O antraz é uma doença infecciosa aguda grave, com uma taxa de mortalidade superior a 90% em sua forma respiratória, causada pelo Bacillus anthracis, uma bactéria altamente virulenta, que está desenvolvendo resistência e que tem potencial aplicação como arma biológica e agente de bioterrorismo. Nesse trabalho, a inibição de deidroshikimato desidratase do B. anthracis foi estuda por meio docking, dinâmica molecular e ensemble docking. Essa enzima é responsável por uma etapa chave na biossíntese de petrobactina, molécula através da qual o B. anthracis adquire ferro – micronutriente essencial para seu desenvolvimento e proliferação no hospedeiro. O docking de 25 compostos com ação inibitória conhecida na estrutura cristalográfica da enzima indicou interações importantes com os resíduos His144, His175, Phe211, Tyr217 (ligações de hidrogênio), Arg102 (ponte salina), His144 e Phe255 (interações π-π). Ligantes estruturalmente semelhantes ao cristalográfico (3,4-DHBA) foram docados adequadamente no sítio ativo, enquanto ligantes mais volumosos foram docados na entrada do sítio, resultando em baixa correlação entre as energias livres de ligação experimentais e os escores de docking (R² = 0,1295; R-Pearson = 0,360) e desvios de 23%, em média, frente ao experimental. Simulações de dinâmica molecular mostraram que essa proteína apresenta uma grande rigidez estrutural intrínseca, porém porções do seu sítio ativo, sobretudo da estrutura em forma de laço que o recobre, apresentaram flexibilidade significativa. A presença de ligantes induz a alterações conformacionais que proporcionam o alargamento do sítio e permitem a entrada de ligantes mais volumosos, indicando que o sítio cristalográfico era, de fato, muito restrito. A atividade inibitória aparenta estar relacionada com a formação de uma rede de ligações de hidrogênio entre os ligantes e resíduos do sítio ativo, sendo as principais entre grupos 3-OH do anel aromático dos ligantes e a His175; entre o grupo carboxílico e a Arg102 (ponte salina); entre o grupo 4-OH e a Phe211 e principalmente entre o grupo 5-OH e a His144, um resíduo importante no mecanismo enzimático. O ensemble docking em três estruturas extraídas das simulações de dinâmica molecular permitiu a aprimorar a correlação entre os escores de docking e atividade inibitória experimental, com R² = 0,363 e R-Pearson = 0,602 considerando a totalidade dos ligantes ou com R² = 0,8157 e R-Pearson = 0,903 considerando-se os dez ligantes mais potentes (contra R² = 0,5683 e R-Pearson = 0,754 na estrutura cristalográfica), evidenciando a necessidade de se considerar a flexibilidade do receptor para o docking adequado. Esse modelo linear juntamente com essa compreensão mais profunda dos mecanismos relacionados com a inibição dessa enzima permitirão o desenho e a triagem in silico de novas moléculas com potência e seletividade aprimoradas e com potencial aplicação como uma nova terapia contra o Bacillus anthracis. / Anthrax is a serious acute infectious disease with a mortality rate higher than 90% in its inhalational form. This disease is caused by Bacillus anthracis, a highly virulent bacterium that is developing resistance and which has potential application as a biological weapon and bioterrorism agent. In this work, the inhibition of dehydroshikimate dehydratase from B. anthracis was studied through docking, molecular dynamics and ensemble docking. This enzyme is responsible for a key step in the biosynthetic pathway of petrobactin, a molecule released by B. anthracis to acquire iron, an essential micronutrient for its development and proliferation within the host. Molecular dockings of 25 compounds with known inhibitory activity against dehydroshikimate dehydratase in the crystallographic structure of this enzyme indicated important interactions with the residues His144, His175, Phe211, Tyr217 (hydrogen bonds), Arg102 (salt bridge), His144 and Phe255 (π-π interactions). Ligands structurally similar to the crystallographic (3,4-DHBA) were appropriately docked within the active site, while bulkier ligands were docked at the site's entrance, resulting in a low correlation between the experimental binding free energies and the docking scores (R² = 0,1295; R-Pearson = 0,360), as well as a deviation of 23%, on average, compared to the experimental data. Molecular dynamics simulations showed that this protein has a high structural rigidity, however portions of its active