Lipopolysaccharide (LPS), one of the main components of Gram-negative bacterial cell walls, is a potent endotoxin. Structures of the unique protective monoclonal antibody (mAb) WN1 222-5 in complex with Escherichia coli R2 and R4 LPS core regions show that recognition occurs in a manner similar to the innate immune receptor Toll-like receptor 4 (TLR4). Inner core LPS is shown to exist in a conserved epitope with multiple intramolecular interactions that allows the conserved epitope to bind strongly to mAb WN1 222-5. The structure of mAb FDP4, directed against truncated E. coli J-5 LPS, shows a deep pocket combining site specific for a terminal epitope that does not allow room for wild type (wt) LPS. Research into these anti-LPS binding mAbs opens up new avenues for potential septic shock therapy. The explosion of new techniques and bright x-ray sources in the 80’s and 90’s led to rapid advancement of protein x-ray crystallography; however, structure determination on some of the most important problems is now stalled due to the general inability to grow crystals of sufficient size. Recent advances in electron microscopy (EM) technology has led to improved beam characteristics, which has allowed the initiation of research to develop EM as a viable alternative to x-ray crystallography. In this research, method development using standard equipment to explore potential avenues for analysing three-dimensional protein crystals via EM has been explored. / Graduate / 0982 / 0487 / 0537 / kgomery@uvic.ca

Two underground concrete manure reservoirs of 70 m$ sp3$ were tested for their infiltration rate to water as well as to 1% and 3% total solid dairy manure. Positive and negative infiltration were measured for the water test while only negative infiltration was measured with the manure. / Positive infiltration is met when the water table into the soil is above the liquid level into the reservoir. In that case, the water is moving from the outside to the inside of the reservoir. An opposite situation is met when the infiltration is negative. / From the results, the infiltration rate varied from 0.00 to 6.684 $ times$ 10$ sp{-7}$ m/s. The minimum value was recorded at the reservoir 55 with 1% of total solid manure and the maximum value was recorded with same reservoir with positive pressure to water.

On-site sewage systems, such as septic tank-absorption trenches, are used by approximately 20 000 people who live within the catchments that supply Sydney??s drinking water. These systems discharge sewage, treated to varying degrees depending on the system type and level of maintenance, to the environment. This can result in contamination of drinking water supplies if systems are not designed or managed appropriately. The aim of the project was to develop a methodology to define appropriate buffer distances between on-site sewage systems and waterways in Sydney??s drinking water catchments, to ensure the protection of drinking water quality. Specific objectives included: identifying the current status of on-site sewage management; assessing the effluent quality and treatment performance of septic tanks, aerated wastewater treatment systems (AWTS) with disinfection and an amended material sand mound; and development of an appropriate methodology for delineating buffer distances and assessing development applications. Viruses were used as a focus for delineating the buffer distances due to their mobility and robustness in the environment, and the potential health consequences of their presence in drinking water. A Quantitative Microbial Risk Assessment (QMRA) model was developed to calculate the cumulative impact of the on-site sewage systems in the Warragamba catchment based on data from literature and experiments, with consideration of virus loads from sewage treatment plants within the catchments. The model enabled consideration of what was a tolerable impact in terms of the resulting infections within the community. The QMRA the tolerable loads of viruses from the Warragamba catchment were 108 viruses per year in raw water and 104 viruses per year in treated water. A log reduction method was developed to facilitate individual site development assessments. This method was compared to other management approaches to development assessment: fixed minimum buffer distances of 100m, reducing failure rates to zero, and the use of a preferred system. Each of these methods had a limit for how much they could reduce virus loads to the catchment due to either failure or short buffer distances at some sites. While the log reduction method is limited by the failure rates, the method provides a quantitative measure of risk by which maintenance inspections can be prioritised.

