Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-15T11:47:50Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese.nelson certa.pdf: 11814547 bytes, checksum: 2cbb2aad17bbcac9f45275deb527071a (MD5) / Made available in DSpace on 2016-04-15T11:47:50Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese.nelson certa.pdf: 11814547 bytes, checksum: 2cbb2aad17bbcac9f45275deb527071a (MD5) Previous issue date: 2015-07-31 / A ocorrência de tolerância por parte dos micro-organismos contaminantes de alimentos a agentes e/ou tratamentos antimicrobianos clássicos, impõe a busca de tecnologias alternativas para emprego na conservação de alimentos, a exemplo dos óleos essenciais, em especial aqueles obtidos da espécie Rosmarinum officinalis L. (OERO). A ação antimicrobiana do OERO tem sido atribuída ao 1,8-cineol (CIN) frequentemente citado como seu constituinte majoritário. O objetivo deste estudo foi investigar o efeito da exposição a concentrações subinibitórias do OERO e CIN na modificação da tolerância direta e cruzada de algumas bactérias contaminantes de alimentos (Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa e Salmonella Typhimurium). Ainda, foi avaliada a influência dos fatores sigma alternativos σS e σB na tolerância de E. coli MG1655 e L. monocytogenes EGDe, e seus mutantes isogênicos E. coli MG1655 ΔrpoS e L. monocytogenes EGD-e ΔsigB, frente ao OERO e ao óleo essencial de Origanum vulgare L. (OEOV) bem como na modificação da tolerância bacteriana direta e cruzada ao campo elétrico pulsado (CEP). Para isso, foram realizados ensaios de determinação da Concentração Inibitória Mínima (CIM) e de modificação da tolerância direta e cruzada após exposição das cepas às concentrações subinibitórias (sub-CIMs) (1/2 CIM e 1/4 CIM) do OERO ou do CIN em caldo base carne (18 h) e em modelo base carne (72 h). Para os ensaios de verificação de injúria subletal e interferência dos fatores sigma na tolerância bacteriana, células de E. coli MG1655 e L. monocytogenes EGD-e foram expostas a concentrações inibitórias do OERO ou OEOV para, em seguida, serem cultivadas em meios adicionados de agentes seletivos (NaCl ou sais de bile). Os valores de CIM variaram entre 20 e 40 μL/mL e 40 e 80 μL/mL para o OERO e CIN, respectivamente. Os ensaios de modificação de tolerância não revelaram aumento de tolerância direta e cruzada nas cepas bacterianas testadas. A CIM do OERO e OEOV frente às células mutantes foi menor daquele encontrado frente às células parentais. As células mutantes também apresentaram maior diminuição da sua viabilidade quando tratadas com os óleos essenciais (OES) em relação às células parentais, bem como maior proporção de células apresentando lesão subletal. Ainda, as células mutantes mostraram maior sensibilidade ao CEP quando comparadas as parentais. A ausência dos fatores sigma diminuiu a tolerância direta e ao CEP nas células pré-expostas ao OES em sub-CIMs. Os resultados obtidos neste estudo demonstram que: i) a exposição das cepas bacterianas a sub-CIMs do OERO ou CIN não aumentou a tolerância direta e cruzada; ii) os fatores sigma σS e σB influenciaram a sensibilidade das cepas de E. coli MG1655 e L. monocytogenes EGD-e, respectivamente, frente ao OERO e OEOV ao CEP; e iii) os fatores sigma não influenciaram no aumento da tolerância bacteriaia direta e cruzada ao CEP. Estes resultados demonstram o potencial do OERO e do CIN para uso como antimicrobiano na conservação de alimentos, quando considerado o seu efeito inibitório sobre bactérias contaminantes de alimentos e a ausência de indução de aumento na tolerância bacteriana direta e cruzada. / The occurence of tolerance in food contaminant microorganisms to agents and/or classic antimicrobial treatments has required the search of alternative technologies for use in food preservation, such as essential oils, especially those obtained from the specie Rosmarinum officinalis L. (ROEO). The antimicrobial action of ROEO has been attributed to 1.8-cineol (CIN), frequently cited as its main constituent. The aim of this study was to investigate the effect of exposure to subinhibitory concentrations of ROEO and CIN on the modification of direct tolerance and cross-tolerance of some food-contaminant bacteria (Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella Typhimurium). Still, the influence of alternative sigma factors σS and σB on tolerance of E. coli MG1655 and L. monocytogenes EGD-e, and its isogenic mutant E. coli MG1655 and L. monocytogenes EGD ΔrpoS and ΔsigB to the ROEO and the essential oil of Origanum vulgare L. (OVEO), as well as direct on the modification of the bacterial direct tolerance and cross tolerance the pulsed electric field (PEF) were investigated. For this, assays to determine the Minimum Inhibitory Concentration (MIC) and the modification of the direct tolerance and the cross-tolerance after exposure of the strains to subinibitory concentrations (sub-MICs) (1/2 MIC and 1/4 MIC) of ROEO or CIN in meat broth (18 h) and model based meat (72 h) were performed. For verification of sublethal injury and interference of sigma factors on bacterial tolerance, E. coli MG1655 cells and L. monocytogenes EGD-e were exposed to inhibitory concentrations of ROEO or OVEO and further cultivated in media added selective agents (NaCl or bile salts). The MIC values ranged between 20 and 40 μL/mL and 40 to 80 μL/mL for ROEO and CIN, respectively. The tolerance modified assays revealed no increase in either the direct tolerance or the cross-tolerance in the tested bacterial strains. The MIC of ROEO and OVEO against the mutant cells was lower than that found against the parental cells. Mutant cells showed also a greater decrease in viability when treated with essential oils (EOS) compared to the parental cells, as well as a higher proportion of cells with sublethal injury. Still, the mutant cells showed higher sensitivity to PEF when compared to parental cells. The absence of sigma factors decreased the direct tolerance and tolerance to the PEF in cells pre-EOS exposed to sub-MIC. The results of this study show that: i) exposure of the bacterial strains to sub-MICs of ROEO or CIN did not increase the direct or the crosstolerance; ii) the sigma σS and σB factors influenced the sensitivity of strains of E. coli MG1655 and L. monocytogenes EGD-e, respectively, to the OVEO and ROEO and PEF; and i) the sigma factors did not influence the increase in direct bacterial tolerance and cross tolerance to the PEF. These results demonstrate the potential of ROEO and CIN for use as antimicrobials in food preservation, when considering their inhibitory effect on bacterial foodcontaminants and the absence of induction to direct or cross bacterial tolerance.

Mycobacterium are important human pathogens and their strength lies in establishing acute infections, latent infections as well as co-existing with other dreadful infectious agents like HIV. The success of mycobacterium infection often relies in its ability to evade immune-surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immune regulatory molecules. In this scenario, the role of pattern recognition receptors (PRRs) in orchestrating host immune responses assumes central importance. Of the PRRs, the Toll-like receptors (TLRs) or intracellular surveillance receptors such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) govern key immune-surveillance mechanisms in recognition as well as control of mycobacterial or viral infections. The first part of this study illustrates the role of SHH signaling in macrophage induced neutrophil recruitment during mycobacterial infections. The present investigation demonstrates that, in response to mycobacterium infection, macrophages displayed robust activation of TLR2 dependent SHH signaling. By utilizing the well-documented experimental air pouch model, we show that the ability of pathogenic mycobacterium infected macrophages to recruit polymorph nuclear leukocytes (PMNs) like neutrophils to the infected site was dependent on SHH signaling. The activated SHH signaling differentially regulated the expression of proteolytic enzymes, MMP-9 and MMP-12 that would contribute to PMN migration. Interestingly, SHH-responsive krüppel-like family (KLF) of transcription factors, KLF4 and KLF5 were found to modulate these chemokine effectors to regulate neutrophil recruitment. Subsequent chapters describe novel functions of SHH signaling during RIG-I mediated anti-viral immunity and RIG-I mediated modulation of TLR2 anti-inflammatory signature in mycobacteria infected macrophages. In this perspective, we demonstrate that RIG-I ligand robustly induces the activation of SHH signaling via the phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. Furthermore, we show that the sustained inhibition of PKA-GSK-3β-SUFU negative regulatory axis upon RIG-I engagement with 5'3pRNA is critical for the activation of SHH signaling. Gain or loss of function studies implicate the necessity of RIG-I triggered MAVS-TBK1 canonical axis in the inhibition of PKA-GSK-3β-SUFU negative regulatory axis that contributes to SHH signaling activation. The RIG-I activated SHH signaling drives the production of anti-viral type 1 interferons leading to the inhibition Japanese encephalitis virus (JEV) replication. Further, RIG-I-mediated anti-viral type 1 interferon production and subsequent control of viral replication suggested the involvement of two transcriptional factors, IRF3 and YY1 in the response along a SHH axis. Further, mounting evidence clearly depicts a significant cross talk among the molecular events initiated by given TLRs and RLRs like RIG-I. Clearly, these studies present an interesting challenge in delineating the events during polymicrobial infection of host immune cells like macrophages or DCs. Altogether, our results improve our understanding of mycobacteria associated confections’ and may add significantly to the current knowledge of the delicate balance that determines a successful mycobacterial infection.

