Utilizzo di composti d'aroma per aumentare la sicurezza igienico-sanitaria di germogli di ravanello

Questo elaborato ha come obiettivo valutare l’efficacia di alcuni composti d’aroma, ossia componenti di oli essenziali la cui attività antimicrobica è nota in letteratura, nell’aumentare la sicurezza igienico-sanitaria di germogli di ravanello Daikon. In particolare sono stati utilizzati timolo e carvacrolo, costituenti di oli di piante tra cui timo e origano. Vista la grande espansione nel mercato alimentare di semi germogliati pronti all’uso, matrice veicolante numerosi casi di tossinfezione alimentare, sono state effettuate prove per valutare il rischio di sviluppo di Listeria. Si è lavorato simulando un processo casalingo, facendo germinare i semi in acqua addizionata o meno di diverse concentrazioni dei due terpeni fenolici. I semi utilizzati venivano precedentemente inoculati con Listeria innocua, utilizzata come surrogato del patogeno Listeria monocytogenes. Il ricambio dell’acqua di ammollo avveniva ogni 24 ore, e dopo 6/24/32/48 ore venivano prelevate quantità note di semi e acqua, al fine di valutarne il contenuto in L. innocua, carica microbica totale ed enterobatteri. La prima prova, che ha previsto l’uso di timolo come unico composto antimicrobico, ha mostrato una riduzione di un ciclo logaritmico di L. innocua ed enterobatteri nei semi, mentre nell’acqua essi erano presenti solo dopo 24 e 48 ore. Risultati analoghi sono stati ottenuti utilizzando solo carvacrolo. Infine è stato testato l’eventuale effetto sinergico di timolo e carvacrolo, che però non ha dato differenze particolari rispetto a campioni trattati coi singoli composti. I risultati ottenuti hanno evidenziato l’elevato rischio microbiologico associato ai semi germogliati con produzione domestica, poiché le condizioni adottate favoriscono lo sviluppo di specie patogene. L’utilizzo dei composti di aroma, se pure in grado di ridurre sia le cinetiche di crescita che la quantità di Listeria, non è tuttavia in grado di controllare in maniera significativa il rischio associato a tali prodotti.

Caractérisation fonctionnelle de facteurs de virulence chez "Streptococcus suis"

Les progrès technologiques dans l’industrie de la viande ont des répercussions considérables sur les agents pathogènes de ces environnements. Parmi ceux-ci, Streptococcus suis occupe une place prédominante dans l’industrie porcine. En effet, S. suis, colonisateur naturel des voies respiratoires et digestives du porc, peut infecter son hôte en provoquant des méningites, septicémies, endocardites, arthrites ou pneumonies. De surcroît, S. suis peut également infecter l’humain en provoquant majoritairement des méningites et septicémies, et a notamment été la cause de deux épidémies en Chine en 1998 et 2005. La pathogenèse des infections à S. suis demeure partiellement connue à l’heure actuelle, rendant difficile le contrôle des infections. Il est par conséquent essentiel de caractériser les facteurs de virulence chez S. suis puisqu’ils pourraient représenter des cibles d’intérêt pour des applications préventives ou thérapeutiques. Ce projet de doctorat consiste donc en la caractérisation fonctionnelle de facteurs de virulence chez S. suis. Dans un premier temps, la capacité de S. suis à moduler son potentiel pro-inflammatoire en présence de concentrations sous-inhibitrices d’amoxicilline a été mise en évidence. Dans un second temps, la caractérisation plus avancée de la hyaluronate lyase de S. suis a permis de démontrer que son activité ne contribue pas à la virulence de la bactérie étant donné son absence au sein de souches les plus virulentes, mais que les interactions avec l’acide hyaluronique pourraient moduler la virulence de S. suis. Par la suite, l’étude fonctionnelle d’une DNase de S. suis a permis de démontrer son implication comme facteur de virulence et suggère son intérêt dans le développement de vaccins. Finalement, le dernier objectif du projet a permis la mise en évidence de la production de microvésicules fortement immunogéniques par S. suis. La présence de facteurs de virulence dans leur contenu protéique représente un élément encourageant dans le développement de vaccins contre l’agent pathogène. Ce projet a donc permis d’élargir les connaissances sur le potentiel néfaste de l’utilisation des antibiotiques à faible concentration dans l’industrie porcine, sur le rôle des activités hyaluronate lyase et DNase dans la virulence de S. suis, et de découvrir un nouveau mécanisme impliqué dans la virulence de la bactérie par le biais des microvésicules.