The prediction of the expected long-term performance of concrete exposed to sewage and similar materials can be difficult as it is affected by a large number of parameters. In addition, the deterioration process in concrete is generally slow. The focus of the study was to ascertain the life expectancy of concrete septic tanks located in rural Victoria. In developing the accelerated test method, ASTM C 192, ASTM C 452, ASTM C 1293 and ASTM C 109 standard procedures were adopted wherever possible. From the analysis of mass change data after 350 days, it was found that the mass change rate for concrete in sulphate solutions increases with the increase of concentration of Na2SO4 solution. The weights of the samples in Na2SO4 solution increased with time, whereas the control specimens lost weight as a result of heating cycles. The probable reason for weight increase in Na2SO4 solution was hypothesized as the formation of gypsum (CaSO4.2H2O) and ettringite (3CaO.Al2O.3CaSO4.32H2O), which is confirmed from microstructural analysis. The rate of weight gain was higher at the beginning and reduced with time. The stronger the concentration of Na2SO4 the stronger was the weight gain or expansion of mass. All the samples in sulphate solutions attained their maximum weight at around 250 days. For the specimens in sulphuric acid solutions weight loss was observed to be higher for higher concentrations. The lower the pH of the acidic solutions, the larger was the weight loss. The weight loss of specimens in acidic solutions exceeded the control specimen after 250 days. The reason for the loss of weight of the samples in sulphuric acid may be the decalcification of C-S-H gel within the concrete, and as a consequence the loss of cementitious structure. Comparison of the corrosion of concrete and also microstructural examination of field samples confirmed that the deterioration mechanism is similar to that observed in the laboratory. The accelerated testing adopted here offers a realistic method of predicting the deterioration of septic tanks under biogenic sulphuric acid corrosion. Two equations have been proposed to predict deterioration due to sulphate attack and sulphuric acid attack as mass loss (or gain) with time.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 118 p. : ill. (some col.), map. Includes abstract. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available online.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1985. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 264-281).

Submitted by ISADORA PIMENTEL DE SOUZA (euisaps@yahoo.com.br) on 2018-08-10T17:48:48Z No. of bitstreams: 1 DISSERTACAO 10.08.pdf: 1322071 bytes, checksum: bb198d40ebd35093636b6713a6bab637 (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-08-13T18:02:13Z (GMT) No. of bitstreams: 1 souza_ip_me_bot.pdf: 1322071 bytes, checksum: bb198d40ebd35093636b6713a6bab637 (MD5) / Made available in DSpace on 2018-08-13T18:02:13Z (GMT). No. of bitstreams: 1 souza_ip_me_bot.pdf: 1322071 bytes, checksum: bb198d40ebd35093636b6713a6bab637 (MD5) Previous issue date: 2018-07-23 / Introdução: O choque séptico é uma importante causa de morbimortalidade em recém-nascidos prematuros. O lactato vem sendo estudado como marcador diagnóstico e prognóstico do choque, mas os estudos no período neonatal são escassos. Objetivo: Investigar se os níveis séricos de lactato podem predizer o risco de morte em prematuros com choque séptico. Métodos: Estudo retrospectivo, longitudinal, realizado na UTI Neonatal do Hospital das Clinicas da Faculdade de Medicina de Botucatu no período de janeiro de 2014 a dezembro de 2016, após aprovação do CEP. Foram incluídos todos os prematuros menores que 37 semanas, internados na UTI, com mais de 72 horas de vida, que apresentaram diagnóstico de choque séptico e dosagem do lactato sérico nas primeiras 48 horas do choque. Foram excluídos aqueles com malformações múltiplas, infecções congênitas e erros