Tumorstroma und Tumor-assoziierte Makrophagen (TAMs) spielen in neoplastischen Erkrankungen eine wichtige Rolle im Bezug auf Tumorwachstum und Progression. In einigen Krebsarten besteht zwischen den Tumor-assoziierten Makrophagen und den Krebszellen eine intensive Interaktion, welche zu vermehrter Angiogenese und zur Unterdrückung lokaler Immunantworten führt. Aus diesem Grund stellen TAMs einen vielversprechenden Angriffspunkt für eine Krebstherapie da. In dieser Arbeit wird gezeigt, dass intrazelluläre Bakterien wie Salmonella und Shigella hauptsächlich TAMs im Tumorgewebe infizierten. Um dieses Verhalten näher zu untersuchen, konstruierten wir einen im Wachstum abgeschwächten Shigella Stamm, welcher jedoch noch die Fähigkeit hat, Apoptose in Makrophagen zu induzieren. Shigellen sind invasive Bakterien, die in das Darmgewebe einwandern und dort eine massive Inflammation induzieren. Intrazelluläre Shigellen aktivieren Caspase-1 und induzieren dadurch Apoptose in Makrophagen durch den sekretierten Virulenzfaktor IpaB. Durch eine Deletion des genomischen aroA-Gens, wurde ein Shigella Stamm konstruiert, der Defekte im intrazellulären Wachstum aufweist. Dennoch war dieser Stamm noch fähig eukaryotische Zellen zu infizieren, sich interzellulär fortzubewegen, Caspase-1 zu aktivieren und Apoptose in Makrophagen zu induzieren. Es wurde gezeigt, dass dieser Shigellen Stamm nach i.v. Injektion hauptsächlich die TAMs im 4T1-induzierten und transgenen MMTV-HER2/neu Brustkrebsmodel infizieren. Diese attenuierten Shigellen wurden im Tumorgewebe hauptsächlich intrazellulär detektiert, im Gegensatz dazu wurden attenuierte Salmonellen zu späten Zeitpunkten (7 d p.i.) auch extrazellulär im Tumorgewebe aufgefunden. Der metabolisch aber nicht in der Virulenz attenuierte Shigella Stamm konnte in beiden Brustkrebsmodellen zu allen Zeitpunkten (4 h, 6h and 7 d p.i.) Caspase-1 in TAMs aktivieren und Apoptose induzieren. Diese Apoptose führte in beiden Brustkrebsmodellen zu einer langandauernden und hoch signifikanten Reduktion der TAMs-Anzahl (bis zu 70 %). Im Gegensatz dazu konnten Salmonellen nur zu frühen Zeitpunkten (6 h p.i.) Apoptose in TAMs induzieren und dies führte in beiden Modellen zu keiner Reduzierung der TAMs-Anzahl. In dem 4T1-induzierten Tumormodel wurden die Mäuse mit dem attenuierten Shigella Stamm behandelt, was zu einer kompletten Blockierung des Tumorwachstums führte, dies traf aber nicht für den avirulenten Stamm zu. Darüberhinaus infizierte Shigella hauptsächlich die Makrophagen Fraktion eines Ovarkarzinoms ex vivo und induzierte in diesen Zellen Caspase- 1 Aktivierung und Apoptose. Zusammenfassend zeigen diese Daten, dass im Wachstum attenuierte intrazelluläre Bakterien dazu fähig sind Apoptose in TAMs zu induzieren. Dadurch werden sie zu einem vielversprechenden Therapeutikum zur Behandlung von betimmten Krebserkrankungen, bei denen TAMs eine erwiesene Rolle im Tumorwachstum und der Tumorprogression spielen. / In neoplastic diseases the tumor stroma and especially tumor-associated macrophages (TAMs) play an important role in tumor growth and progression. TAMs exhibit an intensive cross-talk with tumor cells resulting in the promotion of angiogenesis and the inhibition of local protective immune responses in certain tumor entities. Therefore, TAMs are a potential target for tumor therapy. Here it was shown that intravenously applied intracellular bacteria like Salmonella and Shigella primarily target TAMs. To exploit this feature a growth attenuated Shigella strain with the capacity to induce apoptosis in macrophages was designed. Shigella are invasive bacteria that penetrate the colonic tissue and initiate an acute inflammation. In macrophages, Shigella rapidly induces caspase-1 processing and apoptosis via the virulence factor IpaB. By genomic deletion of the aroA-locus a metabolically attenuated strain defective in intracellular growth but with retained capacity of infection, cell-to-cell spread, caspase-1 processing and apoptosis induction in macrophages was designed. It was shown that this strain primarily targets TAMs in 4T1 cell induced and transgenic MMTV-HER2/new breast cancer models. Shigella were almost exclusively found