site, especially the loop-like structure that covers it, showed a significant mobility. The presence of ligands induced conformational changes that lead to the widening of the site and allowed bulkier ligands to enter it, what indicates the crystallographic site was, in fact, very restricted. The inhibitory activity appears to be related with the formation of a network of hydrogen bonds between ligands and active site residues, mainly between the 3-OH moiety in the aromatic ring of ligands and His175; between the carboxylic group and Arg102 (salt bridge); between the 4-OH moiety and Phe211 and specially between the 5-OH group and His144, a residue with an important role in the enzymatic mechanism. Ensemble docking with three structures extracted from molecular dynamics simulations allowed to improve the correlation between docking scores and experimental inhibitory activity, with R² = 0,363 and R-Pearson = 0,602, when considering all ligands, and R² = 0,8157 and R-Pearson = 0,903 when considering the ten ligands of higher activity (against the values of R² = 0,5683 and R-Pearson = 0,754 for their docking in the crystallographic structure). This point out the need to account for receptor's flexibility for an appropriate docking. This linear model coupled with this deeper understanding about the mechanisms related with enzymatic inhibition will allow the in silico drug design and screening of new molecules with improved potency and selectivity and with potential application as a new therapy against Bacillus anthracis.

Os melanomas malignos em cães são uma das mais frequentes neoplasias diagnosticadas na cavidade oral. Infiltração local, recidiva (15-41%) e o alto potencial para metástases em linfonodos regionais (18-53%) e pulmões (23-27%) nos animais acometidos, conferem uma menor sobrevida (131-818 dias), ressaltando a necessidade e importância do estudo de novas terapias para o tratamento efetivo da doença. As uroquinases (UPA) e metaloproteinases (MMPs) são proteases superexpressas em uma variedade de células tumorais e raramente estão presentes em células fisiologicamente normais. A toxina do Bacillus anthracis é composta por três proteínas chamadas: fator letal (LF), fator de edema (EF) e antígeno protetor (PA). A toxina foi reengenheirada para a formação de dois tipos de PAs chamadas PAU2-R200A e PAL1-I207R, ativadas por UPA e MMPs da superficie das células tumorais, respectivamente, formando um complexo semelhante a um poro celular para permitir a internalização da LF. A citotoxicidade dessa associação reengenheirada PAU2-R200A, PAL1-I207R e LF ocorre quando a LF atinge o meio intracelular e causa a morte celular por interrupção da via de sinalização celular MAPkinase. O objetivo deste estudo é avaliar o potencial terapêutico da toxina reengenheirada do Bacillus anthracis, PAU2-R200A, PAL1-I207R e LF, dependentes de UPA e MMP, em melanomas orais de cães. Três etapas foram propostas para este estudo: o estudo in vitro da citotoxicidade de 5 linhagens de melanomas caninos submetidas à toxina reengenheirada, a avaliação da expressão de UPA e MMP em amostras parafinadas de melanoma oral canino e o tratamento intratumoral com a toxina modificada em cães com melanomas orais espontâneos. A linhagem GMGD2 foi a única que demonstrou sensibilidade à toxina estudada, apesar da concentração inibitória de 50% das células ter sido alta (IC50=4.964,16 mg/dl) em relação a linhagem controle HT29-RJ (IC50=179,47). As demais linhagens não demostraram redução da viabilidade celular com o aumento da concentração da toxina reengenheirada e não atingiram a IC50. Dentre as amostras de melanomas submetidos a imuno-histoquimica, 76,6% expressavam tanto uroquinases quanto metaloproteinases. Melanomas orais espontâneos de cães variando de 231,8 a 18601,6 mm3 em volume, sem evidências de metástases, foram tratados com as aplicações da toxina modificada por via intratumoral, previamente à excisão, realizada nos dias 07 ou 14 do tratamento. Dentre os animais estudados, todos apresentaram evolução favorável classificada como doença estável e resposta parcial. Somente um animal apresentou reação local. Nenhum dos pacientes apresentou efeito colateral sistêmico importante. Os resultados sugerem que existe potencial terapêutico da toxina reengenheirada do Bacillus anthracis sobre os melanomas bucais caninos e futuros ensaios clínicos são possíveis em cães e de extrema importância