Epidemias bacterianas emergentes do século XXI

As doenças infeciosas distantes de serem um problema do passado têm aumentado drasticamente nestes últimos anos, causando epidemias emergentes, quer de origem bacteriana ou vírica ou de outros tipos de microrganismos. Esta dissertação tem como objetivo uma pesquisa atual bibliográfica sobre o estudo de algumas epidemias bacterianas emergentes do século XXI, como a Tuberculose, Cólera, Staphylococcus aureus resistente à meticilina (MRSA) e Meningite Meningocócica, bem como os seus dados epidemiológicos. A Tuberculose é uma das doenças mais antigas, que apresenta uma elevada taxa de mortalidade e com o passar do tempo tem vindo a aumentar a nível mundial. A TB é causada por uma bactéria denominada Mycobacterium tuberculosis que normalmente afeta os pulmões e outros órgãos. O tratamento, a prevenção e o diagnóstico precoce são pontos essenciais, para ter um bom desfecho para o doente. A Cólera tem-se propagado pelo mundo desde o século XX. Esta doença caracteriza-se por uma diarreia aguda grave que é causada pela bactéria Vibrio cholerae. O seu tratamento se for realizado precocemente é tratado facilmente, com apenas hidratação com sais orais. A prevenção é uma medida essencial para ter um bom prognóstico, e evitar surtos emergentes desta infeção. Devido à sua virulência, Staphylococcus aureus é responsável por infeções graves adquiridas em hospital e na comunidade. Na maioria das vezes esta infeção é assintomática, mas pode causar infeções graves até mesmo fatais. Devido às resistências aos antibióticos β-lactâmicos e de outros tipos de antibióticos, e também devido ao aumento do número crescente de quadros infeciosos de MRSA, houve necessidade de novos antibióticos como o linezolide, as cefasloporinas de 5ª geração no combate a estas infeções. As medidas de prevenção são essenciais, visto que se não forem realizadas pode haver progressão da doença. Além de um estudo científico constante dos mecanismos de resistências desta bactéria, ser essencial. A meningite bacteriana é um grave problema de Saúde Pública devido à alta incidência em crianças. A meningite meningocócica é causada pela bactéria Neisseria meningitidis que origina um processo inflamatório das meninges. Há algum tempo atrás a mortalidade era elevada, mas com o advento da antibioterapia reduziu significativamente. As vacinas fizeram com que ocorresse uma mudança bastante significativa na epidemiologia desta patologia, e mais uma vez a prevenção é essencial.

