A própolis é uma substância resinosa, cujas atividades antibacteriana, antiinflamatória, antiviral, fungicida, imuno estimulante, cicatrizante e de anestésico local tem sido valorizadas no uso clínico. O lipopolissacarídeo (LPS) é reconhecidamente uma endotoxina e pode induzir processos inflamatórios. Os objetivos deste trabalho foram: a) analisar in vitro as seguintes propriedades do extrato de própolis verde: 1) perfil físico químico 2) Concentração Inibitória Mínima (CIM) frente à endotoxina da bactéria gram negativa Escherichia coli; e 3) sua atividade imunorregulatória sobre leucócitos de baço de ratos; b) analisar &Prime;in vivo&Prime; a ação do Extrato Etanólico de Própolis (EEP) 10% e 90% e do Extrato de Própolis Puro (EPP), em alvéolos dentais contaminados ou não com lipopolissacarídeo (LPS) bacteriano. Para o estudo in vivo 35 ratos foram submetidos às exodontias dos primeiros molares superiores direito e esquerdo, os quais, imediatamente tiveram o alvéolo dental direito contaminado com 0,1µL de lipopolissacarídeo (LPS) (100µg/kg) e o esquerdo sem tal contaminação. Os grupos com (n=7) para cada tratamento, após 2 semanas: GI- Controle Negativo (CN) - sem tratamento; GII- Tratados com Extrato de Própolis puro (EPP) GIII- Tratados com pasta de própolis a 90% (P90); GIV- Tratados com pasta de própolis a 10% (P10); e, GV- Tratado com veículo das pastas (SB) foram analisados. Os alvéolos foram removidos, desmineralizados, processados pela técnica histológica de rotina, submetidos a secções sistematizadas a 6 µm de espessura e corados em H.E. O volume de osso formado, foi avaliado por contagem de pontos, usando um Sistema Teste sobreposto as imagens capturadas com auxílio de uma câmera acoplada a um microscópio. Observada a normalidade dos dados, procedeu-se o teste ANOVA fatorial e Tukey-Kramer test (p<0,05). In vitro determinou-se a propriedade da própolis, sua CIM e atividade imunorregulatória. In vivo os alvéolos dentais contaminados com lipopolissacarídeo bacteriano e tratados com a própolis verde apresentou maior área de osso neoformado, quando comparado aos demais grupos experimentais. O alvéolo não contaminado e tratado com própolis pura mostrou maior área de fibras colágenas. / Propolis is a resinous substance, whose antibacterial, antiinflammatory, antiviral, antifungal, immune stimulant, and local anesthetic wound healing properties has been considered for clinical practice. The lipopolysaccharide (LPS) is recognized as an endotoxin and can induce inflammatory processes. Our objectives were: a) to analyze in vitro the following properties of green propolis extract: 1) physicochemical profile of green propolis sample, 2) Minimum Inhibitory Concentration (MIC) against endotoxin from gram negative Escherichia coli and 3) its immunoregulatory activity using leukocytes from the spleen of mice,. b) analyze in vivo, the action of propolis ethanol extract (EEP) 10% and 90% and Pure Propolis Extract (EPP) in dental alveoli or not contaminated with lipopolysaccharide (LPS) bacterial. For in vivo study, 35 rats were subjected to extractions of maxillary first molars, right and left, which immediately had the right dental socket contaminated with 0.1 µL of lipopolysaccharide (LPS) (100µg/kg) and left without such contamination. They were divided into groups (n = 7) for each sample after 2 weeks, according to the treatment in the right and left alveoli: GI-Negative Control (NC) - no treatment, GII-Treated Pure Propolis Extract (EPP) GIII-Treated folder with propolis 90% (P90), GIV-Treated folder with propolis 10% (P10) and GV-Treated folder with vehicle (SB). The alveoli were removed, demineralized, processed by routine histologic technique, submitted to systematic sections (6 microns) of thick and stained with HE for histological analysis to assess the new bone tissue volume by point counting method, using a Test system on images captured with the aid of a digital camera attached to a microscope. Observed data normality, we proceeded to ANOVA and Tukey-Kramer tests (p <0.05). In vitro, it was determined the propolis properties, CIM and immunoregulatory activity. In vivo, the alveoli contamined with bacterian lipopolysaccharide and treated with green propolis induced higher bone formation when compared to other groups. The non contamined alveoli and treated with pure propolis showed more quantity of collagen fibers.