para o estudo mais aprofundado da toxina como nova terapia antineoplásica / Malignant melanomas in dogs are one of the most frequent malignancies diagnosed in the oral cavity. Local infiltration, recurrence (15-41%) and the high potential for regional lymph nodes metastases (18-53%) and lungs (23-27%) in the affected animals, confer a lower survival (131-818 days), emphasizing the necessity and importance of the study of new therapies for the effective treatment of the disease. Urokinase (UPA) and metalloproteinases (MMPs) are overexpressed proteases in a variety of tumor cells and are rarely present in normal physiological cells. Bacillus anthracis toxin is composed of three proteins called lethal factor (LF), edema factor (EF) and protective antigen (PA). The toxin was re-engineered for the formation of two types of PAs called PAU2-R200A and PAL1-I207R, activated by UPA and MMPs from the surface of tumor cells, respectively, forming a cell-like complex to allow the internalization of the LF. The cytotoxicity of this association PAU2-R200A, PAL1-I207R and LF occurs when LF reaches the intracellular environment and causes cell death by disruption of the MAPkinase cell signaling pathway. The objective of this study is to evaluate the therapeutic potential of UPA and MMP-dependent Bacillus anthracis toxin (PAU2- R200A, PAL1-I207R and LF) to treat oral melanomas in dogs. Three steps were proposed: cytotoxicity assay of 5 lineages of canine melanomas submitted to the reengineered toxin, immunohistochemistry study for UPA and MMP expression in paraffin samples of canine oral melanoma and intratumoral treatment with toxin in dogs with spontaneous oral melanomas. The lineage GMGD2 was the only one that showed sensitivity to the toxin studied, although 50% inhibitory concentration of the cells was high (IC50 = 4,964.16 mg / dl) in relation to the HT29-RJ control lineage (IC 50 = 179.47). Among the samples of melanomas submitted to immunohistochemistry, 76.6% expressed both urokinase and metalloproteinases. Spontaneous oral melanomas of dogs ranging volume from 231.8 to 18601.6 mm3 with no evidence of distant metastases, were treated with the applications of intratumoral re-engineered toxin prior to surgical excision. All of them has presented favorable evolution classified as stable disease and partial response. Only one animal had a local allergic reaction. None of the patients had a significant systemic side effects. The results suggest that there is a potential therapeutic effect of re-engineered anthrax toxin on canine melanomas and future clinical trials are possible in dogs and extremely important for further studies on the role of the B. anthracis toxin as a new antineoplastic agent

La virulence de la bactérie Gram+ Bacillus anthracis, responsable de la maladie du charbon, est due à la présence d'une capsule et deux toxines. Chaque toxine résulte de l'assemblage de l'antigène protecteur (PA) avec l'un des deux facteurs, létal (LF) ou oedémateux (EF), dans le cytoplasme de la cellule hôte. EF est une adénylyl cyclase, qui transforme l'ATP en AMPc de manière incontrôlée, provoquant des dérèglements cellulaires. Elle est activée par la calmoduline (CaM), impliquée dans de nombreuses voies de signalisation du calcium. Des structures cristallographiques et une étude par RMN ont montré que la stabilité du complexe EF-CaM dépend du niveau de calcium fixé à CAM. Des simulations de dynamique moléculaire du complexe, avec 0, 2 ou 4 ions calcium, ont permis de caractériser l'effet du calcium sur la plasticité conformationnelle des deux partenaires et de proposer un modèle de l'interaction EF-CaM. L'analyse conjointe des corrélations dynamiques et des influences énergétiques a fait émerger le concept de connexité du réseau de résidus comme critère de stabilité. La large transition conformationnelle induite chez EF par la fixation de CaM a été décrite, grâce à la détermination d'un chemin de réaction plausible, par modélisation moléculaire. Les conformations intermédiaires obtenues ont servi à guider la recherche rationnelle d'inhibiteurs de la toxine EF, dans le cadre d'une approche combinant méthodes computationnelles et expérimentales. Une stratégie innovante, impliquant le criblage virtuel d'une poche allostérique plutôt que du site catalytique de l'enzyme, a identifié six molécules actives, inhibant totalement l'activité de EF à des concentrations de 10-100 microM.