No presente estudo, um conjunto de três experimentos foram conduzidos com o objetivo de avaliar a ocorrência de Staphylococcus aureus e Listeria monocytogenes em três lacticínios (A, B e C) localizados na região sudeste do Brasil de dezembro 2013 a abril de 2015 (Experimento 1), a eficiência do tratamento com ácido peracético (APA) e jato de plasma a frio (PF) para inativar os isolados em diferentes tempos (Experimento 2) e a capacidade dos isolados produzir biofilmes na superfície de poliestireno e de aço inoxidável, juntamente com inativação e remoção de células aderidas pelo APA (Experimento 3). No Experimento 1, foram analisadas amostras de leite e queijo, superfícies com e sem contato com alimentos. L. monocytogenes foi isolada em apenas uma amostra (0,3%, N = 349) de ralo no laticínio B, enquanto seis (1,7%, n = 349) S. aureus foram isolados de luvas de manipuladores em laticínio A, salmoura no laticínio B e superfície do queijo, utensílio, bota e mão esquerda de trabalhador no lacticínio C. Apesar das incidências desses dois agentes patogênicos de origem alimentar nos lacticínios avaliados foram baixo, sua presença também indica a necessidade de controle estratégias para impedir a sua persistência e contaminação cruzada. No Experimento 2, tratamento com APA (0,5%) e jato de PF foram aplicados diretamente sobre suspensões de isolados de S. aureus e L. monocytogenes. A inativação bacteriana (aproximadamente de 7 ciclos log) foi alcançada em 120 seg. com o tratamento com APA para todos os isolados, enquanto que o tratamento com plasma a frio reduziu aproximadamente 2 ciclos log nas superficies. Outros estudos usando tratamentos de plasma a frio mais longos são necessários para a total descrição da cinética desta tecnologia para a inativação de importantes patógenos de origem alimentar. No Experimento 3, o tratamento com APA (0,5%) em diferentes tempos (0-controle, 15, 30, 60 e 120 seg.) foi avaliada para a remoção de células aderidas de quatro isolados de S. aureus e um isolado de L. monocytogenes em microplacas de poliestireno, assim como para a inativação de biofilmes dos isolados em aço inoxidável. O tratamento com APA removeu (p<0,05) células aderidas de todos os isoladoas estudados S. aureus da superfície, sem diferenças (p> 0,05) no indice de formação de biofilmes nos tempos de tratamento. No entanto, nenhum efeito (p> 0,05) foi observado em células aderidas de L. monocytogenes. A microscopia de epifluorescência mostrou que todas as bactérias testadas foram parcialmente e completamente inativadas após 15 seg e 30 seg. respectivamente. Os resultados indicam um potencial para a utilização de APA contra biofilmes formados por S. aureus e L. monocytogenes, e da necessidade de novos estudos com a PF para determinar os parâmetros ideais para a inativação dos patógenos de origem alimentar. / In the present study, a set of three experiments were conducted aiming to evaluate the occurrence of Staphylococcus aureus and Listeria monocytogenes in three dairy plants (A, B and C) from Southeast region of Brazil from December 2013 to April 2015 (Experiment 1), the efficiency of peracetic acid (PAA) and cold plasma (CP) jet treatment to inactivate the isolates at different times (Experiment 2) and the ability of the isolates to produce biofilms on polystyrene and stainless steel surface, along with inactivation and removal of biofilms by PAA (Experiment 3). In Experiment1, samples of milk and cheese, food contact surfaces and non-food contact were analyzed. L. monocytogenes was isolated in only one sample (0.3%, N=349) of drain sponge swab in dairy plant B, while 6 (1.7%, N=349) S. aureus strains were isolated from handlers\' glove in dairy plant A, brine in dairy plant B and cheese surface, cheese utensil, worker\'s boot and worker\'s left hand in dairy plant C. Although the incidences of those two food-borne pathogens in the dairy plants evaluated were low, their presence also indicates the need for control strategies to prevent their persistence and cross-contamination. In Experiment 2, PAA (0.5%) and CP jet treatment were applied directly on suspensions of S. aureus and L. monocytogenes strains. Reduction of bacterial load (nearly 7 log cycles) was achieved with 15 sec. of PAA treatment of all strains, whereas