intracellularly, whereas growth attenuated Salmonella were also found extracellularly at late time points. The metabollically attenuated Shigella strain with retained virulence, but not avirulent Shigella strains, was able to activate caspase-1 and induce apoptosis in TAMs at all time points (4 h, 6 h and 7 d p.i.) in both breast cancer models. This unrestricted apoptosis induction translated into a substantial, long-lasting and highly significant reduction of TAMs number (up to 70 %) in both models. In contrast, Salmonella could only induce apoptosis in TAMs at early time points (6 h p.i.) and failed to reduce TAMs in both models. In the 4T1 model, the effect on tumor size was monitored and treatment of the mice with the attenuated Shigella strain resulted in a complete block of tumor growth. Finally, Shigella primarily infected the macrophage fraction, activated caspase-1 and induced apoptosis in cells derived from a human ovarian carcinoma ex vivo. Taken together, this data suggests that growth attenuated intracellular bacteria capable of inducing apoptosis in TAMs are a promising therapeutic option for certain cancer diseases where TAMs have a proven role for tumor growth or progression.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, 2008. / Includes "Thesis Amendments" attached to the inside back pages. "October 2007" Bibliography: leaves 108-121. Also available in print form.

Not available / text

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Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998? / Includes bibliography (27 leaves).

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1999. / Corrigenda pasted onto back end-papers. Bibliography: leaves 163-182.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-08-27T11:49:12Z No. of bitstreams: 1 texto completo.pdf: 676884 bytes, checksum: e1f9724ccc2f0d91eee10a5d92c70b8a (MD5) / Made available in DSpace on 2018-08-27T11:49:12Z (GMT). No. of bitstreams: 1 texto completo.pdf: 676884 bytes, checksum: e1f9724ccc2f0d91eee10a5d92c70b8a (MD5) Previous issue date: 2017-02-22 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A bioconservação é uma das técnicas atuais utilizadas na conservação de alimentos, que se baseia na utilização de micro‐organismos e, ou de seus metabólitos, como por exemplo, bacteriocinas. Nisina é uma bacteriocina produzida por Lactococcus lactis subsp. lactis, usada na conservação de alimentos em mais de 50 países, incluindo o Brasil. Nisina é altamente ativa contra ampla gama de bactérias gram-positivas, mas bactérias gram-negativas, como Shigella, são naturalmente resistentes à sua ação. Esta resistência está relacionada à presença da membrana externa, que atua como barreira impedindo a difusão da bacteriocina até a membrana celular, seu local de ação. O uso de estratégias para desestabilizar a membrana externa, como o ultrassom (US), pode favorecer a ação da nisina contra bactérias gram-negativas. Este estudo teve como objetivo avaliar o efeito da nisina associada ao US contra Shigella flexneri 2a em caldo infusão de cérebro e coração (BHI). Foi utilizado o delineamento composto central rotacional (DCCR) e a metodologia de superfície de resposta (MSR) com três variáveis independentes: tempo de sonicação (X 1 : 5 a 20 min), pH (X 2 : 4,0 a 7,0) e concentração de nisina (X 3 : 29,1 μM a 291,1 μM). A diferença entre o logaritmo do número de células viáveis (log UFC/mL) no início e ao final de cada tratamento foi utilizada para construção do modelo preditivo. Análise de variância (ANOVA) e oito condições adicionais foram usadas para validação do modelo. Foi avaliado a sobrevivência de S. flexneri após a sonicação na presença de nisina e estocagem sob refrigeração (7 oC) por 120 h. Para isso, duas condições foram estabelecidas (10 min de US, pH 6,0 e 175 μM de nisina e 20 min de US, pH 4,5 e 175 μM de nisina). O extravasamento de ATP também foi avaliado nestas duas condições. Para comparações dos dados foi utilizado o teste Tukey para verificar a existência de diferenças entre o tratamento combinado (nisina + US) e os grupos controles. O nível de significância adotado foi de 5%. Os resultados indicaram que o US é uma estratégia eficaz para sensibilizar