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

Microcantilever sensors for biochemical detection and analysis

Biochemical agents, including bacteria and toxins, are potentially dangerous and responsible for a wide variety of diseases. Reliable detection and characterization of small samples is necessary in order to reduce and eliminate their harmful consequences. Microcantilever sensors offer a potential alternative to the state of the art due to their small size, fast response time, and the ability to operate in air and liquid environments. At present, there are several technology limitations that inhibit application of microcantilever to biochemical detection and analysis, including difficulties in conducting temperature-sensitive experiments, material inadequacy resulting in insufficient cell capture, and poor selectivity of multiple analytes. This work aims to address several of these issues by introducing microcantilevers having integrated thermal functionality and by introducing nanocrystalline diamond as new material for microcantilevers. Microcantilevers are designed, fabricated, characterized, and used for capture and detection of cells and bacteria. The first microcantilever type described in this work is a silicon cantilever having highly uniform in-plane temperature distribution. The goal is to have 100 μm square uniformly heated area that can be used for thermal characterization of films as well as to conduct chemical reactions with small amounts of material. Fabricated cantilevers can reach above 300C while maintaining temperature uniformity of 2−4%. This is an improvement of over one order of magnitude over currently available cantilevers. The second microcantilever type is a doped single crystal silicon cantilever having a thin coating of ultrananocrystalline diamond (UNCD). The primary application of such a device is in biological testing, where diamond acts as a stable, electrically isolated reaction surface while silicon layer provides controlled heating with minimum variations in temperature. This work shows that composite cantilevers of this kind are an effective platform for temperature-sensitive biological experiments, such as heat lysing and polymerase chain reaction. The rapid heat-transfer of Si-UNCD cantilever compromised the membrane of NIH 3T3 fibroblast and lysed the cell nucleus within 30 seconds. Bacteria cells, Listeria monocytogenes V7, were shown to be captured with biotinylated heat-shock protein on UNCD surface and 90% of all viable cells exhibit membrane porosity due to high heat in 15 seconds. Lastly, a sensor made solely from UNCD diamond is fabricated with the intention of being used to detect the presence of biological species by means of an integrated piezoresistor or through frequency change monitoring. Since UNCD diamond has not been previously used in piezoresistive applications, temperature-denpendent piezoresistive coefficients and gage factors are determined first. The doped UNCD exhibits a significant piezoresistive effect with gauge factor of 7.53±0.32 and a piezoresistive coefficient of 8.12×10^−12 Pa^−1 at room temperature. The piezoresistive properties of UNCD are constant over the temperature range of 25−200C. 300 μm long cantilevers have the highest sensitivity of 0.186 m-Ohm/Ohm per μm of cantilever end deflection, which is approximately half that of similarly sized silicon cantilevers. UNCD cantilever arrays were fabricated consisting of four sixteen-cantilever arrays of length 20–90 μm in addition to an eight-cantilever array of length 120 μm. Laser doppler vibrometry (LDV) measured the cantilever resonant frequency, which ranged as 218 kHz−5.14 MHz in air and 73 kHz−3.68 MHz in water. The quality factor of the cantilever was 47−151 in air and 18−45 in water. The ability to measure frequencies of the cantilever arrays opens the possibility for detection of individual bacteria by monitoring frequency shift after cell capture.

Influência das etapas do processo de abate de suínos na prevalência de patógenos e níveis de microrganismos indicadores de qualidade e higiene

The pig slaughter process involve different steps that can influence the microbiological quality of carcasses. At this, the understanding of the slaughter process on the microbiological aspects is necessary for the implementation and evaluation of critical control points. The microbiological control of the slaughter process should involve the evaluation of pathogens prevalence and levels of quality and hygiene indicator microorganisms. This study aimed at investigating the influence of steps slaughter process on the microbiological levels of pig carcasses, and evaluate if there is correlation between pathogens (Salmonella spp. and Listeria monocytogenes) and indicators (aerobic mesophilic counts, total coliforms, Escherichia coli and Enterobacteriaceae) microorganisms. A high Salmonella soroprevalence in pigs were founded before the slaughter (57.49 %). While the Salmonella prevalence in carcasses at the initial stage of the slaughter was 26.67 % and in the final stage 1.11 %, L. monocytogenes was detected only in the final washing and cooling steps, with a prevalence of 21.11 and 8.89 %, respectively. The aerobic mesophilic counts, Enterobacteriaceae, total coliforms and E. coli levels in initial steps of slaughter process were 4.25 ± 0.37; 1.25 ± 0.38; 1.10 ± 0.35 and 0.86 ± 0.36, respectively. At the end of slaughter process the results were lower (ranging from 0.16 at 2.70 log CFU/cm2). The step that most reduced microbiological levels was the scalding. The dehairing was a critical step that led to a significant increase of microorganisms levels in the process (p < 0.05). The evisceration not proved to be a critical step on the increase of microbial levels, differently of the final washing, which showed significant increases (p < 0.05) over the levels of aerobic counts, total coliforms, E. coli and enterobacterias (0.30; 0.36; 0.27 and 0.42 log respectively) and Salmonella spp. and L. monocytogenes. The chilling contributes significantly to the reduction of microbiological levels of carcasses, bringing them to levels below the all process stages, with the exception of scalding. No correlation between the hygiene indicator microorganisms used and presence of Salmonella spp. and L. monocytogenes were obtained (p < 0.05). The results show that steps in the process are critical to the sanitary profile, which implies the need to implement actions in the process to reducing the microbiological levels.

