Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2016-10-06T17:42:59Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao revisao FINAL.pdf: 661499 bytes, checksum: d43fc86e349e9c7d614609bf8c904ca4 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-10-11T20:38:40Z (GMT) No. of bitstreams: 2 dissertacao revisao FINAL.pdf: 661499 bytes, checksum: d43fc86e349e9c7d614609bf8c904ca4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-11T20:38:40Z (GMT). No. of bitstreams: 2 dissertacao revisao FINAL.pdf: 661499 bytes, checksum: d43fc86e349e9c7d614609bf8c904ca4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-22 / Sem bolsa / A busca crescente dos consumidores por praticidade destaca-se dentre as principais tendências de consumo de alimentos no mundo. Neste contexto, enquadra-se o Beef Jerky, um produto cárneo produzido através de processo térmico, que não necessita de refrigeração no ponto de venda. Entretanto, por ser um alimento pronto para o consumo, deve-se garantir sua segurança e uma das bactérias patogênicas que, uma vez presente neste alimento, pode causar danos à saúde dos consumidores, é Listeria monocytogenes. O objetivo deste estudo foi avaliar a ocorrência de L. monocytogenes em uma linha de produção de Beef Jerky localizada no município de Bagé, Rio Grande do sul- Brasil. Foram realizadas sete coletas, nas quais se avaliaram 12 pontos ao longo da linha de processamento, dentre eles a matéria-prima, superfícies com e sem contato com o produto, bem como o produto final. As amostras foram analisadas no equipamento Mini Vidas® bioMérieux e, aquelas que apresentaram resultado positivo para L. monocytogenes, foram submetidas a PCR, utilizando-se o gene prs para confirmação de gênero, e os genes inlA, inlC e inJ para confirmação da espécie L. monocytogenes. A ocorrência global de L. monocytogenes foi de 7,14%, sendo 21,42% em superfícies sem contato com produto, 1,78% em superfícies de contato com produto, e 28,57% na matéria-prima, entretanto, não se identificou o patógeno no produto final. Observa-se que a matériaprima é uma importante fonte de contaminação e introdução de L. monocytogenes na indústria produtora de Beef Jerky e que a contaminação por esse microorganismo em superfícies de contato e sem contato com o alimento, reforça a importância dos programas de higienização como forma de evitar contaminação cruzada para o produto final. Entretanto, como o produto final (Beef Jerky) não apresentou contaminação por L. monocytogenes, as etapas do processo de produção que envolvem barreiras à multiplicação de patógenos e morte microbiana, podem ter sido efetivas no controle do patógeno. / The growing demand of consumers for convenience, stands out among the main trends in food consumption in the world. In this context is the beef jerky a meat product produced by thermal process that does not require refrigeration at the point of sale, ensuring practicality of use. However, being a food ready for consumption, should ensure its security and one pathogenic bacteria that, once present, can harm the health of consumers is Listeria monocytogenes. The objective of this research was to identify the occurrence of L. monocytogenes in a Beef Jerky production line located in the municipality of Bage, Rio Grande do Sul- Brazil. We evaluated 12 points along the processing line, including the raw materials, surfaces with and without contact with the product as well as the final product, in seven collections. Samples were analyzed in Mini Vidas® bioMérieux equipment and those that tested positive for L. monocytogenes, were subjected to PCR using the prs gene for gender confirmation and the inlA genes, inlC and inlJ to confirm the species L. monocytogenes. The overall occurrence of L. monocytogenes was 7.14% and 21.42% on surfaces without contact with product, 1.78% on product contact surfaces, and 28.57% in the raw material, however, it did not identify the pathogen in the final product. It is observed that the raw material is a major source of contamination and introduction of L. monocytogenes in producer Beef Jerky industry and the contamination by that micro-organism contact surfaces without contact with food, reinforces the importance of cleaning programs in order to avoid cross-contamination of the final product. However, since the final product (Beef Jerky) showed no contamination by L. monocytogenes, the steps of the production process involving barriers to multiplication of pathogens and microbial death, may have been effective in pathogen control.

