Mobilização inadequada de células progenitoras hematopoéticas (CPH) tem sido observada em 10 - 30% dos pacientes submetidos a transplante de medula óssea (TMO) autogênico para tratamento de doenças onco-hematológicas. Os fatores relacionados com má resposta à mobilização ainda não estão totalmente estabelecidos. Apresentamos uma análise retrospectiva de pacientes submetidos à TMO autogênico com o objetivo de identificar variáveis associadas com resposta ruim ao regime de mobilização utilizado. Casuística e Métodos: Fizeram parte desta análise 307 pacientes com diferentes diagnósticos, tratados com TMO autogênico em uma única Instituição, no período de Abril de 2001 a Abril de 2007. Todos os pacientes incluídos no estudo foram submetidos a um único regime de mobilização baseado na administração de ciclofosfamida (dose total de 60-120 mg/kg de peso IV) e fator estimulador de colônias de granulócitos (G-CSF) (dose diária de 6 - 17 ug/(kg de peso)/dia SC). O sucesso na resposta ao regime de mobilização foi definido quando um número maior ou igual a 2,0x10 (6) células CD34 + /(kg de peso) foi coletado do sangue periférico com até três procedimentos de leucaférese. Resultados: Dos pacientes analisados, 260 apresentaram sucesso na mobilização (84,7%). Nestes pacientes, um número mediano de 3,67 (2,0 - 46,0) células CD34+ /(kg de peso) foi coletado por paciente com um número mediano de 1 (1-3) procedimento de leucaférese. O insucesso na mobilização foi observado em 47 pacientes (15,3%): 24 (7,8%) que foram submetidos à coleta de CPH de sangue periférico, porém não coletaram número maior ou igual 2,0x10 (6) células CD34+/(kg de peso) com pelo menos três procedimentos de leucaférese; e, 23 (7,5%) foram submetidos à coleta de CPH por punção da medula óssea, por não terem atingido número mínimo de 10 células CD34+/mm3 no sangue periférico para realização de leucaférese. De acordo com análise univariada, os fatores associados com o insucesso foram: diagnóstico (P < 0,0001), tempo de doença (P < 0,0001), número prévio de ciclos de quimioterapia (P = 0,0001), exposição prévia a agentes alquilantes (P = 0.0003) e a mitoxantrone (P = 0,0006), contagem de plaquetas pré-mobilização <150.000/mm3 (P = 0,0006) e intervalo entre o início da mobilização e o pico de células CD 34+ no sangue periférico (P < 0,0001). Idade, sexo, atividade da doença e envolvimento medular ao início da mobilização, tratamento prévio com radioterapia e exposição a análogos da platina não mostraram correlação significativa na resposta à mobilização. Após análise multivariada, as variáveis que permaneceram associadas com insucesso na mobilização foram: diagnóstico (P = 0,0232), número prévio de ciclos da quimioterapia (P = 0,0167), tratamento prévio com mitoxantrone (P = 0,0285) e contagem de plaquetas pré-mobilização < 150.000/mm3 (P = 0,0423). Conclusão: A carga cumulativa de quimioterapia administrada, exposição prévia à mitoxantrone, contagem de plaquetas pré-mobilização e diagnóstico foram os fatores independentes relacionados com a falha na resposta à mobilização. Os achados obtidos podem auxiliar no reconhecimento de pacientes de risco para resposta ruim à mobilização e permitir um planejamento alternativo ou mais agressivo no regime de mobilização para este grupo de pacientes. / Inadequate stem cells mobilization is seen in 10-30% of patients undergoing autotransplantation for hematologic malignancies. Factors affecting peripheral blood progenitor cell (PBSC) mobilization have not been clearly established. We retrospectively reviewed the data of patients treated by autologous bone marrow transplantation (BMT) with the aim to identify factors associated with poor PBSC mobilization. Design and Methods: We evaluated 307 patients with different diagnoses, submitted to autologous BMT between April 2001 and April 2007. PBSC were collected following mobilization with cyclophosphamide (60-120 mg/kg of weight IV) and granulocyte-colony stimulating factor (G-CSF) (dose of 6-17 ug/kg of weight/day SC). Success in mobilization was defined when > ou = a 2,0x10(6) CD34+ cells/(kg weight) could be collected from the peripheral blood with a maximum of three leukapheresis procedures. Clinical and laboratory parameters at the time of mobilization were analyzed for correlations with the number of CD34+ cells collected. Results: Two hundred and sixty patients (84.7%) presented success in mobilization. In this group, a median of 