Development and Application of Predictive Models for Survival, Growth, and Death of Enteric Pathogens in Leafy Greens Supply Chain

Leafy greens are essential part of a healthy diet. Because of their health benefits, production and consumption of leafy greens has increased considerably in the U.S. in the last few decades. However, leafy greens are also associated with a large number of foodborne disease outbreaks in the last few years. The overall goal of this dissertation was to use the current knowledge of predictive models and available data to understand the growth, survival, and death of enteric pathogens in leafy greens at pre- and post-harvest levels. Temperature plays a major role in the growth and death of bacteria in foods. A growth-death model was developed for Salmonella and Listeria monocytogenes in leafy greens for varying temperature conditions typically encountered during supply chain. The developed growth-death models were validated using experimental dynamic time-temperature profiles available in the literature. Furthermore, these growth-death models for Salmonella and Listeria monocytogenes and a similar model for E. coli O157:H7 were used to predict the growth of these pathogens in leafy greens during transportation without temperature control. Refrigeration of leafy greens meets the purposes of increasing their shelf-life and mitigating the bacterial growth, but at the same time, storage of foods at lower temperature increases the storage cost. Nonlinear programming was used to optimize the storage temperature of leafy greens during supply chain while minimizing the storage cost and maintaining the desired levels of sensory quality and microbial safety. Most of the outbreaks associated with consumption of leafy greens contaminated with E. coli O157:H7 have occurred during July-November in the U.S. A dynamic system model consisting of subsystems and inputs (soil, irrigation, cattle, wildlife, and rainfall) simulating a farm in a major leafy greens producing area in California was developed. The model was simulated incorporating the events of planting, irrigation, harvesting, ground preparation for the new crop, contamination of soil and plants, and survival of E. coli O157:H7. The predictions of this system model are in agreement with the seasonality of outbreaks. This dissertation utilized the growth, survival, and death models of enteric pathogens in leafy greens during production and supply chain.

Avaliação da atividade antimicrobiana de filmes protéicos a base de anchoita (engraulis anchoita) incorporados com ácidos orgânicos.

Os filmes são produzidos a partir de macromoléculas, que podem ser utilizados como embalagem, como os polissacarídeos, lipídeos e proteínas. As proteínas se destacam dos demais, pois possuem uma estrutura com 20 monômeros diferentes, que confere um amplo potencial de ligações intermoleculares. A incorporação de agentes ativos em filmes é uma alternativa como embalagem, para inibir ou retardar a multiplicação de microrganismos patógenos e deteriorantes em alimentos. O objetivo deste trabalho foi avaliar a atividade antimicrobiana de filmes à base de isolado protéico de anchoita (Engraulis anchoita) – IPA adicionados de ácidos orgânicos. Para tanto, foi elaborado o IPA, pela solubilização alcalina da proteína e precipitação no ponto isoelétrico a partir de carne mecanicamente separada. O IPA foi avaliado quanto a sua composição proximal, aminoacídica e por DSC. A solução formadora dos filmes foi elaborada a partir de IPA, água, glicerol e hidróxido de sódio. As formulações dos filmes foram elaboradas segundo um planejamento fatorial 23 . Foram avaliadas as propriedades físico-químicas de resistência a tração (RT) e elongação (E); espessura, solubilidade e permeabilidade ao vapor de água (PVA); a diferença de cor (∆E*) e opacidade (Y) e microscopia eletrônica de varredura (MEV) de filmes à base de IPA. Os filmes com diferentes concentrações de ácido sórbico (AS) ou ácido benzóico (AB) foram desenvolvidos a partir da condição cujo as propriedades físico-químicas foram as melhores, sendo comparados aos filmes controles. Estes, foram avaliados quanto a sua atividade antimicrobiana frente aos microrganismos Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus e Salmonella Enteritidis pelo método de difusão em disco, além das propriedades físico-químicas, MEV e FT-IV. Os filmes com maior atividade antimicrobiana e os filmes controle foram aplicados sobre carne bovina, inoculados com os microrganismos inibidos no método de difusão em disco e armazenados a 5°C. Estes, foram avaliados a cada 2 dias durante 12 dias de armazenamento, pela método de contagem em gotas. O IPA apresentou 88,8% de proteína e 53,3% de aminoácidos polares e temperatura de desnaturação de 62,2°C. A espessura, PVA, ∆E* e Y dos filmes não foram afetados pelas variáveis estudadas no experimento. A menor solubilidade e maior RT dos filmes ocorreram em baixa concentração de IPA, glicerol e tratamento térmico, mas a E aumentou com o acréscimo dessas variáveis. As MEV das superfícies dos filmes foram homogêneas, para aqueles com leve tratamento térmico. O aumento da concentração de AS e AB na faixa de 0,50 a 1,50% resultou na diminuição da RT e aumento da E, solubilidade, ∆E* e Y. Houve mudança da organização molecular e interações intermoleculares entre as moléculas de IPA e AB testados pela avaliação do FT-IV. As MEV revelaram microporos em filmes com 1,50% de AS, o que resultou em filmes com menor homogeneidade. A maior atividade antimicrobiana foi verificada nos filmes com 1,50% de AS e AB frente a E. coli O157:H7, L. monocytogenes e S. Enteritidis. Estes filmes foram aplicados sobre carne bovina inoculada com E. coli O157:H7 e L. monocytogenes. Os filmes de AS frente a E. coli O157:H7 e L. monocytogenes apresentaram uma redução de 5 e 4 log UFC.g-1, respectivamente, em relação ao filme controle. O efeito do AB frente a estas bactérias, apresentou uma redução de 6 e 5 log UFC.g-1, ao final do 12° dia de armazenamento, respectivamente. Os filmes elaborados à base de IPA, adicionados de AS ou AB podem ser eficazes contra os patógenos alimentares testados.