Bacillus anthracis, agent de la maladie du charbon, est aussi un agent majeur de la menace biologique. Sa virulence est liée à deux principaux facteurs : une capsule et deux toxines, la toxine oedémateuse (ET = EF + PA) et la toxine létale (LT = LF + PA). EF est une adénylate cyclase, calcium et calmoduline dépendante, produisant une élévation de la concentration en AMPc intracellulaire tandis que LF est une métalloprotéase à zinc clivant la majorité des Mitogen Activated Protein Kinase Kinases. Les toxines jouent un rôle central dans la pathogénie de la maladie du charbon et dans la dérégulation des fonctions des cellules du système immunitaire. Le cytosquelette d'actine participe activement aux fonctions de phagocytose et de migration des macrophages et des cellules dendritiques.Cependant, peu d'études analysent l'implication du cytosquelette d'actine des cellules immunitaires dans la physiopathologie des toxines. ET induit une rétraction temps-dépendant des cellules dendritiques et des macrophages normalisés sur des micropatterns de fibronectine, s'accompagnant d'une dépolymérisation de l'actine et d'une perte des points d'ancrage des cellules dendritiques. Précocement, ET active la cofiline par l'activation de la voie de signalisation AMPc – PKA – Protéines phosphatases. Malgré ces altérations du cytosquelette d'actine, ET n'induit pas de modification des capacités phagocytaires des cellules dendritiques, à l'exception d'une dérégulation de la maturation des phagosomes. ET conduit également à une augmentation de la migration des cellules dendritiques in vitro par activation et expression de CCR7 et CXCR4 à la surface des cellules dendritiques.A l'inverse, LT conduit à un étalement temps-dépendant des cellules dendritiques normalisées, accompagné d'une dérégulation de la dynamique de l'actine provoquant des regroupements anormaux d'actine filament. LT active les myosines phosphatases via la voie RhoA-ROCK pour déphosphoryler les myosines II. A la différence de ET, LT inhibe les capacités phagocytaires des cellules dendritiques mais ne conduit pas à une modification de la migration des cellules dendritiques in vitro. / Bacillus anthracis, the agent of anthrax, is also a major agent of biological warfare threat. Its virulence is caused by two main factors : the capsule and two toxins, edema toxin (ET = PA + EF) and lethal toxin (LT = LF + PA). EF is a calcium and calmodulin-dependent adenylate cyclase, producing a rise in intracellular cAMP concentration, while LF is a zinc metalloprotease cleaving the majority of Mitogen Activated Protein Kinase Kinases. The toxins play a central role in the pathogenesis of the disease and the deregulation of the functions of immune cells. The actin cytoskeleton is actively participating in the phagocytosis and the migration of macrophages and dendritic cells.However, few studies analyze the involvement of the actin cytoskeleton of immune cells in the pathogenesis of toxins. ET induces a time-dependent retraction of dendritic cells and macrophages on fibronectin micropatterns, accompanied by actin depolymerization and a loss of the anchor points of dendritic cells. ET early activates cofilin by activating the cAMP - PKA - Protein phosphatases signaling pathway. Despite these alterations of the actin cytoskeleton, ET does not induce any change in the phagocytic capacity of dendritic cells, except for a deregulation of the phagosomes maturation. ET also leads to an increase in the migration of dendritic cells in vitro by activation and expression of CCR7 and CXCR4 on the surface of dendritic cells.In contrast, LT results in a time-dependent spreading of micropatterned dendritic cells, accompanied by a dysregulation of actin dynamics causing abnormal combinations of actin filament. LT activates myosin phosphatase via the RhoA-ROCK pathway to dephosphorylate myosin II. Unlike ET, LT inhibits the dendritic cells phagocytosis but does not lead to a change in dendritic cells migration in vitro.