3.67 (2.0-46.0) CD34+ cells/(kg weight) was collected per patient in a median of 1(1-3) leukapheresis procedure. Poor response to mobilization was observed in 47 patients (15.3%): 24 (7.8%) were submitted to PBSC collection but didn\'t collected at least 2.0 x 106 CD34+ cells/(kg weight) with three leukapheresis procedures and 23 (7.5%) didn\'t reach an absolute number count of 10 CD34+ cells/mm3 in the peripheral blood to start collection by leukapheresis. In univariate analysis poorer PBSC mobilization was associated with diagnosis (Pp < 0.0001), time interval from the diagnosis to mobilization (P < 0.0001), number of cycles of previous chemotherapy (P = 0.0001), previous treatment with alkylating agents (P = 0.0003) and mitoxantrone (P = 0.0006), platelet count <150.000/mm3 before mobilization (P = 0.0006) and interval between mobilization and peak of CD34+ cells in peripheral blood (P < 0.0001). No significant correlation was found with age, gender, disease status, marrow involvement at mobilization, prior radiation therapy and exposition to platin analogues. In the stepwise regression model, diagnosis (P = 0.0232), number of cycles of previous chemotherapy (P = 0.0167), previous treatment with mitoxantrone (P = 0.0285) and platelet count <150.000/mm3 before mobilization (P = 0.0423) were found to be independent negative predictive factors for CD34+ cells mobilization. Conclusion: Cumulative load of chemotherapy, exposition to Mitoxantrone, platelet count just prior to mobilization and diagnosis were independent factors related to poor progenitor cells mobilization. These results could help in the previously recognition of patients at risk for poor or no response to mobilization and allow to plan an alternative or more aggressive regimen for this group of patients.

Introdução: O leite pode ser considerado um dos alimentos mais completos por apresentar alto teor de proteínas e sais minerais, porém, também é considerado excelente meio de cultura para microrganismos. Objetivos: Identificar o número de UFC de Staphylococcus spp. e Klebsiella pneumoniae, cultivados isoladamente ou em associação, em leite integral estéril, a 6 oC, 27 oC e 37 oC e descrever a curva de morte térmica, quando submetidas ao processo de pasteurização lenta isoladamente ou em associação. Material e Métodos: Avaliação através da contagem das UFC, do comportamento de Staphylococcus spp. e K. pneumoniae, isoladas de tanque de refrigeração de leite, submetidas a temperaturas que simulam o leite em condições de refrigeração (6 °C), condições ambientais (25 °C) e na temperatura ideal de crescimento de patógenos mesófilos (36°C). Avaliação da sensibilidade de Staphylococcus spp. e K. pneumoniae isoladas e em associações ao processo de pasteurização lenta. Resultados: Na escala 1 de Mac Farland a média de UFC de K. pneumoniae foi maior que a de Staphylococcus spp. A 6 °C as bactérias no leite, isoladas ou em associação crescem na mesma velocidade. A 25 °C a K. pneumoniae cresce mais que o Staphylococcus spp. A 25 °C K. pneumoniae associada ao Staphylococcus spp. cresce mais do que quando encontra-se isolada. A 36 °C K. pneumoniae associada ao Staphylococcus spp. cresce mais do que quando encontra-se isolada e ainda mais que a 25 °C. A pasteurização lenta foi efetiva, pois reduziu em no mínimo 90% as UFC no leite após 30 minutos a 65 °C. Conclusão: a medida que aumenta a temperatura, até 36 °C, a Klebsiella pneumoniae apresenta crescimento superior ao Staphylococcus spp., a contaminação de leite por Klebsiella pneumoniae (contaminação ambiental) irá influenciar mais na qualidade do produto final, comparando-se à contaminação por Staphylococcus spp., oriundo de mastite e a interação de microrganismos altera a morte dos mesmos, recomendando-se que novos estudos sejam realizados para que se entenda melhor esse processo. / Introduction: The milk can be considered one of the most complete food for its high contents of protein and minerals, but also it is considered an excellent medium of culture for microorganisms. Objective: To identify the number of the Colony Forming Units of Staphylococcus spp. and Klebsiella pneumoniae, in integral sterile milk, at different temperatures (6ºC, 27ºC e 37ºC) and to describe a thermal curve death, when submitted to the process of slow pasteurization isolated or in association. Materials and methods: Evaluation by counting of the colony forming units and the behavior of the