Selon un rapport de l’OMS, les maladies infectieuses figurent parmi les 3 causes les plus fréquentes de mortalité en début de vie. En effet, les nouveau-nés présentent une sensibilité particulièrement importante aux infections, de par leur système immunitaire toujours en développement et donc immature. Parmi les particularités de l’immunité néonatale, l’absence de cDCs CD11chigh CD8α+ durant la 1ère semaine de vie rend compte de l’incapacité du nouveau-né à développer des réponses de type Th1 et T CD8+ cytotoxiques, essentielles à la protection contre certains pathogènes comme Listeria monocytogenes (Lm). Mon travail de thèse a porté sur l’ontogénie des DCs conventionnelles CD11chigh CD8α+ et plus particulièrement sur la fonction de cette lignée de DCs en début de vie lors d’une infection par Lm. Au cours de cette étude, nous avons identifié, chez le nouveau-né, une population splénique de cDCs CD11chigh qui expriment les marqueurs CD24, CD205 et Clec9A mais pas le CD8α. Cette population, qui dépend du facteur de transcription Batf3, acquière le CD8α une fois transférée dans une souris adulte. Ces DCs néonatales, que nous nommerons DCs pre-CD8α Clec9A+, constituent donc les précurseurs des DCs CD8α+. L’étude fonctionnelle de ces DCs pre-CD8α Clec9A+ a montré qu’elles étaient capables de phagocyter Lm et de générer des réponses T CD8+ contre cette bactérie. De plus, ces cellules sécrètent de l’IL-12p40 et de manière unique de l’IL-10 en réponse à une stimulation par Lm. Par contre, contrairement aux cDCs CD8α+ adultes, nous n’avons observé aucune production d’IL-12p70 par les DCs pre-CD8α Clec9A+ en conditions physiologiques. Elles ne sécrètent pas non plus d’IL-23. Nous avons également montré que ces sécrétions d’IL-12p40 et d’IL-10 jouaient respectivement un rôle positif et négatif dans l’induction des réponses T CD8+ contre Lm. Ainsi, la génération des réponses T CD8+ contre Lm semble résulter, en début de vie, d’une balance entre la sécrétion de ces 2 cytokines aux propriétés antagonistes. Par ailleurs, nous avons démontré que ces cellules constituaient une cible privilégiée en vue d’améliorer les stratégies vaccinales en début de vie. En effet, l’administration à des nouveau-nés d’une construction anti-Clec9A/OVA, associée au poly(I:C), induit des réponses T CD8+ anti-Lm mémoires protectrices à l’âge adulte. Finalement, nous montrons que le TNF-α produit par les monocytes et les neutrophiles joue un rôle essentiel dans la génération des réponses T CD8+ en régulant notamment le statut et la fonction des DCs pre-CD8α Clec9A+ en début de vie. En effet, cette cytokine modifie, en faveur d’une production d’IL-12p40, la balance IL-12p40/IL-10 sécrétée par celles-ci. L’inhibition de la production d’IL-10 par le TNF-α pourrait s’expliquer au moins en partie par une inhibition de la β-caténine au sein des DCs pre-CD8α Clec9A+. En conclusion, nous avons caractérisé un précurseur des DCs CD8α+ biologiquement actif au sein de la rate du nouveau-né de 3 jours. Celui-ci représente une cible potentielle pour l’amélioration des stratégies vaccinales contre des bactéries intracellulaires ou des virus en début de vie, et ce pour autant que l’on puisse contrôler ses propriétés régulatrices. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Memoria para optar al Título Profesional de Médico Veterinario / Listeria monocytogenes es la bacteria causante de la enfermedad transmitida por alimentos conocida como listeriosis, la cual se ha visto asociada principalmente a alimentos listos para consumo (LPC). Dentro de los brotes de listeriosis se han visto involucrados los productos hidrobiológicos. En los últimos años, Chile ha aumentado la producción y exportación de este tipo de productos, llegando a diferentes mercados que tienen requisitos sanitarios, los cuales Chile debe cumplir. Los laboratorios, para demostrar que son capaces de realizar un método de análisis microbiológico de manera satisfactoria, pueden realizar un Proceso de Verificación, a través del cual se evalúa el rendimiento del método en un laboratorio específico. Para analizar L. monocytogenes en alimentos, existen diferentes métodos, entre los cuales se encuentran los descritos en las normas ISO 11290-1 para análisis cualitativo, e ISO 11290-2 para análisis cuantitativo. Ambos métodos fueron implementados y verificados en Salmón del Atlántico y en Chorito, en el Laboratorio de Inocuidad de los Alimentos del Departamento de Medicina Preventiva Animal de la Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile. El proceso de verificación se realizó en base a la norma ISO/WD 16140-3. En el método cualitativo, se