Os melanomas malignos em cães são uma das mais frequentes neoplasias diagnosticadas na cavidade oral. Infiltração local, recidiva (15-41%) e o alto potencial para metástases em linfonodos regionais (18-53%) e pulmões (23-27%) nos animais acometidos, conferem uma menor sobrevida (131-818 dias), ressaltando a necessidade e importância do estudo de novas terapias para o tratamento efetivo da doença. As uroquinases (UPA) e metaloproteinases (MMPs) são proteases superexpressas em uma variedade de células tumorais e raramente estão presentes em células fisiologicamente normais. A toxina do Bacillus anthracis é composta por três proteínas chamadas: fator letal (LF), fator de edema (EF) e antígeno protetor (PA). A toxina foi reengenheirada para a formação de dois tipos de PAs chamadas PAU2-R200A e PAL1-I207R, ativadas por UPA e MMPs da superficie das células tumorais, respectivamente, formando um complexo semelhante a um poro celular para permitir a internalização da LF. A citotoxicidade dessa associação reengenheirada PAU2-R200A, PAL1-I207R e LF ocorre quando a LF atinge o meio intracelular e causa a morte celular por interrupção da via de sinalização celular MAPkinase. O objetivo deste estudo é avaliar o potencial terapêutico da toxina reengenheirada do Bacillus anthracis, PAU2-R200A, PAL1-I207R e LF, dependentes de UPA e MMP, em melanomas orais de cães. Três etapas foram propostas para este estudo: o estudo in vitro da citotoxicidade de 5 linhagens de melanomas caninos submetidas à toxina reengenheirada, a avaliação da expressão de UPA e MMP em amostras parafinadas de melanoma oral canino e o tratamento intratumoral com a toxina modificada em cães com melanomas orais espontâneos. A linhagem GMGD2 foi a única que demonstrou sensibilidade à toxina estudada, apesar da concentração inibitória de 50% das células ter sido alta (IC50=4.964,16 mg/dl) em relação a linhagem controle HT29-RJ (IC50=179,47). As demais linhagens não demostraram redução da viabilidade celular com o aumento da concentração da toxina reengenheirada e não atingiram a IC50. Dentre as amostras de melanomas submetidos a imuno-histoquimica, 76,6% expressavam tanto uroquinases quanto metaloproteinases. Melanomas orais espontâneos de cães variando de 231,8 a 18601,6 mm3 em volume, sem evidências de metástases, foram tratados com as aplicações da toxina modificada por via intratumoral, previamente à excisão, realizada nos dias 07 ou 14 do tratamento. Dentre os animais estudados, todos apresentaram evolução favorável classificada como doença estável e resposta parcial. Somente um animal apresentou reação local. Nenhum dos pacientes apresentou efeito colateral sistêmico importante. Os resultados sugerem que existe potencial terapêutico da toxina reengenheirada do Bacillus anthracis sobre os melanomas bucais caninos e futuros ensaios clínicos são possíveis em cães e de extrema importância para o estudo mais aprofundado da toxina como nova terapia antineoplásica / Malignant melanomas in dogs are one of the most frequent malignancies diagnosed in the oral cavity. Local infiltration, recurrence (15-41%) and the high potential for regional lymph nodes metastases (18-53%) and lungs (23-27%) in the affected animals, confer a lower survival (131-818 days), emphasizing the necessity and importance of the study of new therapies for the effective treatment of the disease. Urokinase (UPA) and metalloproteinases (MMPs) are overexpressed proteases in a variety of tumor cells and are rarely present in normal physiological cells. Bacillus anthracis toxin is composed of three proteins called lethal factor (LF), edema factor (EF) and protective antigen (PA). The toxin was re-engineered for the formation of two types of PAs called PAU2-R200A and PAL1-I207R, activated by UPA and MMPs from the surface of tumor cells, respectively, forming a cell-like complex to allow the internalization