Staphylococcus spp. and Klebsiella pneumoniae, isolated and in association occurred in the cooling tank, submitted to temperatures that simulate the conditions of milk in cooling (6ºC), environmental condition (25ºC) and the optimal temperature for the growth of microorganisms (36ºC). Evaluation of the sensibility of Staphylococcus spp. and Klebsiella pneumoniae isolated and in association during the pasteurization process. Results: In Scale 1 of Mac Farland the average of the colony form units of the K. pneumoniae was greater than the colony of Staphylococcus spp. At 6ºC the microorganisms in the milk isolated or in association grew at the same speed. At 25ºC K. pneumoniae associated with Staphylococcus spp. grew more than when they were isolated. At 36ºC K. pneumoniae associated with Staphylococcus spp. grew more then when it was isolated and more than at 25ºC. So we can conclude that the slow pasteurization was effective, because it has reduced at least 90% of the colony form unity in the milk after 30 minutes at 65ºC. Conclusion: By raising the temperature up to 36ºC, a Klebsiella pneumoniae had a superior growth when compared with Staphylococcus spp.; the contamination of milk by Klebsiella pneumoniae (environmental contamination) influences the most the quality of the final product when compared to the contamination by Staphylococcus spp. from mastitis, we can also conclude that the interaction of microorganisms modify the death rate of them. New studies and researches are recommended to a deeper and better comprehension of this process.

Objetivo: Considerando que a precisão na adaptação do implante ao intermediário transmucoso é fator importante para a longevidade da prótese e que os sistemas de implantes possuem origens diversas na sua fabricação, nos propusemos a verificar a infiltração da bactéria Enterococcus faecalis na interface entre intermediário transmucoso e implantes do tipo Cone Morse e Hexágono Interno no sistema Neodent . Material e Método: A amostra foi composta por 20 implantes do tipo Cone Morse com Index Protético, 20 implantes Titamax Cone Morse e 20 implantes de Hexágono Interno e 20 intermediários transmucosos do tipo Munhão Universal CM Exact, 20 Munhão Universal em corpo único de 3,3 x 5,5 mm e 20 Munhão Universal II Plus com Parafuso. Com o auxílio da Micropipeta o Inóculo foi aspirado, imediatamente após, inserido no interior do implante que se encontrava estático na placa do Dispositivo desenvolvido para este experimento. Os intermediários foram então conectados e apertados com chave digital própria, em seguida, foi dado o torque ao parafuso do intermediário de acordo com as recomendações do fabricante. Após o torque o conjunto, implante-intermediário foi removido da placa do Dispositivo com pinça de titânio e inserido dentro do tubo de ensaio que continha o meio de cultura estéril. Diante do conhecimento da área interna de cada implante, o volume do Inóculo a ser utilizado foi determinado em 1L. Todos os tubos contendo os conjuntos foram numerados e incubados a 37oC por um período de 14 dias. Foi realizado acompanhamento diário para verificação da possível passagem de bactérias do interior do implante para o caldo. O indicativo da passagem de bactéria para o meio foi a turvação do meio de cultura (caldo BHI). Decorrido os 14 dias de observação, os dados foram tabulados e submetidos à Análise de Variância (ANOVA) com nível de significância de 5%, quando detectada diferença estatística, os valores foram submetidos ao Teste de Tuckey para comparações múltiplas. Resultados: O Teste de Tukey evidenciou que houve diferença estatisticamente significante entre o grupo de implantes Cone Morse, Cone Morse Índex e Hexágono Interno, onde o grupo de implantes Cone Morse diferiu estatisticamente do grupo Cone Morse Índex e também do grupo de implantes de Hexágono Interno. O grupo de implantes Cone Morse Índex não diferiu estatisticamente do grupo de implantes de Hexágono Interno, porém o grupo Cone Morse Índex presentou os melhores resultados. / Objective: Whereas the accuracy adaptation of the implant abutment is an important factor for the longevity of the prosthesis, and implant systems have diverse backgrounds in its manufacture, we set out to verify the infiltration of Enterococcus faecalis bacteria at the interface between the abutment and transmucosal implants of Morse Taper and Internal Hex of Neodent system. Material and Methods: The sample