S. flexneri à ação da nisina. A combinação de nisina com US resultou em sinergismo na inativação deste patógeno, sendo que a maior inativação foi observada em baixos valores de pH, concentrações altas de nisina e maior tempo de sonicação, com redução máxima de, aproximadamente, 5 ciclos logarítmicos. O modelo preditivo da combinação (nisina + US) apresentou coeficiente de determinação (R 2 ) de 0,942 e ajustado de 0,916 e o modelo foi validado. A estocagem sob-refrigeração após a sonicação na presença de nisina resultou no declínio da população de S. flexneri ao final do tempo de armazenamento que reduziu de 5,42 para 1,64 ciclo logarítmico em pH 6,0 após tratamento por 10 min de sonicação e 5,69 para 0,80 ciclo logarítmico em pH 4,5 quando tratada por 20 min de sonicação. O tratamento de nisina com US provocou o maior extravasamento de ATP intracelular em ambas as condições testadas. Estes resultados demonstram o potencial uso da nisina combinada ao US como estratégia de sanitização e conservação de alimentos. / Bioconservation is one of the current techniques used in food preservation, which is based on the use of microorganisms and their metabolites such as bacteriocins. Nisin is a bacteriocin produced by Lactococcus lactis subsp. lactis, used as a food preservative in more than 50 countries, including Brazil. Nisin is highly active against a wide range of gram-positive bacteria, but gram-negative bacteria, such as Shigella, are naturally resistant to its action. This resistance is related to the presence of the outer membrane, that acts as barrier preventing the diffusion of the bacteriocin to its site of action. The use of strategies to destabilize the outer membrane, such as ultrasound (US), can favor the action of nisin against gram-negative bacteria. This study aimed to evaluate the effect of nisin associated with US against Shigella flexneri 2a in brain and heart infusion broth (BHI). A Central Composite Rotated Design (CCRD) and response surface methodology (RSM) was performed with three parameters: sonication time (X 1 : 5 to 20 min), pH (X 2 : 4.0 to 7.0) and nisin concentration (X 3 : 29.1 μM to 291.1 μM). The difference among the logarithm of the viable cell number (log CFU/mL) at the beginning of the experiment and at the end of each treatment was used for the construction of predictive model. Analysis of variance (ANOVA) and eight additional conditions were used for model validation. The behavior of S. flexneri after sonication in the presence of nisin and storage on refrigeration (7 oC) for 120 h was evaluated. For this, two conditions were established (10 min of US, pH 6.0 and 175 μM nisin and 20 min of US, pH 4.5 and 175 μM nisin). The leakage of ATP was also evaluated in these two conditions. For comparisons of the data, the teste Tukey was used to verify the existence of differences between the combined treatment (nisin + US) and the control groups. The level of significance was set at 5%. The results indicated that US is an efficient strategy for sensitize S. flexneri to the action of nisin. The combination of nisin with US resulted in synergism in the inactivation of this pathogen being that the higher inactivation observed was low pH, high nisin concentrations and longer sonication time, with a maximum reduction of approximately 5 logarithms cycles. The predictive model of the combination (nisin + US) showed coefficient of determination (R 2 ) of 0.942 and adjusted of 0.916, and the model was validated. Refrigerated storage after sonication in the presence of nisin resulted in the decline of the S. flexneri population at the end of storage time decreasing from 5.42 to 1.64 logarithm cycle at pH 6.0 treated with 10 min of sonication and 5.69 to 0.80 logarithm cycle at pH 4.5 when treated for 20 min sonication. The treatment of nisin with US caused the highest leakage of intracellular ATP, combined with US in both conditions tested. These results demonstrate the potential use of nisin combined to US as strategy for sanitation and food preservation.