CP treatment reduced approximately 2 log cycles after 2 min. Hence, plasma treatment has a potential for reducing the bacterial load on surfaces, although further studies using longer CP treatment times are necessary to fully describe the kinetics of this technology for inactivation of important food pathogens. In Experiment 3, PAA (0.5%) treatment at different times (0-control, 15, 30, 60 and 120 sec.) was evaluated for removing of adherent cells of 4 strains of S. aureus and one strain of L. monocytogenes on polystyrene plates, as well as for inactivation of biofilms of those strains on stainless steel. PAA treatment removed (p<0.05) all the S. aureus cells from the surface, with no difference (p>0.05) in the reduction of the biofilm-forming index at the treatment times. However, no effect (p>0.05) was observed on L. monocytogenes adhered cells. Epifluorescence microscopy showed that all bacterial strains tested were partially and completely inactivated after 15 sec. and 30 sec., respectively. Results indicate a potential use of PAA against biofilms formed by S. aureus and L. monocytogenes, and the need of further studies with CP to determinate the ideal parameters for inactivation of food-borne pathogens.

Pela utilização de métodos bioquímicos, biofísicos, de tipagem sorológica e de visualização das proteínas totais bacterianas, isolados de surubim Pseudoplatystoma corruscans, tilápia Oreochromis niloticus e pacu Piaractus mesopotamicus, foram caracterizados, identificados e sua virulência determinada. Dentre as linhagens de referência, o isolado de surubim caracterizou-se como sendo Plesiomonas shiguelloides e os demais isolados de tilápia e pacu foram identificados como Aeromonas hydrophila, todos pertencentes à família Vibrionaceae. Os isolados de tilápia e pacu caracterizaram-se como linhagens virulentas, resistentes aos antibióticos ampicilina, amoxicilina, lincomicina, novobiocina, oxacilina, penicilina, rifampicina e trimetoprim+sulfametoxazol, em ensaios de antibiograma realizados em meio YEA que evidenciaram que as linhagens isoladas de peixes são resistentes a oito das dezessete substâncias antimicrobianas testadas pelo método de difusão em disco. Essas características são compatíveis com as apresentadas pelo espécime tipo de A. hydrophila. Ambas as linhagens quando cultivadas em meio YEA compartilharam a mesma banda de aproximadamente 33,61kDa com o espécime tipo para A. hydrophila. Em meio enriquecido com glucose, a banda compartilhada entre elas teve peso molecular aproximado de 144,28kDa. Os testes de aglutinação sorológica evidenciaram nestas duas linhagens a presença de antígenos estáveis ao calor do tipo O. A técnica de dupla imunodifusão de Ouchterlony demonstrou que o antígeno preparado a partir do isolado de tilápia é a linhagem de A. hydrophila mais indicada para ser utilizada em estudos visando o desenvolvimento de uma vacina polivalente. / Bacteria isolated from surubim Pseudoplatystoma corruscans, tilapia Oreochromis niloticus e pacu Piaractus mesopotamicus, were characterized and identified by biochemical, biophysical, serology, and SDS-PAGE, and their virulence observed. The strain isolated from surubim was characterized as Plesiomonas shigelloides. The other strains isolated from tilapia and pacu were Aeromonas hydrophila. The isolated A. hydrophila strains presented virulence and resistance against the follow antibacterial substances: ampicillin, amoxicillin, lincomicin, novobiocin, oxacillin, penicillin, rifampin and trimetoprim+sulfametoxazole. Both strains when cultivated in YEA medium shared with the A. hydrophila type strain a similar protein band of 33,61kDa. In a medium supplemented with glucose, only one protein exhibiting relative molecular mass of 144.28 kDa, was shared by the type strains isolated from fish and the type strain. The serology tests revealed that all isolated strains presented heat-stable O-antigens. The Ouchterlony double-immunodiffusion showed that the antigen prepared from the tilapia strain possessed surface antigens similar to A. hydrophila type strain and the strains isolated from pacu. This suggested the possibility of development and usage of a common or polyvalent vaccine for A. hydrophila among tilapia and pacu or other freshwater fish species.