consisted of 20 Morse Taper implants, 20 Titamax Morse Taper implants, 20 Internal Hexagon implants and 20 transmucosal Exact CM Universal Post abutment, 20 Universal Post 3 in one body , 3 x 5.5mm and 20 Universal Post II Plus with Screw . With the aid of a micropipette the inoculum was aspirated, immediately thereafter, inserted into the implant that was in the static device board developed for this experiment. The abutments were then connected and tightened to own digital key, then the torque was given to the screw of the abutment according to the manufacturer\'s recommendations. After the torque, deploy abutment was removed from the plate clamp device of titanium and inserted into a test tube containing sterile culture. According to the internal area of each implant, the inoculum size was determined to be 1L. All the tubes were numbered and incubated at 37 ° C for a period of 14 days. Daily monitoring to check the possible passage of bacteria into the interior of the implant broth was carried out. The indication of the passage of bacteria was turbidity of the culture (BHI). Upon expiry of the 14 days of observation, the data were tabulated and an analysis of variance (ANOVA) with a significance level of 5 % was done. The values were submitted to Tukey test for multiple comparisons. Results: The Tukey test showed a statistically significant difference between the group of Morse Taper implants, Cone Morse Index and Internal Hex, where the group of implants differed from Morse Taper, Morse Taper Index group and also the group of Internal Hexagon implants. The group of Morse Taper implants Index did not differ statistically from the group of Internal hexagon implants, but Morse Taper Index group showed the best results

The cytokines GM-CSF, IL-3 and IL-5 are central regulators of haemopoietic cell functions and are pivotal in the regulation of haemopoiesis and inflammatory responses of myeloid cells. In particular, these cytokines have been shown to perform essential functions in host defence against foreign pathogens through their ability to regulate innate immune responses in myeloid cells. As key regulators of such important processes, these cytokines play an important role in human inflammatory pathologies such as rheumatoid arthritis, asthma, multiple sclerosis and psoriasis as well as a number of leukemias such as JML and CMML. GM-CSF, IL-3 and IL-5 signal through receptors containing α subunits specific to each cytokine and a common β subunit (βc). Cytokine stimulation leads to tyrosine phosphorylation of the βc and promotes specific responses such as proliferation, survival and activation of haemopoietic cells. Mouse knockout studies identified a key function of these cytokines in the activation of effector functions of myeloid cells, including production of reactive oxygen species (ROS) and phagocytosis. These earlier studies provide a link between cytokine signalling and inflammation, but the molecular mechanisms by which βc activation regulates effector cell functions, and the receptor motifs involved, are unknown. The aim of this thesis was to address two broad questions with regard to βc signalling: (1) Does βc regulate specific cellular responses by phosphotyrosine-independent mechanisms? (2) What are the molecular mechanisms by which βc initiates signalling to promote specific biological responses such as activation of effector cell functions? To address the first question, we have focussed on Serine 585, a potential 14-3-3 binding site which lies in the cytoplasmic potion of huβc. Out results show that the mutation huβc S585G disrupted the interaction of 14-3-3ζ with βc, whilst not affecting receptor tyrosine phosphorylation. Both mouse and human βc were shown to interact with 14-3-3 proteins, indicating that this interaction is conserved between these species. Significantly, a huβc S585G mutant was unable to promote haemopoietic cell survival in response to IL-3. These results identify a new mechanism by which cytokine receptors are able to couple to downstream signalling pathways that regulate cell survival. An approach was developed and optimised to analyse specific GM-CSF-mediated responses in monocytes/macrophages expressing wildtype or mutant huβc, (including huβc S585G that was defective in regulating survival). Bone marrow-derived muβc -/-;muβIL-3 -/- monocytes/macrophages were retrovirally transduced with constructs expressing wildtype or mutant