In June 2008, the Cedar River crested flooding more than 5,000 Cedar Rapids homes. Residents whose homes were flooded were invited to participate in this study. We characterized exposures and symptoms experienced by individuals inhabiting 73 flood-damaged homes. Exposures and questionnaire-based health assessments were compared at two levels of remediation, in-progress and completed. Homes with remediation in-progress (n=24), as compared to the completed homes (n=49), had significantly higher airborne concentrations of mold, bacteria, iPM, endotoxin and glucan. Residents of in-progress homes had a significantly higher prevalence of doctor diagnosed allergies (adjusted OR=3.08; 95%CI: 1.05-9.02) and all residents had elevated prevalence of self-reported wheeze (adjusted OR=3.77; 95%CI: 2.06-6.92) and prescription medication use for breathing problems (adjusted OR=1.38; 95%CI: 1.01-1.88) after the flood as compared to before. Proper post-flood remediation led to improved air quality and lower exposures among residents living in flooded homes. Recognition of endotoxin as a proinflammatory ligand for pattern recognition receptors has increased the demand for endotoxin assessment in studies of environmental lung disease. Measurements using the Limulus amebocyte lysate (LAL) assay of air and reservoir dust samples are routinely incorporated into epidemiologic studies. However, it is unknown if endotoxin reactivity in the LAL assay varies by its physical presentation as aggregates, as membrane components of whole bacteria or as shed membrane blebs or if this parallels differences in the inflammatory potency of endotoxin in vivo. Endotoxins as14C-labeled-lipooligosaccharide (14C-LOS) and 14C- labeled-lipopolysaccharide (14C-LPS) were produced from Neisseria meningitidis and Escherichia coli. The reactivity of the endotoxin presentations was assessed in the LAL assay and in vivo using a murine model. The LAL assay significantly underestimated the quantity of endotoxin in the whole bacteria form whereas there was no significant difference in detecting endotoxin in aggregate and bleb forms. The failure of the LAL assay to equally quantify endotoxin was not mirrored in vivo where all three presentations of endotoxin were equally inflammatory. The inability of the LAL assay to detect the full quantity of endotoxin presented in the whole bacteria form has troubling implications for exposure assessment studies. Various extraction methods were applied to samples of known endotoxin quantity to improve the detection ability of the LAL assay. Extraction using EDTA and Tris/EDTA significantly improved the detection of endotoxin compared to the reference method of extracting in pyrogen-free water. These extraction methods also significantly increased the quantity of endotoxin measured in house and barn dust samples. A higher quantity of endotoxin measured in the LAL assay corresponded to a higher neutrophilic response in vivo. A standardized methodology for endotoxin detection that mimics the in vivo response is necessary for accurate and consistent endotoxin analysis.

Chapter II describes results from 197 rural households that were sampled over five continuous days for indoor and outdoor PM10, PM2.5, and endotoxin. Geometric mean indoor concentrations of PM10 and PM2.5 (21.2 πg m-3, 12.2 πg m-3) were larger than outdoor concentrations (19.6 πg m-3, 8.2 πg m-3; p =0.072, p<0.001). While geometric mean endotoxin levels were almost six times larger in outdoor air compared to indoor (1.47 EU m-3, 0.23 EU m-3; p <0.001). Airborne PM10 and endotoxin concentrations in a rural county were elevated compared to those previously reported in certain urban areas. Furthermore, during the harvest season, concentrations of endotoxin in ambient air approached levels that have been shown to cause decreased respiratory function in occupational workers. Chapter III evaluated the effectiveness of using Radiello passive monitors to measure hydrogen sulfide (H2S) in close proximity (<40 m) to a medium sized CAFO. A total of eight passive H2S monitors were deployed 7-14 days around a swine confinement for seven months. Additionally, a separate laboratory study was carried out to determine the monitor's H2S uptake rate. Concentrations of H2S measured near the confinement were varied and ranged from 0.6 to 95 ppb depending on the sampling period and proximity to the lagoon .The uptake rate provided by the supplier (0.096 ng ppb-1 min-1) was significantly larger (p=0.002) than the rate determined experimentally (0.062 ng ppb-1 min-1). In Chapter IV we evaluated the association between residential proximity to swine operations and childhood asthma. A metric was created to determine children's relative environmental exposure to swine CAFOs which incorporated facility size and distance and direction of the CAFO to the home. When controlling for six significant asthma risk factors, children with a larger relative environmental exposure to CAFOs had a significantly increased risk of physician-diagnosed asthma (OR=1.20, p=0.009). In stratified analysis that adjusted for a respiratory infection before the age of two years, the association between relative exposure and childhood asthma was significantly increased in children with a respiratory infection (OR=1.45, p=0.001) but not in children free from respiratory infection (OR=1.12, p=0.355).