evaluó si este es capaz de detectar un nivel suficientemente bajo de L. monocytogenes. Como resultado se obtuvo la detección positiva de la bacteria en un 100% de las muestras contaminadas. En el método cuantitativo, las muestras fueron contaminadas con tres niveles de inóculos (102, 103 y 104) de L. monocytogenes, y se evaluó la repetibilidad, reproducibilidad intralaboratorio, exactitud e incertidumbre del método. Los resultados obtenidos en ambos métodos cumplieron con los criterios para la verifiación de éstos. Así, el Laboratorio de Inocuidad de los Alimentos del Departamento de Medicina Preventiva Animal de la Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile, demuestra que tiene las capacidades para realizar ambos métodos en las matrices que fueron probadas, siendo capaz de entregar resultados confiables. / Listeria monocytogenes is the bacterium that causes the foodborne disease known as listeriosis, which has been mainly linked to ready-to-eat (RTE) foods. Hydrobiological products have been involved in listeriosis outbreaks. In recent years, Chile has increased production and export of this type of products, reaching various markets with their own health requirements, which Chile must meet. In order to prove they are capable of performing a microbiological analysis method, laboratories can go through a Verification Process, which evaluates the method’s performance in a specific lab. There are various methods to analyze L. monocytogenes in food, among which are those described in the ISO 11290-1 standard, for qualitative analysis, and ISO 11290-2, for quantitative analysis. Both methods were implemented and verified in the Atlantic Salmon and the Mussel, in the Food Harmlessness Laboratory of the Department of Preventative Animal Medicine of the Faculty of Veterinary and Animal Sciences of the University of Chile. The verification process was carried out according to the ISO/WD 16140-3 standard. It evaluated whether the qualitative method is able to detect a low enough level of L. monocytogenes. The results showed positive bacteria detection in 100% of the contaminated samples. For the quantitative method, the samples were contaminated with three inocula levels (102, 103 and 104) of L. monocytogenes, and it evaluated the method’s repeatability, within-laboratory reproducibility, accuracy and uncertainty. The results obtained for both methods satisfied their verification criteria. This way, the Food Harmlessness Laboratory of the Department of Preventative Animal Medicine of the Faculty of Veterinary and Animal Sciences of the University of Chile demonstrates it has the capabilities to perform both methods in the tested matrices.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

Natural Killer (NK) cells, lymphocytes of the innate immune response, play a vital role in controlling infections and in tumor surveillance. NK cells provide protection by direct cytolysis of infected cells and by the production of pro-inflammatory cytokines such as, IFN-γ and TNF-α. Notably, NK cells have recently been identified to regulate the immune response by producing the anti-inflammatory cytokine IL-10. Several other cells can produce IL-10 during infections, however NK cell derived IL-10 can be critical in regulating immune response during early phases of infection and therefore protecting the host from excessive immunopathology. Although the regulatory role of NK cells seems to be plausible, the physiological relevance of NK cell mediated immune regulation during infections has not been demonstrated in detail. To investigate the immunoregulatory function of NK cells, I used Murine Cytomegalovirus (MCMV) infection induced by a high dose challenge and demonstrated that NK cells are a major IL-10 producer during acute stage of the infection. To elucidate the role of NK cell derived IL-10 during infections, I generated NK cell specific IL-10 knockout, NKp46iCre  Il-10flox/flox mice (NK-Il-10-/-) by crossing Il-10flox/flox mice with mice expressing Cre recombinase exclusively under the NK cell specific promoter, NKp46 (NKp46iCre knock-in mice). My results indicated that Cre mediated Il-10 genomic deletion occurred predominantly in NK cells but not in NKT, T and B cells. Enriched NK cells from NK-Il-10-/- mice failed to produce IL-10 upon ex vivo IL-2/IL-12 stimulation. Furthermore, histological analysis of the colon indicated that NK-Il-10-/- mice are free from aberrant inflammation. During sustained MCMV infection, significantly higher production of IFN-γ by CD8+ T cells of NK-Il-10-/- mice in salivary glands indicates that NK cell derived IL-10 contributes to the establishment of the immune suppressive environment in the organ. NK-Il-10-/- mice also demonstrated increased susceptibility to acute Listeria monocytogenes (LM) infection based on enhanced body weight loss. Taken together, I have successfully generated NK-Il-10-/- mice that lack the Il-10 gene exclusively in NK cells. The NK-Il-10-/- mouse can be used as an ideal model to dissect the immunoregulatory role of NK cells during various microbial infections and tumorogenesis.