huβc, along with huGMRα, then purified by FACS. Two assays were established to measure effector functions in the transduced monocyte/macrophages; (1) a flow cytometry assay for ROS production, and (2) an assay for phagocytosis. The capacity for GM-CSF to prime (i.e. enhance effector functions) ROS production and phagocytosis was investigated in huGMRα-transduced monocytes/macrophages. Our results have identified two key residues in the cytoplasmic domain of βc subunit: Tyrosine 577 (required for huβc interaction with the adaptor protein Shc) and serine 585 (required for 14-3-3 association), that are essential for the ability of GM-CSF to regulate key effector functions in monocytes/macrophages. These novel findings are significant in that they establish a molecular link between the GM-CSF/IL-3/IL-5 receptor and the regulation of both haemopoietic cell survival and inflammatory responses, and therefore have important implications in our understanding of inflammatory diseases such as rheumatoid arthritis and asthma. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317007 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

In 1878, Britain occupied Cyprus to protect imperial interests in the Near East and India, interests, both strategic and economic, that Russian expansion threatened and Ottoman weakness undermined. By 1912, Cyprus had become a pawn. The island had not been converted into the strategic, economic or political base to protect and extend British interests in the Near East. The policy of 1878 had failed because it was perceived rather than actual benefits that underlay the imposition of British rule. / The primary aim of this study is to present Cyprus as a failed case of imperialism. Historians have traditionally claimed that Cyprus was a strategic asset within the British imperial structure throughout British rule (1878-1960). That notion is challenged for the first phase of British rule – from the occupation of Cyprus in 1878 to when it was annexed in 1914 and then offered to Greece in October 1915. The approach is to situate the island within the British imperial imagination, which will help to understand why the island was occupied, and then to situate it within the British imperial structure after it was occupied to determine its place, value and viability. Understanding British politics and imperial policy is vital when trying to grasp the complexities of the imperial imagination(s) and the role of Cyprus within the imperial structure. This dissertation will show that perceptions generate reality and inform policy and that often these perceptions are imagined and exaggerated and thus, not based on evidence or reality. / This study will show that the British perceived Cyprus within two competing imaginaries that were at the heart of an imagined European spiritual identity: the Christian/Crusader/Holy Land tradition and that of Ancient Greece. The first tradition helps to explain why Cyprus was occupied; understanding the second provides one of the main reasons why the British failed in their imperial venture in Cyprus. Many British Conservative politicians and those that knew the Near East, through their imagined view of the Holy Land and their travels, diplomatic and military careers, situated Cyprus within the first tradition. They considered it strategically vital to the Levant and beyond to Armenia and Mesopotamia. Liberal leaders perceived Cyprus to be apart of Europe and, more significantly, within the unitary ideal of Modern Greece that the British had fashioned in continuum of the unitary ideal of Ancient Greece. Although the identity of the Cypriots was complex, the British imposed – unwittingly – modernity on the Cypriots. / Ultimately, it was the latter imagination that became dominant and with the failure of Cyprus to have a place within imperial strategy, it became a pawn to be parted to Greece with.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719