inatos do metabolismo. Variáveis estudadas: maternas, gestacionais, neonatais, agente etiológico e dosagem do lactato. Os prematuros foram comparados em dois grupos: sobrevida e óbito. Desfecho: óbito. Análise estatística: testes paramétricos e não paramétricos com significância estatística se p<0.05. Acurácia do lactato: sensibilidade, especificidade, valor preditivo positivo e negativo. Resultados: Dentre os 456 prematuros sobreviventes por mais de 72 horas, 130 apresentaram sepse tardia (28,5%) e destes, 36 choque séptico (28%). A mortalidade no choque foi de 42%. A positividade em hemocultura foi de 36% com predomínio de Gram-positivos no grupo sobrevida e de Gram-negativos no grupo óbito. A comparação entre os grupos sobrevida e óbito, respectivamente mostrou: idade gestacional 29,5sem vs 27,5sem (p=0.05); peso de nascimento 950g vs 900g (p=0.386), idade no diagnóstico do choque 11 dias vs 7 dias (p=0.071), uso de drogas vasoativas 52% vs 93% (p=0.011). Os níveis de lactato foram maiores no grupo óbito (1,2mmol/L vs 1,7 mmol/L; p=0.043). O lactato  4mmol/L apresentou boa acurácia na predição de morte diretamente relacionada ao choque (89%) com baixa sensibilidade, 33%, mas com especificidade e valor preditivo positivo de 100% e valor preditivo negativo de 88%. Conclusão: A incidência e mortalidade do choque séptico em prematuros foram altas, sendo os muito prematuros e os de extremo baixo peso os mais acometidos. Valores de lactato  4 mmol/L apresentaram boa acurácia na predição de morte, alta especificidade e alto valor preditivo positivo. / Introduction: Septic shock is an important cause of morbidity and mortality in premature infants. Lactate has been studied as a diagnostic and prognostic marker of shock, but studies in the neonatal period are scarce. Objective: To investigate if serum lactate levels can predict the risk of death in preterm infants with septic shock. Methods: Retrospective, longitudinal study performed at the Neonatal Intensive Care Unit (NICU) of the Clinics Hospital – Botucatu School of Medicine, from January 2014 to December 2016, after approval of the Ethics Committee. All preterm infants less than 37 weeks gestational age, with more than 72 hours of life, admitted at the NICU with diagnosis of septic shock and serum lactate dosage in the first 48 hours of shock were included. Those with multiple malformations, congenital infections and inborn errors of metabolism were excluded. Variables studied: maternal, gestational, neonatal, etiologic agent and lactate dosage. The preterm infants were compared in two groups: survival and death. Outcome: death. Statistical analysis: parametric and non-parametric tests with statistical significance if p<0.05. Lactate accuracy: sensitivity, specificity, positive and negative predictive value. Results: Among the 456 preterm infants who survived for more than 72 hours, 130 had late onset sepsis (28.5%) and of these 36 septic shock (28%). The shock mortality was 42%. The positivity in blood cultures was 36%, with a predominance of Gram-positive in the survival group and Gram-negative in the death group. The comparison between survival and death groups, respectively, showed: gestational age 29.5weeks vs 27.5 weeks (p=0.05); birth weight 950g vs 900g (p=0.386), age at shock diagnosis 11 days vs 7 days (p=0.071), vasoactive drugs 52% vs 93% (p=0.011). Lactate levels were higher in the death group (1.2mmol/L vs 1.7 mmol/L, p=0.043). Lactate ≥4 mmol/L showed good accuracy in predicting death directly related to shock (89%) with low sensitivity 33%, but with specificity and a positive predictive value of 100% and a negative predictive value of 88%. Conclusion: The incidence and mortality of septic shock in premature infants were high, and very premature and extremely low birth weight were the most affected. Values of lactate  4 9 mmol/L showed good accuracy in predicting death, high specificity and high positive predictive value.