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Escherichia coli enteroinvasora (EIEC) possuem características bioquímicas e genéticas semelhantes às de Shigella, porém para que ocorra um processo infeccioso são necessárias 102 células de Shigella em relação a 106 células de EIEC. A patogenicidade de Shigella e EIEC se dá pela presença de um plasmídio de virulência denominado pInv, que contêm os genes necessários para a invasão e disseminação bacteriana nas células do hospedeiro. Estudos anteriores relataram que os genes ipaA, ipaB, ipaC e ipaD de EIEC e Shigella não possuem diferenças genéticas que possam explicar sua diferença de patogenicidade. No presente trabalho, foram avaliados os níveis transcricionais dos genes envolvidos na invasão e disseminação dessas bactérias. Pela técnica de RT-PCR semi-quantitativo, pode-se observar diferenças nos níveis de transcrição para a maioria dos genes de virulência selecionados, quando as bactérias estavam em contato com os macrófagos. Porém, sem o contato com estas células, o nível de transcrição dos genes foi o mesmo entre as espécies, com exceção do gene ipaD. Foi verificada que a transcrição deste gene em contato com macrófago é praticamente a mesma entre as bactérias, enquanto que na ausência de macrófagos, o nível de transcrição é bem menor em EIEC, quando comparado no mesmo intervalo de tempo. Após estes resultados foram selecionados os principais genes para estudo por PCR em tempo real. Foi possível observar que o nível de transcrição de EIEC é menor, em relação a Shigella. Mais ainda, a análise dos genes dentro do mesmo operon mostrou uma cinética de transcrição distinta do icsB em relação a outros genes do operon das ipas. Os resultados obtidos sugerem que esta diferença de transcrição possa estar relacionada com a virulência mais branda em EIEC. Ainda mais, Os genes pertencentes ao operon icsB-ipaCB parecem ser regulados de forma distinta. Além disso, a transcrição de ipaD em EIEC, provavelmente, depende de uma via de sinalização distinta de Shigella flexneri. Diante desses resultados, novos estudos estão sendo propostos em nosso laboratório para melhor compreensão do mecanismo de virulência desse enteropatógeno. / Enteroinvasive Escherichia coli (EIEC) serotypes described so far share antigenic, biochemical, genetic and pathogenetic properties with Shigella sp. However, in order for an infectious process to occur, an inoculum of 102 Shigella cells is needed in contrast to as much as 106 EIEC cells. The characteristic ability of S. flexneri and EIEC to enter epithelial cells, multiply intracellularly and spread from cell to cell is uniquely encoded by their 220-kb virulence plasmid. Previous studies realized in our laboratory showed that the genes ipaA, ipaB, ipaC and ipaD do not possess molecular alterations in the nucleotides sequences that can explain the difference in the pathogenicity between EIEC and Shigella spp. In the present work, the transcription levels of the bacterial genes involved in the invasion and escape from host cells were evaluated. Through reverse transcription-polymerase chain reaction (RT-PCR) analysis, differences in the transcription levels for the majority selected virulence genes could be observed when bacteria were in contact with the macrophages. However, without the contact with those cells, the transcription levels of the genes were the same between both bacteria species, with the exception of ipaD. When the bacteria is in contact with macrophages, the transcription of ipaD is the same in both species whereas in the absence of macrophages, the transcription level is lower in EIEC than Shigella, when compared in the same period of time. Those results provided the selection of the genes for the real time PCR analysis. In general, the EIEC transcription levels genes are lower than Shigella. More still, the icsB showed a distinct kinetic of transcription from the others genes in the same operon. All results suggest that the lower pathogenicity due to EIEC can partially have to the differences of the virulence genes transcription. Still more, the genes-encoded by operon icsB-ipaCB seem to be regulated of a distinct form. Therefore, transcription of the ipaD in EIEC probably depends on distinct signaling way of S. flexneri. New studies shows to be necessary for the better understanding of the pathogenic mechanism of EIEC.