of the LF. The cytotoxicity of this association PAU2-R200A, PAL1-I207R and LF occurs when LF reaches the intracellular environment and causes cell death by disruption of the MAPkinase cell signaling pathway. The objective of this study is to evaluate the therapeutic potential of UPA and MMP-dependent Bacillus anthracis toxin (PAU2- R200A, PAL1-I207R and LF) to treat oral melanomas in dogs. Three steps were proposed: cytotoxicity assay of 5 lineages of canine melanomas submitted to the reengineered toxin, immunohistochemistry study for UPA and MMP expression in paraffin samples of canine oral melanoma and intratumoral treatment with toxin in dogs with spontaneous oral melanomas. The lineage GMGD2 was the only one that showed sensitivity to the toxin studied, although 50% inhibitory concentration of the cells was high (IC50 = 4,964.16 mg / dl) in relation to the HT29-RJ control lineage (IC 50 = 179.47). Among the samples of melanomas submitted to immunohistochemistry, 76.6% expressed both urokinase and metalloproteinases. Spontaneous oral melanomas of dogs ranging volume from 231.8 to 18601.6 mm3 with no evidence of distant metastases, were treated with the applications of intratumoral re-engineered toxin prior to surgical excision. All of them has presented favorable evolution classified as stable disease and partial response. Only one animal had a local allergic reaction. None of the patients had a significant systemic side effects. The results suggest that there is a potential therapeutic effect of re-engineered anthrax toxin on canine melanomas and future clinical trials are possible in dogs and extremely important for further studies on the role of the B. anthracis toxin as a new antineoplastic agent

<p>Bacillus anthracis is a risk class III organism and needs to be handled inside a biosafety level 3-laboratory. A major problem when working with airborne, spore-forming bacteria like B. anthracis are the hazardous aerosols created when using an automated DNA-extraction method to prepare samples suspected to contain the organism. This study has therefore evaluated the possibility of enclosing a DNA-extraction-robot inside an air tight container (glove box). A prototype of a class III safety cabinet (also known as a glove box) was designed and built to enclose a BioRobot EZ1 from Qiagen. The purpose of this prototype was to evaluate the measurements needed to and also the feasibility of working with the robot inside the cabinet. During the manual DNA-extractions, there was some contamination found on the glove box gloves, probably due to the significantly lowered dexterity that was seen with the thick gloves. The enclosing of the robot revealed no obstacles as the machine was very easy to operate. In addition, protocols have been created for the operation of a transportable class III safety cabinet from Germfree available at SVA. The protocols include the different pressure tests that needed before every experiment take place and also decontamination steps before and after each run. Bacillus cereus was used as a model organism for different DNA-extractions, i.e. automated and manual extractions. The extracted DNA was analysed by real-time polymerase chain reaction (PCR). DNA was also extracted and analysed from B. cereus-spores. When using a manual DNA-extraction kit, B. cereus-DNA was detected at the femtogram level, i.e. 10-15 g DNA / PCR. When using the automated BioRobot EZ1, detection level was found to be at 10-16 g DNA / PCR. The PCR-efficiency for the manual kit was 89-90 % for all samples, whereas with the EZ1, efficiency was 99 %, showing the strengths of the magnetic bead separation used by the machine. A novel PCR-machine, the AlphaHelix QuanTyper™, was evaluated and compared to an ABI 7500 with regards to efficiency, speed and consistency. The QuanTyper™ was found to be superior in ramping