Pacientes críticos são indivíduos internados em Unidades de Terapia Intensiva (UTI) e se caracterizam por apresentar um quadro patológico crítico e complexo, decorrente de fragilidades fisiológicas graves, podendo evoluir para sepse, sepse severa, choque séptico ou óbito. Muitos recursos financeiros são investidos no controle da sepse em hospitais públicos e privados, e a sepse é a maior causa de morte entre as UTIs brasileiras. O desfecho de um paciente crítico é influenciado por vários fatores, entre eles, os genéticos. A molécula HLA-G apresenta variabilidade proteica limitada e expressão tecidual restrita. A interação entre moléculas de HLA-G e os receptores KIR e LILR desencadeia diversas atividades imunomodulatórias. Na região 3’ UTR localizam-se os polimorfismos +2960INDEL, +3142C>G e +3187A>G que podem regular a expressão de HLA-G. Até o momento, apenas um estudo investigou a expressão de sHLA-G5 em pacientes críticos com choque séptico, e se observou que o aumento dos níveis de sHLA-G5 foram preditivos de sobrevivência entre os pacientes que evoluíram para choque séptico. O objetivo do presente trabalho foi determinar as frequências alélicas do polimorfismo +2960INDEL e dos seguintes SNPs: +3003C>T, +3010C>G, +3027A>C,+3142 C>G e +3187A>G, bem como avaliar a influência dessas variantes na evolução para sepse, choque séptico e óbito entre pacientes críticos. Foram analisadas 698 amostras provenientes de pacientes críticos do Hospital São Lucas – PUCRS. O éxon 8 da região 3’ UTR foi amplificado por PCR e encaminhado para sequenciamento direto (ABI 3730 XL DNA Sequencer). A inferência haplotípica foi determinada pelo software PHASE versão 2.1. A frequência haplotípica, desequilíbrio de ligação (DL), Equilíbrio De Hardy-Weinberg (EHW) e teste de heterozigosidade foram estimados pelo software ARLEQUIN versão 3.5. Os dados foram submetidos ao pacote estatístico SPSS versão 18.0. Observaram-se as seguintes frequências alélicas e genotípicas, respectivamente: +2960INDEL, DEL= 0,578 e IN= 0,421, DELDEL= 0,310, DELIN= 0,536 e ININ= 0,152; +3003C>T, C= 0,109 e T= 0,890, CC= 0,012, CT= 0,194 e TT= 0,793; +3010C>G, CC= 0,521 e G= 0,478, CC= 0,250, CG= 0,540 e GG= 0,208; +3027A>C, A= 0,034 e C= 0,965, AA= 0,001, AC= 0,006 e CC= 0,932; +3035C>T, C= 0,861 e T= 0,141, CC= 0,733, CT= 0,255 e TT= 0,010; +3142C>G, C= 0,463 e G= 0,536, CC= 0,180, CG= 0,565 e GG= 0,253 e +3187A>G, A= 0,699 e G= 0,304, AA= 0,448, AG= 0,503 e GG= 0,048. Os polimorfismos +2960INDEL, +3010C>G, +3142C>G e +3187A>G não corresponderam ao EHW e apresentaram heterozigosidade observada maior do que a esperada. Os dois haplótipos mais frequentes foram DTGCCCG (27,90%) e ITCCCGA (26,65%), confirmando a heterogeneidade da população brasileira. A região 3’ UTR está sob seleção balanceadora, em que a seleção dos heterozigotos pode ser vantajosa, permitindo o balanço entre altas e baixas expressões de HLA-G, conforme o contexto biológico. Todos os sítios polimórficos apresentaram forte DL entre si. Entre os pacientes críticos que evoluíram para sepse, observou-se uma associação entre os portadores do alelo +2960IN e choque séptico (Chi-Square, p= 0,036). Entre os pacientes críticos, observou-se uma associação entre os portadores do haplótipo +2960IN_+3142G_+3187A e choque séptico (Chi-Square, p=0,038). E os pacientes críticos com sepse e portadores do haplótipo +2960IN_+3142G_+3187A (Chi-Square, p=0,023) também foram mais suscetíveis a evoluírem para choque séptico. Observou-se, pela primeira vez, uma associação entre o haplótipo +2960IN_+3142G_+3187A e os desfechos de pacientes críticos. Mais estudos são necessários para confirmação desses resultados e outros parâmetros poderiam ser considerados, como níveis de IL-10 e glicocorticóides endógenos, além do perfil de citocinas liberado por pacientes críticos. Devido às propriedades imunomodulatórias descritas para a molécula de HLA-G, futuramente os seus polimorfismos e sua expressão poderiam ser empregadas em abordagens clínicas nos mais variados contextos imunológicos. A amostra estudada está de acordo com as freqüências alélicas, genotípicas e haplotípicas observadas na população brasileira