A mussarela de leite de búfala, principal queijo obtido a partir desse leite no Brasil, é um produto praticamente novo no mercado, com alta aceitação pelos consumidores e excelentes perspectivas. Seguindo tecnologia de produção tradicional italiana, caracteriza-se pela intensa manipulação durante a sua elaboração. No presente trabalho, avaliou-se a qualidade microbiológica de duas marcas comerciais de queijo mussarela de leite de búfala, sendo uma das marcas comercializada em embalagem com soro (A) e a outra em embalagem sem soro e a vácuo (B), adquiridas no comércio varejista da cidade de Piracicaba/SP. As análises microbiológicas compreenderam a determinação do NMP de coliformes totais e fecais, a pesquisa de Listeria spp., a contagem de Staphylococcus coagulase-positiva e a pesquisa de Salmonella spp. Com base nos resultados obtidos, pode-se afirmar que as duas marcas analisadas encontram-se em acordo com os padrões microbiológicos legais vigentes. No entanto, pôde-se notar que a qualidade microbiológica dos queijos comercializados em embalagem com soro mostrou-se inferior à dos oferecidos ao consumo em embalagem sem soro e a vácuo. / Buffalo mozzarella cheese, main cheese obtained from buffalo milk in Brazil, is practically a recent product in the market, showing high acceptance by consumers and excellent perspectives. Following traditional italian production tecnology, this cheese is intensely manipulated during its manufacture. In this study, the microbiology quality of two commercial brands of buffalo mozzarella cheese was evaluated, being one of the brands presented in bag with whey (A) while the other one is presented in bag without whey and under vacuum (B). The samples were acquired in the Piracicaba city commerce. Microbiology analysis comprehended the determination of the MPN of total and fecal coliforms, the Listeria spp. presence / absence, the coagulase-positive Staphylococcus accounting and the Salmonella spp. presence / absence. Based on the analysis results, both brands are according to current legal microbiology standards specifications. However, the microbiology quality of the cheeses packed in bag with whey was lower than the microbiology quality of those offered in bag without whey and under vacuum.

O carrapato Rhipicephalus microplus é o principal ectoparasita de bovinos. O controle de R. microplus baseia-se principalmente no uso de acaricidas químicos, o que contribuiu para o problema emergente da seleção de populações de carrapatos resistentes. Portanto, há a necessidade do desenvolvimento de métodos mais eficientes/sustentáveis de controle, como o controle biológico utilizando fungos acaropatogênicos. A eficácia do fungo Metarhizium anisopliae de forma isolada ou em associação com acaricida químico para controle do carrapato bovino já foi evidenciada em condições de campo utilizando uma cepa de carrapatos resistente à acaricidas. No entanto, pouco se sabe sobre os mecanismos moleculares de R. microplus envolvidos na sua interação com M. anisopliae e com acaricidas. Assim, o objetivo do presente estudo foi avaliar a resposta de larvas de R. microplus expostas ao fungo, ao acaricida e à associação de ambos. Primeiramente foi realizado um estudo para determinar a metodologia mais indicada para avaliar o efeito in vitro do fungo M. anisopliae sobre larvas de R. microplus. Para isso, comparamos o Teste de Pacote de Larvas modificado (TPL) e o Teste de Imersão de Larvas (TIL). Os valores de tempo letal mediano (TL50) obtido na maior concentração de M. anisopliae (108conídios/mL) foram 24,8 e 9,2 dias para o TPL e TIL, respectivamente. A mortalidade após 21 dias foi de 38% e 98% para o TPL e TIL respectivamente, na mesma concentração. O TIL demonstrou ser o teste mais indicado a ser utilizado, sendo, portanto, escolhido para realização dos experimentos futuros. Em seguida foi realizado um estudo para comparar a suscetibilidade de diferentes isolados de R. microplus ao fungo M. anisopliae. Foram avaliados 67 isolados de campo. Para tanto, as larvas de R. microplus foram imersas em uma suspensão de M. anisopliae (108conídios/mL) durante 5 min. Os tempos letais medianos (TL50) variaram de 2,6 a 24,9 dias A mortalidade observada no 15º dia após o tratamento variou de 26,3 a 100% nas amostras testadas. Esses resultados demonstraram que as populações de campo de R. microplus apresentam uma alta variação em sua suscetibilidade a M. anisopliae. Por fim, foi realizada uma análise transcricional (RNAseq) de larvas de R. microplus expostas a M. anisopliae, cipermetrina, associação de ambos e do controle (não-tratado). A análise dos transcritos dos quatro grupos gerou um total de 507.792 sequências com um tamanho total de 303.160.891 pb. Foram encontrados 31 genes diferencialmente expressos no grupo controle quando comparado com M. anisopliae, 39 com cipermetrina e 73 com M. anisopliae + cipermetrina. M. anisopliae e o grupo da associação