Valor diagnóstico y pronóstico de la medición de la presión intraabdominal transvesical en pacientes con abdomen agudo en el H.V.C.M. Cuenca febrero a septiembre del 2000

A 60 pacientes ingresados en emergencia y cirugía se realizó la medición de la presión intraabdominal [P.I.A.] por vía vesical a través de una sonda Foley. Clasificados en dos grupos de estudio: 30 del grupo de control; sin patología abdominal: 22 hombres y 8 mujeres con un promedio de edad 49 años, diagnosticados de; T.E.C., trauma raquimedular, meningitis, absceso cerebral y mal formación arteriovenoso, que necesitaban de sonda vesical. Obteníendose una media normal o valor referencial de normalidad de 2.7 cm. H20 o 1.98 mmHg. El segundo grupo experimental: 30 pacientes diagnosticados de abdomen agudo quirúrgico: 21 hombres y 9 mujeres, con un promedio de edad de 46 años, clasificados en 5 tipos de abdomen agudo: AAOI, AAT,AAP AAI. AAOV. se les mediÓ la presión intraabdominal transvesical, obteniéndose los siguientes resultados. P.I.A.: Prequirúrgico medio: 19.29 cm H20 [14.18 mmHg.]. P.IA.: Posquirúrgico media: 8.97 cm H20 [6.59 mmHg.]. Todos sometidos a laparotomía exploratoria. Mejorando 28 pacientes, uno empeoró y solicitÓ el alta, un paciente falleció. En el grupo de control los 30 pacientes presentaron la presiÓn intraabdominal en valores normales

Ultra high pressure homogenization (UHPH) inactivation of Bacillus amyloliquefaciens spores in phosphate buffered saline (PBS) and milk

Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7), and whole milk (3.5%, pH 6.7) at initial concentration of similar to 10(6) CFU/mL were subjected to UHPH treatments at 200, 300, and 350 MPa with an inlet temperature at similar to 80 degrees C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using first-order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125 degrees C caused no reduction of spores. A reduction of 3.5 log(10)CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150 degrees C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation.