A fim de garantir a qualidade final de produtos os laboratórios de análise microbiológica fornecem dados sobre a qualidade dos mesmos em todas as suas etapas de produção. A crescente preocupação com a saúde dos pacientes conduz à busca de métodos que forneçam resultados precisos e rápidos, pois possibilitam que ações corretivas sejam tomadas em tempo real. O presente trabalho teve por objetivo avaliar o potencial de tecnologia alternativa no monitoramento de endotoxina bacteriana na água tratada para diálise e dialisato e avaliar o potencial da citometria de fluxo na análise de água. Para isso utilizou-se Portable Test System (PTS®) como método alternativo para detecção de endotoxina bacteriana no monitoramento da água tratada para diálise e dialisato, o qual foi validado frente ao método convencional farmacopeico. Paralelamente realizou-se revisão narrativa da literatura a fim de avaliar a aplicabilidade da citometria de fluxo em análises de água. A análise dos diferentes parâmetros de validação para endotoxina bacteriana no método alternativo mostrou que, exceto para a menor diluição analisada, houve linearidade e precisão nos resultados. Por outro lado a concentração de 0,25 UE/mL foi a menor que apresentou exatidão e especificidade. Observou-se ainda, que o limite de detecção foi de 0,125UE/mL e o de quantificação de 0,25 UE/mL, portanto o intervalo foi de 0,25-1,0 UE/mL. Adicionalmente pela análise de resistência pode-se perceber que ao variar analistas não houve diferença significativa. Em relação ao tempo de análise em uma condição de rotina laboratorial com muitas amostras, o PTS® mostrou-se demorado. Ressalta ainda, que seria importante que a legislação vigente deternimasse a análise mensal de endotoxinas no dialisato. A revisão da literatura evidencia o potencial da tecnologia de citometria de fluxo, pois a mesma mostrou-se satisfatória quando comparada a metodologias convencionais para análise de água. O trabalho desenvolvido permitiu concluir que o PTS®) mostrou-se adequado para analisar amostras in loco, permitindo análises em tempo real, que para as quais haja a expectativa de ausência de endotoxinas ou de concentração respeitando o intervalo de 0,25 UE/mL a 1,0 UE/mL. Quanto a citometria de fluxo, esta mostrou-se uma tecnologia promissora em analisar amostras de água, sendo portanto recomendável proceder a estudos de validação e aplicabilidade. / In order to guarantee the final quality of products, the microbiological analysis laboratories provide data about their quality at all production stages. The growing concern for patients\' health leads to the search for methods that provide accurate and fast results, as they enable corrective actions to be taken in real time. The present work aimed to evaluate the alternative technology potential in the monitoring of bacterial endotoxin in treated water for dialysis and dialysate and to evaluate the potential of flow cytometry in water analysis. The different validation parameters analysis for bacterial endotoxin in alternative method showed that, except for the lowest dilution analyzed, there was linearity and precision in the results. On the other hand, the concentration of 0.25 EU / mL was the lowest that presented accuracy and specificity. It was further observed that the detection limit was 0.125UE / mL and the quantification limit was 0.25 EU / mL, so the range was 0.25-1.0 EU / mL. Additionally by the ruggedness analysis it was possible to perceived that when varying analysts there was no significant difference. Regarding the analysis time in a laboratory routine condition with many samples, the PTS® was was time consuming. It was also observed that it would be important to determine monthly analysis of endotoxins in dialysate. The literature review evidence the flow cytometry technology potential of the because it was satisfactory when compared to conventional methodologies for water analysis. The research showed that the PTS® was suitable for analyzing samples in loco, allowing real-time analyzes, for which there is expectation of endotoxins absence or concentration respecting the range of 0.25 EU / mL to 1.0 EU / mL. For the flow cytometry, it was shown to be a promising technology for analyzing water samples, and it is therefore advisable to carry out validation and applicability studies.