apresentaram 81 genes diferencialmente expressos e M. anisopliae e cipermetrina, 46. Houve 177 genes diferencialmente expressos ao comparar M. anisopliae + cipermetrina com larvas expostas à cipermetrina Os resultados deste estudo demonstraram que a associação M. anisopliae + cipermetrina aumentou a expressão de genes relacionados a resposta à agressão, provavelmente pelo fato da associação provocar uma agressão maior ao carrapato e este responder de forma mais rápida, levando à superexpressão de genes de defesa. As descobertas deste estudo fornecem informações sobre as vias e mecanismos moleculares de R. microplus ativados em resposta à interação de M. anisopliae e a acaricida. Além disso, esses estudos estabelecem as bases para pesquisas futuras sobre genes-chave que controlam a suscetibilidade de R. microplus a M. anisopliae. O sinergismo evidenciado em nossos estudos comprova a ideia de que o controle integrado, utilizando o controle biológico com o controle químico é uma opção tanto para o controle de cepas de carrapatos resistentes à acaricidas, quanto para um controle mais rápido de cepas de carrapatos que não apresentam resistência ou que apresentem resistência intermediária. / The tick Rhipicephalus microplus is the major cattle ectoparasite. The control of R. microplus is based mainly on the use of chemical acaricides, which contributed to the emerging problem of the selection of tick populations resistant to acaricides. Therefore, there is a need for the development of more efficient/sustainable methods of control, such as biological control using acari pathogenic fungi. The effectiveness of the fungus Metarhizium anisopliae alone or in association with chemical acaricide to control the bovine tick has already been evidenced under field conditions using an acaricide resistant tick strain. However, to date, little is known about the molecular pathways of R. microplus involved with its interaction with M. anisopliae and with acaricides. Thus, the aim of the present study is to evaluate the response of R. microplus larvae exposed to the fungus, the acaricide and the association of both. Firstly, a study was carried out to determine the most appropriate methodology to evaluate the in vitro effect of the fungus M. anisopliae on larvae of R. microplus tick. For this, we compared the Modified Larval Packet Test (LPT) and the Larval Immersion Test (LIT). The values of lethal time (LT50) obtained in the highest concentration of M. anisopliae (108conidia / mL) were 24.8 and 9.2 days for LPT and LIT, respectively. Mortality after 21 days was 38% and 98% for LPT and LIT, respectively, at the same concentration. The LIT proved to be the most indicated test to be used and was therefore chosen to carry out the following experiments Then, a study was performed to compare the susceptibility of different R. microplus isolates to M. anisopliae fungus. Sixty-seven field isolates were evaluated. For this, R. microplus larvae were immersed in a suspension of M. anisopliae (108conidia/mL) for 5 min. Lethal times (LT50) ranged from 2.6 to 24.9 days. Mortality observed on the 15th day after treatment ranged from 26.3 to 100% in the tested samples. These results demonstrated that R. microplus field populations showed a high variation in their susceptibility to M. anisopliae. Finally, a RNAseq analysis of R. microplus larvae exposed to M. anisopliae, cypermethrin, association of both or untreated (control) was performed. Analysis of the transcripts of the four groups generated a total of 507,792 sequences with a total length of 303,160,891 bp. We found 31 differentially expressed genes in the control group when compared to M. anisopliae, 39 with cypermethrin and 73 with M. anisopliae + cypermethrin. M. anisopliae and the association group had 81 differentially expressed genes and M. anisopliae and cypermethrin, 46 There were 177 differentially expressed genes when comparing M. anisopliae + cypermethrin with cypermethrin exposed larvae. The results of this study demonstrated that the association of M. anisopliae + cypermethrin increased the expression of genes related to response to aggression, possibly because the M. anisopliae + cypermethrin association causes a greater aggression to the tick and, then it could respond quickly, leading to overexpression of defense genes. The findings of this study provide information on the pathways and molecular mechanisms of R. microplus in response to the interaction of M. anisopliae and acaricides. In addition, these studies establish the basis for future research on key genes that control the susceptibility of R. microplus to M. anisopliae. The synergism evidenced in our studies confirms the idea that integrated control using biological control and chemical control is an option both for the control of acaricide resistant tick strains and for a faster control of tick strains that do not show resistance or exhibit intermediate resistance.