e novos polimorfismos poderiam ser investigados no éxon 8 da região 3’ UTR do HLA-G. / Critically ill patients are individuals hospitalized in Intensive Care Units (ICU) and are characterized by presenting pathologic complex conditions due to serious physiological weaknesses that may evolve to sepsis, septic shock or even death. Massive resources have been invested in sepsis control both in public and private sectors, although sepsis is still the major cause of death in Brazilians ICUs. Several factors influence the outcome of a critically ill patient, including genetic factors. HLA-G molecule is characterized by limited protein variability and restricted tissue expression. HLA-G binding to KIR and LILR receptors triggers various immunoregulatory activities. The HLA-G gene 3’ UTR presents a 14 bp insertion/deletion at +2960, a SNP at +3142C>G and a SNP at +3187A>G that may regulate HLA-G expression. So far, only one study has investigated HLA-G expression in critically ill patients with septic shock, observing that increased sHLA-G5 levels were predictive of survival among septic shock patients. The aim of this study was to assess the frequencies of 14 pb insertion/deletion polymorphism at +2960INDEL, and the followings SNPs: +3003C>T, +3010C>G, +3027A>C, +3035C>T, +3142C>G and +3187A>G in exon 8 at the 3’ UTR of the HLA-G gene and correlate these variants with different clinical outcomes in critically ill patients. 698 samples were obtained from ICU patients from São Lucas-PUCRS Hospital and the exon 8 at the 3’ UTR of the HLA-G gene was sequenced. The haplotype inference was determinated by PHASE version 2.1 software. Haplotype frequency, linkage disequilibrium (LD), Hardy-Weinberg Equilibrium (HWE) and heterozigosity test were estimated by ARLEQUIN version 3.5 software. The data were submitted to the package SPSS version 18.0 software. The followings allelic and genotypic frequencies were observed, respectively: +2960INDEL, DEL= 0.578 and IN= 0.421, DELDEL= 0.310, DELIN= 0.536 and ININ= 0.152; +3003C>T, C= 0.109 and T= 0.890, CC= 0.012, CT= 0.194 and TT= 0.793; +3010C>G, CC= 0.521 and G= 0.478, CC= 0.250, CG= 0.540 and GG= 0.208; +3027A>C, A= 0.034 and C= 0.965, AA= 0.001, AC= 0.006 and CC= 0.932; +3035C>T, C= 0.861 and T= 0.141, CC= 0.733, CT= 0.255 and TT= 0.010; +3142C>G, C= 0.463 and G= 0.536, CC= 0.180, CG= 0.565 and GG= 0.253 and +3187A>G, A= 0.699 and G= 0.304, AA= 0.448, AG= 0.503 and GG= 0.048. The +2960INDEL, +3010C>G, +3142C>G and +3187A>G polymorphisms were not on HWE and had higher observed heterozygosity than expected. The two most frequent haplotypes were DTGCCCG (27.90%) and ITCCCGA (26.65%). The Brazilian population is a heterogeneous population, and these data agree with this statement. The 3’ UTR is under balancing selection and heterozygote selection is advantageous, allowing the balance between high and low expression of HLA-G, according to the biological context. All polymorphic sites showed strong LD between them. Among critically ill patients who developed sepsis, there was an association between the allele +2960IN and septic shock (Chi-Square, p = 0.036). Among all critically ill patients, there was an association between carriers of the haplotype +2960IN_+3142 G_+3187A and septic shock (Chi-Square, p = 0.038). Critically ill patients with sepsis who carrier the haplotype +2960IN_+3142 G_+3187A were also more susceptible to progression to septic shock (Chi-Square, p = 0.023). For the first time, an association between +2960IN_+3142G_+3187A haplotype and outcomes of critically ill patients was observed. More studies are needed to confirm these results and other parameters could be considered, as IL-10 and endogenous glucocorticoids levels and cytokine profile released by critically ill patients. Because of the immunomodulatory properties described for the HLA-G molecule, in future its polymorphisms and its expression could be used in clinical approaches in various immune contexts. These results are according to allelic, genotypic and haplotypic frequencies observed in Brazilian population, and new polymorphisms could be investigated in exon 8 at the 3 'UTR of HLA-G gene.