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a wide ecological niche and causes a number of diseases in human and animals. It invades mammalian host cells and escapes from the vacuoles prior to replication in the host cell cytoplasm and infecting adjacent cells via actin-based mobility. Phosphoinositide (PIP) metabolism is essential to mammalian cells in signal transduction, actin remodelling, endosome dynamics and membrane trafficking. Modulation of host PIP metabolism by bacteria PIP phosphatases is important for pathogenicity and virulence of many human pathogens. In this study the function of two L. monocytogenes tyrosine and inositol phosphatases LipA and LipB were studied in vitro. The lipA and lipB deletion mutants generated in EGDe and InlA strains were not affected in invasion but were attenuated in intracellular growth in Caco-2 and Hela M cell lines but not in mouse macrophages. Deletion of lipA or lipB did not affect the actin polymerisation but caused reduced plaque number in the plaque assay. The turnover of five PIPs in Hela M cells during L. monocytogenes infection were studied by expression of fluorescent protein tagged domains that specifically recognizes individual PIPs. L. monocytognenes did not affect the metabolism of PI4P, PI(4,5)P2, PI(3,4,5)P3 but co-localised with PI3P at 1.5 hr post-infection and with PI(3,4)P2 at 6 hr to 24 hr post-infection. The PI(3,4)P2 effector protein lamellipodin was discovered to be recruited to actin-associated L. monocytogenes at 4 hr to 24 hr post-infection in Hela M cells. This discovery leads to the hypothesis of a novel mechanism of lamellipodin-dependant cell-to-cell spread. The lipA mutant was found to be attenuated in PI(3,4)P2 recruitment and therefore hypothesized to participate in the proposed lamellipodin pathway by converting PI(3,5)P2 into PI5P, leading to the activation of PI3K and subsequent production of PI(3,4)P2. LipB showed partial localisation at the Golgi complex when over-expressed in Hela M cells, and it was assumed to act mainly as a protein-tyrosine phosphatase. In summary, this study provides some evidence on L. monocytogenes modulating host PIP metabolism by the production of inositol phosphatases. It gives us a better understanding on the intracellular growth of this pathogenic bacterium, and on the interaction between host and parasite.

Thirteen South African medicinal plants which are used traditionally to treat symptoms associated with Listeria monocytogenes infections, were screened for activity against the pathogen. Different plant parts were extracted separately with ethyl acetate or chloroform. All the extracts were first screened against the bacteria using the disc diffusion method. Zones of inhibition observed in the presence of the chloroform extracts of Eucomis autumnalis, ethyl acetate extracts of Acacia karroo and Plectranthus ecklonii (50 mg/ml) were 12 mm, 14 mm and 15 mm respectively. Active extracts were further tested against the bacteria for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the microtitre dilution method. Ethyl acetate extracts of A. karroo exhibited MIC of 3.1 mg/ml and MBC of 6.25 mg/ml. Ethyl acetate extracts of P. ecklonii showed MIC of 0.5 mg/ml and MBC of 1.0 mg/ml. Five samples namely A. karroo (ethyl acetate extract), P ecklonii (ethyl acetate extract), Senecio inonartus (ethyl acetate extract), S. inonartus (chloroform extract) and Aloe arborescens (ethyl acetate extract) showed good MIC against L. monocytogenes and a MBC range from 1.0 to 12.5 mg/ml. The two plants, A. karroo and P. ecklonii were further selected for the isolation of the active compound(s). Column chromatographic purification of ethyl acetate extracts of the leaves of A. karroo led to the isolation of three known pure compounds namely â-sitosterol, epigallocatechin and epicatechin. The MICs of the â-sitosterol and epigallocatechin that were isolated from A. karrroo were found to be 31.25 µg/ml and 62.5 µg/ml respectively against L. monocytogenes. The confocal scanning laser microscopy (CSLM) showed that the biomass of the listerial biofilms were reduced when the isolated