Revisiting molecular diagnostics of Streptococcus pneumoniae

Streptococcus pneumoniae is a human pathobiont that colonizes the nasopharynx. S. pneumoniae is responsible for causing non-invasive and invasive disease such as otitis, pneumonia, meningitis, and sepsis, being a leading cause of infectious diseases worldwide. Due to similarities with closely related species sharing the same niche, it may be a challenge to correctly distinguish S. pneumoniae from its relatives when using only non-culture based methods such as real time PCR (qPCR). In 2007, a molecular method targeting the major autolysin (lytA) of S. pneumoniae by a qPCR assay was proposed by Carvalho and collaborators to identify pneumococcus. Since then, this method has been widely used worldwide. In 2013, the gene encoding for the ABC iron transporter lipoprotein PiaA, was proposed by Trzcinzki and collaborators to be used in parallel with the lytA qPCR assay. However, the presence of lytA gene homologues has been described in closely related species such as S. pseudopneumoniae and S. mitis and the presence of piaA gene is not ubiquitous between S. pneumoniae. The hyaluronate lyase gene (hylA) has been described to be ubiquitous in S. pneumoniae. This gene has not been used so far as a target for the identification of S. pneumoniae. The aims of our study were to evaluate the specificity, sensitivity, positive predicted value (PPV) and negative predicted value (NPV) of the lytA and piaA qPCR methods; design and implement a new assay targeting the hylA gene and evaluate the same parameters above described; analyze the assays independently and the possible combinations to access what is the best approach using qPCR to identify S. pneumoniae. A total of 278 previously characterized strains were tested: 61 S. pseudopneumoniae, 37 Viridans group strains, 30 type strains from other streptococcal species and 150 S. pneumoniae strains. The collection included both carriage and disease isolates. By Mulilocus Sequence Analysis (MLSA) we confirmed that strains of S. pseudopneumoniae could be misidentified as S. pneumoniae when lytA qPCR assay is used. The results showed that as a single target, lytA had the best combination of specificity, sensitivity, PPV and NPV being, 98.5%, 100.0%, 98.7% and 100.0% respectively. The combination of targets with the best values of specificity, sensibility, PPV and NPV were lytA and piaA, with 100.0%, 93.3%, 97.9% and 92.6%, respectively. Nonetheless by MLSA we confirmed that strains of S. pseudopneumoniae could be misidentified as S. pneumoniae and some capsulated (23F, 6B and 11A) and non-capsulated S. pneumoniae were not Identified using this assay. The hylA gene as a single target had the lowest PPV. Nonetheless it was capable to correctly identify all S. pneumoniae.

Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Frecuencia del síndrome meningeo: estudio descriptivo comparativo en el departamento de pediatría del Hospital Vicente Corral Moscoso de Cuenca y en el departamento de pediatría del Hospital Homero Castanier de Azogues entre Abril de 1977-Abril 1982

Doctor en Medicina y Cirugía

Optimization of growth and bacteriocin activity of the food bioprotective Carnobacterium divergens V41 in an animal origin protein free medium

Optimization of Carnobacterium divergens V41 growth and bacteriocin activity in a culture medium deprived of animal protein, needs for food bioprotection, was performed by using a statistical approach. In a screening experiment, twelve factors (pH, temperature, carbohydrates, NaCl, yeast extract, soy peptone, sodium acetate, ammonium citrate, magnesium sulphate, manganese sulphate, ascorbic acid and thiamine) were tested for their influence on the maximal growth and bacteriocin activity using a two-level incomplete factorial design with 192 experiments performed in microtiter plate wells. Based on results, a basic medium was developed and three variables (pH, temperature and carbohydrates concentration) were selected for a scale-up study in bioreactor. A 23 complete factorial design was performed, allowing the estimation of linear effects of factors and all the first order interactions. The best conditions for the cell production were obtained with a temperature of 15°C and a carbohydrates concentration of 20 g/l whatever the pH (in the range 6.5-8), and the best conditions for bacteriocin activity were obtained at 15°C and pH 6.5 whatever the carbohydrates concentration (in the range 2-20 g/l). The predicted final count of C. divergens V41 and the bacteriocin activity under the optimized conditions (15°C, pH 6.5, 20 g/l carbohydrates) were 2.4 x 1010 CFU/ml and 819200 AU/ml respectively. C. divergens V41 cells cultivated in the optimized conditions were able to grow in cold-smoked salmon and totally inhibited the growth of Listeria monocytogenes (< 50 CFU g-1) during five weeks of vacuum storage at 4° and 8°C.