Silicon-based electric DNA chip arrays were utilized to fast identify pathogenic microorganisms with respect to the capacity to produce toxins involved in foodborne poisoning and infections. Bacteria of the B. cereus and the enterohemorrhagic E. coli (EHEC) groups contain different set-ups of various virulence factors that are encoded by the corresponding genes. The purpose of this work was to develop a fast and simple method for determination of the presence of these virulence genes in a colony from primary enrichment cultures. A target gene is detected through hybridization to a surface-immobilized specific capture probe and biotin-labeled detection probe. Following binding of an enzyme conjugate to this sandwich hybrid complex, a current signal is generated by electronic redox recycling of the enzymatic product paminophenol (pAP). Two versions of the assay were developed. In the first version the capture probes were immobilized on magnetic beads, which carried out all reactions until the pAP generation, while the final electric signal was created by transferring pAP to a single-electrode chip surface. In the second version a silicon chip array with 16 parallel sensing electrode positions each of them functionalized by capture probes, carried out all assay steps on the chip surface. This instrument can realize automatic and multiplexed gene detection. The kinetics of bacterial cell disruption and impact of DNA fragmentation by ultrasound were determined. The experimental data suggested that the increased signal after first minutes of ultrasonication were due to the accumulation of released DNA amount, while the further signal increase resulted from the improved hybridization with the shortened target DNA strands. Studies on probe localization on the 16-electrode chip assays indicated that the probe-targeting site, which was located at the 5’-end of strands, gave rise to the highest signal level due to the efficient targetprobes hybridization and the following enzyme binding. When these functionalized chip arrays were exposed to the cell homogenates, the sensing electrodes were fouled by cellular proteins and therefore led to dramatically decreased redox-recycling current. To circumvent this, samples were treated by DNA extraction after the 1st sonication and then DNA fragmentation by a 2nd time sonication. The DNA extract removed most of the interfering components from bacterial cell. This sample treatment was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the chip arrays functionalized by eight DNA probes. The signal patterns of eight virulence genes from chip assays agreed well with PCR control analyses for both strains. By simply adding the SDS detergent to cell homogenates, chip surface blocking effect can be significantly reduced even without DNA extraction treatment. After optimization of some critical factors, the 16-electrode DNA chips with the improved sensing performance can directly detect multiple virulence genes from a single E. coli colony in 25 min after the introduction of supernatant of ultrasonicated cell lysate. / QC 20100824

Steel space frames having irregular shapes when subjected to lateral loads caused by wind or earthquakes undergo twisting as a result of their unsymmetrical topology. As a result, torsional moment comes out which is required to be resisted by the three dimensional frame system. The members of such frame are generally made out of steel I sections which are thin walled open sections. The simple beam theory is not adequate to predict behavior of such thin-walled sections under torsional moments due to the fact that the large warping deformations occur in the cross section of the member. Therefore, it is necessary to consider the effect of warping in the design of the steel space frames having members of thin walled steel sections is significant. In this study the optimum design problem of steel space frames is formulated according to the provisions of LRFD-AISC (Load and Resistance factor design of American Institute of Steel Construction) in which the effect of warping is also taken into account. Ant colony optimization and harmony search techniques two of the recent methods in stochastic search techniques are used to obtain the solution of the design problem. Number of space frame examples is designed by the algorithms developed in order to demonstrate the effect of warping in the optimum design.

<p>Silicon-based electric chip arrays were developed for characterization of Bacillus</p><p>cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all</p><p>chemical reactions until and including the enzymatic reaction took place in an</p><p>eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control</p><p>cells of B. subtilis. The 16 positions of the silicon chip array were activated by</p><p>immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation.</p>

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.