compounds were added and slightly reduced when the crude extract was added. The aggregation of cells which were exposed to â-sitosterol and epigallocatechin was reduced from 25 µm as observed in untreated cells to < 10 µm in length. Therefore as one of the local South African plants identified in the present study, the pure compounds isolated from A. karroo, could be used as a potential natural alternative for eliminating L. monocytogenes biofilms from food processing surfaces. This could help in combating the problem of food contamination and food poisoning caused by the pathogen. It could also help in preparing antibiofilm agents that are cost effective and easily accessible to the public. A. karroo should be further be explored in this regard. The present study reports for the first time the isolation of the three compounds, â-sitosterol, and epicatechin and epigallocatechin from A. karroo. Bioassay-guided fractionation of the P. ecklonii ethyl acetate extract led to the isolation of two known compounds, parvifloron D and parvifloron F. Parvifloron D and F exhibited a minimum inhibitory concentration (MIC) of 15.6 and 31.25 µg/ml respectively against L. monocytogenes. The MICs of parvifloron D and F against a drug-sensitive strain of Mycobacterium tuberculosis were found to be 190 and 95 µg/ml respectively. The ethyl acetate extract of P. ecklonii and its isolated compounds were tested for their activity on tyrosinase inhibition. The concentration of plant extract at which half the tyrosinase activity was inhibited (IC50) was found to be 61.73 ± 2.69 µg/ml. The antibacterial activity of the extract of P. ecklonii and its isolated compounds correlates with the traditional use of the plant for various ailments such as stomach-aches, diarrhoea and skin diseases. This is the first report on the bioactivity of an extract of P. ecklonii and its two compounds. The antibacterial activity of the extracts of A. karroo, P. ecklonii and their isolated compounds correlates with the traditional use of these plants for symptoms associated with listeriosis. / Thesis (PhD)--University of Pretoria, 2011. / Plant Science / unrestricted

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-23T22:49:42Z (GMT). No. of bitstreams: 1 Quadros_MarleneRochade_D.pdf: 3269198 bytes, checksum: 8cc8a1f7d91182cc3f49ebb1b0940318 (MD5) Previous issue date: 1998 / Resumo: Neste trabalho, investigamos os efeitos do extrato hidroalcoólico da Petiveria alliacea (EHA Pa) sobre o crescimento e diferenciação de células progenitoras hematopoiéticas da medula óssea para a série granulócito-macrófagos (CFU-GM) e sobre a atividade estimuladora de colônias do soro de animais infectados com a Listeria monocytogenes. Além disso, investigamos nestes animais os efeitos do EHA Pa sobre a atividade das células "natural killer" (NK) e sobre a resposta específica à infecção através das citocinas produzidas pelas subpopulações de células T auxiliares denominadas "T helper-l" (Thl- IL-2 e IFN-y) e Th2 (IL-4 e IL-I0). Para a realização dos experimentos os animais foram tratados por cinco dias com o EHA Pa na dose de 1000 mg/Kg/dia. Ao final do tratamento, os animais foram infectados com uma dose subletal de Listeria monocytogenes. Após a infecção ocorre uma diminuição do CFU-GM na medula óssea dos animais e um aumento na atividade estimuladora de colônias do soro destes animais. Os animais previamente tratados com com o EHA Pa apresentaram um aumento no CFU-GM da medula óssea 48 e 72 horas após a infecção, assim como, um aumento na atividade estimuladora de colônias do soro, em relação aos grupos infectados. Com relação à atividade das células NK, os animais infectados pela L. monocytogenes apresentaram um aumento da atividade destas células 48 e 72 horas após a infecção. Quando os animais foram previamente tratados com o EHA Pa observamos um aumento da atividade das células NK nas 48, 72 e 120 horas após a inoculação da bactéria, em relação aos animais infectados. o ERA Pa também demonstrou ter um efeito estimulador sobre a produção de citocinas. Com relação à resposta ThI, os camundongos tratados com o ERA Pa demonstraram um aumento na produção de IL-2. Quando os animais foram tratados com o ERA Pa e infectados com a L. monocytogenes observamos um aumento na produção de IFN-y e de IL-2 , 72 horas após a inoculação da bactéria em relação aos animais infectados. Com relação à resposta Th2 não observamos modificações na produção de IL-4 e IL-IO nos animais infectados em relação aos controles. O tratamento prévio dos animais com o ERA Pa não modificou esta resposta nos animais infectados. Para avaliar a resistência