abstract: The recent proliferation of online platforms has not only revolutionized the way people communicate and acquire information but has also led to propagation of malicious information (e.g., online human trafficking, spread of misinformation, etc.). Propagation of such information occurs at unprecedented scale that could ultimately pose imminent societal-significant threats to the public. To better understand the behavior and impact of the malicious actors and counter their activity, social media authorities need to deploy certain capabilities to reduce their threats. Due to the large volume of this data and limited manpower, the burden usually falls to automatic approaches to identify these malicious activities. However, this is a subtle task facing online platforms due to several challenges: (1) malicious users have strong incentives to disguise themselves as normal users (e.g., intentional misspellings, camouflaging, etc.), (2) malicious users are high likely to be key users in making harmful messages go viral and thus need to be detected at their early life span to stop their threats from reaching a vast audience, and (3) available data for training automatic approaches for detecting malicious users, are usually either highly imbalanced (i.e., higher number of normal users than malicious users) or comprise insufficient labeled data. To address the above mentioned challenges, in this dissertation I investigate the propagation of online malicious information from two broad perspectives: (1) content posted by users and (2) information cascades formed by resharing mechanisms in social media. More specifically, first, non-parametric and semi-supervised learning algorithms are introduced to discern potential patterns of human trafficking activities that are of high interest to law enforcement. Second, a time-decay causality-based framework is introduced for early detection of “Pathogenic Social Media (PSM)” accounts (e.g., terrorist supporters). Third, due to the lack of sufficient annotated data for training PSM detection approaches, a semi-supervised causal framework is proposed that utilizes causal-related attributes from unlabeled instances to compensate for the lack of enough labeled data. Fourth, a feature-driven approach for PSM detection is introduced that leverages different sets of attributes from users’ causal activities, account-level and content-related information as well as those from URLs shared by users. / Dissertation/Thesis / Doctoral Dissertation Computer Science 2020

Syftet med studien är att synliggöra hur pedagoger ser på barns hälsa i relation till utevistelse i förskolan. I studien användes metoden kvalitativa intervjuer. Åtta yrkesverksamma förskollärare intervjuades. I studien användes salutogent och patogent perspektiv på hälsa som teoretisk utgångspunkt. Det framkom i resultatet att de verksamma pedagogerna erbjuder barnen att vistas i olika utomhusmiljöer som främjar deras hälsa. Det görs genom att de får utöva fysisk aktivitet och de får lära sig med alla sina sinnen. Detta är ett salutogent synsätt att se på hälsa eftersom pedagogerna fokuserar på att främja barnens hälsa i förskolan. Det framkom även i resultatet att pedagogerna har bedrivit mer verksamhet utomhus på grund av de rådande omständigheterna kring covid- 19. Här framkom det att pedagogerna fokuserade på att försöka förhindra och hantera smittspridningen på förskolan, vilket är ett patogent synsätt att se på barns hälsa. Slutsatsen som drogs var att både det salutogen och det patogena synsättet behövs då de kompletterar varandra. / The purpose of the study is to make visible how educators view children's health in relation to outdoor activities in preschool. In the study, the method of qualitative interviews was used. Eight professional preschool teachers were interviewed. The study used a salutogenic and pathogenic perspective on health as a theoretical starting point. It emerged from the results that the active educators offer the children to stay in different outdoor environments that promote their health. This is done by allowing them to exercise physically and they learn with all their senses. This is a salutogenic approach to look at health where educators focus on promoting children's health in preschool. It also emerged from the results that the educators have conducted more activities outdoors due to the counseling circumstances surrounding covid-19. Here it emerged that the educators focused on trying to prevent and manage the spread of infection in preschool, that it is a pathogenic approach to look at children's health. The conclusion that was drawn that both the salutogenic and the pathogenic approach are needed as they complement each other.