speeds, performing a 40-cycle real-time PCR-run with melting point analysis in only 14 minutes. The fastest run accomplished on the ABI 7500 took 1 h 40 min. A ready made master mix for PCR was used for most tests (Platinum® SYBR® Green qPCR SuperMix-UDG), but faster and more robust enzymes are available and further studies need to be performed on the QuanTyper™ to fully evaluate the platform. Three target genes in Bacillus anthracis-DNA were analysed in only 38 minutes with efficiencies between 96-104 % for the virulence plasmids and detection at femtogram amount of DNA. This master thesis has addressed rapid pathogen-detection with automated DNA-extraction and novel PCR-technology, coupled with a strong biosafety aspect. The thesis will hopefully contribute to the surprisingly small area of biosafety and safety cabinet research.</p>

Bacillus anthracis is a risk class III organism and needs to be handled inside a biosafety level 3-laboratory. A major problem when working with airborne, spore-forming bacteria like B. anthracis are the hazardous aerosols created when using an automated DNA-extraction method to prepare samples suspected to contain the organism. This study has therefore evaluated the possibility of enclosing a DNA-extraction-robot inside an air tight container (glove box). A prototype of a class III safety cabinet (also known as a glove box) was designed and built to enclose a BioRobot EZ1 from Qiagen. The purpose of this prototype was to evaluate the measurements needed to and also the feasibility of working with the robot inside the cabinet. During the manual DNA-extractions, there was some contamination found on the glove box gloves, probably due to the significantly lowered dexterity that was seen with the thick gloves. The enclosing of the robot revealed no obstacles as the machine was very easy to operate. In addition, protocols have been created for the operation of a transportable class III safety cabinet from Germfree available at SVA. The protocols include the different pressure tests that needed before every experiment take place and also decontamination steps before and after each run. Bacillus cereus was used as a model organism for different DNA-extractions, i.e. automated and manual extractions. The extracted DNA was analysed by real-time polymerase chain reaction (PCR). DNA was also extracted and analysed from B. cereus-spores. When using a manual DNA-extraction kit, B. cereus-DNA was detected at the femtogram level, i.e. 10-15 g DNA / PCR. When using the automated BioRobot EZ1, detection level was found to be at 10-16 g DNA / PCR. The PCR-efficiency for the manual kit was 89-90 % for all samples, whereas with the EZ1, efficiency was 99 %, showing the strengths of the magnetic bead separation used by the machine. A novel PCR-machine, the AlphaHelix QuanTyper™, was evaluated and compared to an ABI 7500 with regards to efficiency, speed and consistency. The QuanTyper™ was found to be superior in ramping speeds, performing a 40-cycle real-time PCR-run with melting point analysis in only 14 minutes. The fastest run accomplished on the ABI 7500 took 1 h 40 min. A ready made master mix for PCR was used for most tests (Platinum® SYBR® Green qPCR SuperMix-UDG), but faster and more robust enzymes are available and further studies need to be performed on the QuanTyper™ to fully evaluate the platform. Three target genes in Bacillus anthracis-DNA were analysed in only 38 minutes with efficiencies between 96-104 % for the virulence plasmids and detection at femtogram amount of DNA. This master thesis has addressed rapid pathogen-detection with automated DNA-extraction and novel PCR-technology, coupled with a strong biosafety aspect. The thesis will hopefully contribute to the surprisingly small area of biosafety and safety cabinet research.

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.