination with controlled atmospheres storage in order to better understand what is the relationship among these parameters and if is possible to maximize the 1-MCP treatment to meet the market/consumer needs and then in order to put in the market excellent fruit. To achieve this purpose an incomplete three-level three-factor design was adopted. During the storage were monitored several quality parameters: firmness, ripening index, ethylene and carbon dioxide production and were also performed a sensory evaluations after 6 month of storage. In this study the higher retention of firmness (at the end of storage) was achieved by applying the greatest 1-MCP concentration to fruits with the lowest maturity stage. This finding means that in these semi-commercial conditions we may considerate completely blocked the fruit softening. 1-MCP was able to delay also the ethylene and CO2 production and the maturity parameters (soluble solids content and total acidity). Only in some cases 1-MCP generate a synergistic effect with the CA storage. The results of sensory analyses indicated that, the 1-MCP treatment did not affect the sweetness and whole fruit flavour while had a little effect on the decreasing cut fruit flavour. On the contrary the treated apple was more sour, crisp, firm and juicy. The effects of some treatment (dipping and MAP) on the nutrient stability were also investigated showing that in this case study the adopted treatments did not have drastic effects on the antioxidant compounds on the contrary the dipping may enhance the total antioxidant activity by the accumulation of ascorbic acid on the apple cut surface. Results concerning the effects of 1-MCP in combination with MAP on the quality parameters behaviour of the kiwifruit were not always consistent and clear: in terms of colour maintenance, it seemed to have a synergistic effect with N2O MAP; as far as ripening index is concerned, 1-MCP had a preservative effect, but just for sample packed in air.

Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Elf magnetic field influence on ION Channels studied by Patch Clamp Technique: exposure set up and "Whole Cell" measurements on Potassium currents

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.