246 samples of bulk and packaged spices from retail stores in the western, southeastern, southern, midwestern, and northeastern areas of the U.S. were examined for the presence of Clostridium -perfringens. Isolates were checked for the presence of the lecithinase gene (cpa) and enterotoxin genes (cpe) by PCR. Enterotoxin formation during sporulation was investigated using the Oxoid Toxin Detection Kit. Forty-three confirmed isolates (from 17% of total samples) were cpa-positive. Of those, 27 were cpe-positive. Together, levels of C. perfringens spores ranged from 3.6-2400/gm. The amount of enterotoxin in cell extracts ranged from 2-16 ng/ml. Some of the SEM images of isolated spore (# 78) and one plasmid-borne ent control (FD-153) showed an organized surface structure termed “candy-wrapper”. This extracellular structure remained after treatment with 0.1 % SDS for 1 hr, suggesting it was not composed of membrane debris from the mother cell. The D values of spores ranged from 1.19- 3.31 min. The addition of lysozyme in the plating medium elevated the recovery rate of heat-treated spores. The growth rate of a cocktail of spores from spices (# 31, # 32, # 45) between 4 to 5 hr after inoculation was determined with a doubling time of 6.82 min in hamburger. A cocktail of spores of plasmid-borne ent control showed an optimum growth rate between 5 to 8 hr after inoculation with doubling time of 15.98 min. However; spice isolate cocktail, plasmid-borne ent control cocktail (FD-5603 and FD-153), and a chromosome-borne ent control (NTCT 8239) were unable to germinate and outgrowth at 20oC. Inoculation in laboratory medium FTG indicated the same result as hamburger at 20oC. The ability of C. perfringens spores in spices to potentially survive cooking procedures can be followed by germination and growth of vegetative cells during improper cooling to levels associated with foodborne illness caused by this organism. Our results suggest that retail spices are potential vehicles of transmission of enterotoxin-positive C. perfringens.

Bakgrund Forskning visar att delar av samhället anser att idrott och hälsa (IDH) är ett redskap, vars syfte är att eliminera fetma och andra former av sjukdomar. Vilket tyder på att vissa anser att IDH bör förhålla sig till det patogena hälsoperspektivet. Detta däremot, ställer sig flera forskare kritiska till, då de anser att IDH bör förhålla sig till vad som främjar och utvecklas hälsa, vilket innefattar ett salutogent hälsoperspektiv. Syftet med studien Syftet med detta arbete är att undersöka hur lärare undervisar hälsa och dess dimensioner, samt hur undervisningen förhåller sig till salutogent- och patogent hälsoperspektiv. Metod Empirin samlades genom fyra semi-strukturerade kvalitativa intervjuer, ungefär 40 minuter vardera. Dessa intervjuer genomfördes via konferensprogrammet ”Zoom”. Resultat och diskussion Det framkommer att fysiska aktiviteter är grunden till all hälsoundervisning, vilket tillför möjligheten för lärare att sätta icke-fysiska aspekter av hälsa i kontext. Dessutom framkommer det att lärare nästan forcerar samarbete hos eleverna, vilket ska utveckla eleverna förmåga att bygga relationer, minimera exklusion och förmågan att samverka. Lärare för det mesta implementerar flertalet lärdomar inom en aktivitet. Eleverna ges möjligheten attreflektera kring aktivitetens övriga effekter på hälsa, bortsett från de fysiska aspekterna. Det visade sig att det patogena- och det salutogena hälsoperspektivet framkommer i hälsoundervisningen. Detta för att de både kompletterar varandra och tillför viktiga aspekter till både lärares undervisning metoder och kunskap till eleverna. / Background Research suggests that parts of society view physical education (PE) as a tool, the purpose of which is to eliminate obesity and other kinds of illness. The result is that some argue that PE should have a pathogenic view on health. This view however is criticized by some researchers who suggest that PE should focus on what improves health, in other words a salutogenic view on health. Aim of this study The aim of this study is to investigate how teachers educate health and its dimensions, and how health education adheres to the salutogenic- and pathogenic health perspectives. Method Empirical data were collected from four semi-structured qualitative interviews, approximately 40 minutes each. Interviews were conducted with the conference program “Zoom”. Result and discussion Participants argue that physical activities are the foundation of all health education, this provides teachers with an opportunity to put the non-physical aspects of health into context. These activities of which can be used to enhance and develop psychological and social health aspects. Through almost forced co-operations, students improve their relation building competence, reinforces inclusion and ability to co-operate. Moreover, teachers mostly implement several teachings within one activity. They do this by making the students reflect on “spin-off” effect from the activity, where other health developments other than physical aspects within an activity are required. It is revealed that both pathogenic and salutogenic perspective is present in the health education, and that they complement each other and bring different important aspects to teachers’ methods and knowledge to students.