destes animais à infecção pela L. monocytogenes realizamos uma curva de sobrevida em animais infectados com a dose letal da bactéria. Os animais tratados previamente com o ERA Pa, 30 % sobreviveram à dose letal da L. monocytogenes enquanto 100 % dos animais do grupo controle infectado morreram em seis dias. Diante dos resultados obtidos neste trabalho, podemos sugerir que o EI{A Pa aumenta a resistência dos camundongos Balblcj infectados com a L. monocytogenes através da modulação da resposta imunológica na sua fase inespecífica através do aumento dos precursores hematopoiéticos da medula óssea, através do aumento da atividade das células NK; como também, aumento da produção de IL-2 e IFN-y / Abstract: Not ionformed. / Doutorado / Doutor em Ciências Biológicas

Orientador: Mary Luci de Souza Queiroz / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-18T19:24:46Z (GMT). No. of bitstreams: 1 Bincoletto_Claudia_M.pdf: 1273903 bytes, checksum: fff2b6abb690ce4dc271000f5b6653a7 (MD5) Previous issue date: 1993 / Resumo: Neste trabalho, investigamos os efeitos da exposição ao chumbo sobre o crescimento e diferenciação de células hematopoiéticas, as chamadas células formadoras de colônias (CFCs) da medula óssea de animais infectados e tratados com chumbo. Estudamos também os efeitos da exposição ao metal sobre a atividade dos fatores de crescimento de colônias (CSFs) no soro, assim como a sobrevida deste animais após infecção. Para a realização dos experimentos através da técnica de cultura clonal, em meio semi-sólido, os animais foram infectados com a bactéria Listeria monocytogenes após final do tratamento com acetato de chumbo. Após infecção com esta bactéria ocorre um aumento no número de células formadoras de colônias (CFCs) no baço, assim como nos níveis séricos de fatores estimuladores de colônias (CSFs). Utilizamos duas linhagens de camundongos: Balb\cj, susceptível a Listeria monocytogenes, e C57BI10 resistente a esta infecção. As doses de acetato de chumbo utilizadas foram: 1300, 130 e 13 ppm por períodos de 70, 30 e 10 dias. Ao final do tratamento os animais foram inoculados com doses de 3x102 - Balb\cj e 3x106 - C57BI10 e sacrificados 24, 48 e 72 horas após inoculação. A sobrevida destes animais foi determinada após observação destes camundongos por um período de 10 dias. Nossos resultados demonstraram que o efeito supressor do chumbo foi evidente em ambas linhagens. Na linhagem susceptível à infecção os efeitos da exposição ao chumbo ficou evidente em todos os grupos expostos, infectados ou não, nos três intervalos de tempo estudados após infecção. Nos animais resistentes a esta infecção o efeito supressor do acetato de chumbo também ficou evidente. Nesta linhagem, nas primeiras 24 horas após infecção tanto o chumbo como a infecção apresentaram efeitos supressores. Entretanto após 48 horas o efeito supressor da infecção foi superado, permanecendo apenas o efeito supressor induzido pelo chumbo. Não observamos alterações na atividade dos fatores estimuladores de colônias no soro dos animais em decorrência da administração do chumbo, sugerindo que este metal atue através de ação direta sobre os precursores hematopoiéticos. Observamos também um aumento na mortalidade em animais infectados com doses sub-Ietais de Listeria monocytogenes em ambas linhagens estudadas, quando expostas ao metal / Abstract: In this work we have investigated the effects of lead exposure on the growth and differentiation of hematopoietic cells from bone marrow, the so called colony forming cells (CFCs), in normal and infected mice. We also studied the effects of this exposure the serum activity of hemopoietic colony stimulating factors (CSFs), as well as, the survival of these mice after the infection. For this purpose, we used the technique for the clonal culture of hemopoietic cells in semi-solid medium. Mice were infected with the bacteria Listeria monocytogenes after treatment with lead acetate. Two strains of mice were used: Balb\cj (susceptible to Listeria monocytogenes) and C57BI10 (resistent to this bacteria). The doses of lead acetate were: 1300, 130 and 13ppm in periods of 70, 30 and 10 days. At end of this treatment, mice were infected and killed 24, 48 and 72 hours after the inoculation of the bacteria. The survival of these mice was determineted after a period of ten days. The suppressives effects of lead were observed in both strains in the three different periods studied. The dose-response relationship was observed with the 3 doses of lead used in relation to the effects of the infection, however, we observed that in the resistant strain the suppressives effects were overcome 48 hours after the administration of the baçteria. In the susceptible strain the suppressives effects of the infection were evident in the 3 periods studied. No changes were observed in the serum activity of CSFs due to the administration of lead, thus suggesting that this metal acts by a direct action on the myelopoietic cells. A significant decrease in host resistence, as measured by the mortality rate, was found when both strain of mice, after treatment with 1300ppm of lead for 30 days, were challenged with sub-lethal doses of Listeria monocytogenes / Mestrado / Mestre em Ciências Médicas

<p>Retail environments are critical transmission points for <i>Listeria monocytogenes</i> to humans. Past studies have shown <i>L. monocytogenes</i> contamination varies widely across retail environments. <i>L. monocytogenes</i> can transmit among environmental surfaces and subsequently from environment to food via cross-contamination. Modified SSOPs (sanitation standard operating procedures) have been shown to have limited impact on reducing <i>L. monocytogenes</i> prevalence in retail deli environments. Food safety culture and climate, such as beliefs, values, and hygiene behaviors, have been identified as factors impacting food safety performance and microbial outputs. Handwashing and its compliance are among the most prominent personal hygiene aspects subjected to investigation in the past decade, illustrating hygiene behavior as a risk factor and an important consideration to ensure food safety. Additionally, effective management and well-designed infrastructure, such as vertical and lateral communication, employees’ training, accountability, and equipment designed to prevent cross-contamination, have also been described as critical contributors to a sustainable food safety program. However, given such a deadly foodborne pathogen as <i>L. monocytogenes</i>, the correlation between food safety culture and its prevalence remains unknown. We hypothesized that there was a relationship among food safety culture management, infrastructure, and <i>L. monocytogenes</i> prevalence at retail. Our goal is to identify additional risk factors on <i>L. monocytogenes </i>control, develop feasible recommendations, and direct resources to enhance food safety. </p> <p>In the present dissertation, we developed and implemented a predictive risk model, along with employee- and management-implemented SSOPs, in 50 deli establishments across six U.S. states to evaluate and control <i>L. monocytogenes</i> contamination risk and prevalence (Chapter 2). The predictive risk model, based on logistic regression, uses five environmental sites to predict <i>L. monocytogenes</i> prevalence in the entire deli environment. It identified 13 high-risk stores, seven of which were confirmed during subsequent monthly sampling. We found that deep clean intervention reduced <i>L. monocytogenes </i>prevalence on non-food contact surfaces both immediately after the intervention and during follow-up, with marginal significance (α_adj=0.0125). The employee- and management-implemented deep clean can control <i>L. monocytogenes</i> prevalence in retail delis; the predictive risk model, though conservative, will require further validations and can be useful for surveillance purposes. </p> <p>Complementary to the above study, we tackled the <i>L. monocytogenes</i> challenge via food safety culture and climate approach (Chapter 3). Concurrently to the monthly environmental sampling, we distributed food safety culture and climate survey to the 50 stores, with one manager and up to five associates from each establishment, over a 12-month period and overlapped with before, after, and follow-up deep clean. We found that stores with lower <i>L. monocytogenes</i> contamination risk had better food safety culture, including greater sense of commitment to food safety program (p_adj=0.0317) and more complete training (p_adj=0.0117). Deep clean improved managers’ (p_adj=0.0243) and associates’ (p_adj=0.0057) commitment to food safety. This study indicates that food safety culture and climate are crucial component in building a viable, sustainable food safety program. </p> <p>Another survey tool was used to evaluate infrastructure designs, management strategies, and sanitation practices in relation to <i>L. monocytogenes</i> control in retail produce environments (Chapter 4). We distributed the survey to 30 retail produce departments across seven U.S. states. Hand hygiene, minimizing cross-contamination, and maximizing equipment cleanability were the most prominent factors in <i>L. monocytogenes</i> control.</p>