ABSTRACT Occupational exposure to wood dust and biohazards associated with wood dust (endotoxins, (1->3)-b-D-glucans, Gram (-)ve bacteria and fungi), their correlation to respiratory function, and symptoms among woodworkers have been investigated in the present study. Wood dust, endotoxins, and allergenic fungi are the main hazards found in woodworking environments. Relatively very few studies have been done on wood dust exposure. The present study was designed to comprehensively investigate the health effects of wood dust exposure, and in particular provide new information regarding: Exposure to (1->3)-b-D-glucans in an occupational environment; Levels of exposure to wood dust and biohazards associated with wood dust in different woodworking environments; Correlations among personal exposures, especially correlations between (1->3)-b-D-glucans and fungi exposures, and endotoxins and Gram (-)ve bacteria exposures; Effects of personal exposure to biohazards on lung function; Effects of personal exposure to biohazards on work-related symptoms; and Determinants of inhalable exposures (provide which factors in the environment influence the personal inhalable exposures). Workers at four different woodworking processes; two logging sites, four sawmills, one major woodchipping operation and five joineries situated in the state of New South Wales in Australia were studied for personal exposure to inhalable dust (n=182) and respirable dust (n=81), fungi (n=120), Gram (-)ve bacteria (n=120), inhalable endotoxin (n=160), respirable endotoxin (n=79), inhalable (1->3)-b-D-glucan (n=105), and respirable (1->3)-b-D-glucan (n=62). The workers (n=168) were also tested for lung function. A questionnaire study (n=195) was carried out to determine the prevalence of work-related symptoms. The geometric mean inhalable exposure at logging sites was 0.56 mg/m3 (n=7), sawmills 1.59 mg/m3 (n=93), the woodchipping mill 1.86 mg/m3 (n=9) and joineries 3.68 mg/m3 (n=66). Overall, sixty two percent of the exposures exceeded the current standards. Among joineries, 95% of the hardwood exposures and 35% of the softwood exposures were above the relevant standards. Compared with green mills, the percentage of samples, which exceeded the hardwood standard was high for dry mills (70% in dry mills, 50% in green mills). The respirable dust exposures were high at the joineries compared with the other worksites. Exposure levels to fungi at logging sites and sawmills were in the range 103-104 cfu/m3, woodchipping 103-105 cfu/m3 and joineries 102-104 cfu/m3. The predominant fungi found at sawmills were Penicillium spp. High exposure levels of Aureobasidium pullulans were also found at two sawmills. At the woodchipping mill the predominant species were Aspergillus fumigatus, Penicillium spp., and Paecilomyces spp. The sawmills, which employed kiln drying processes, had lower exposure levels of fungi compared with the green mills. Those workplaces which had efficient dust control systems showed less exposure to fungi and bacteria. Although mean endotoxin levels were lower than the suggested threshold value of 20 ng/m3, some personal exposures at sawmills and joineries exceeded the threshold limit value. The mean inhalable (1->3)-b-D-glucan level at the woodchipping mill was 2.32 ng/m3, at sawmills 1.37 ng/m3, at logging sites 2.02 ng/m3, and at joineries 0.43 ng/m3. For the respirable size fraction, mean endotoxin and mean (1->3)-b-D-glucan concentrations were much lower, being similar to observed dust concentrations. Significant correlations were found between mean inhalable endotoxin and Gram (-)ve bacteria levels (p<0.0001), and mean airborne inhalable (1->3)-b-D-glucan and fungi levels (p=0.0003). The correlations between mean respirable endotoxin levels vs Gram (-)ve bacteria exposure levels (p=0.005), and respirable (1->3)-b-D-glucan exposure levels vs total fungi levels (p=0.005) were also significant. Significant correlations were found between lung function and personal exposures. Multivariate analyses showed that the effect of all the personal exposures on cross-shift decrements in lung function was more prominent among sawmill and chip mill workers compared with joinery workers. Woodworkers had markedly high prevalence of cough, phlegm, chronic bronchitis, frequent headaches, throat and eye irritations, and nasal symptoms compared with controls. Among the woodworkers, smokers had a high prevalence of chronic bronchitis (20%) compared with non-smokers (10%). Some workers also reported a variety of allergy problems due to exposure to various types of wood dust. Both joinery workers and sawmill and chip mill workers revealed significant correlations between work-related symptoms and personal exposures. Chronic bronchitis was significantly correlated with personal exposure to wood dust, endotoxin, (1->3)-b-D-glucan, fungi, and Gram (-)ve bacteria among joinery workers. Whereas among sawmill workers chronic bronchitis was significantly correlated with personal exposure to endotoxin, (1->3)-b-D-glucan, and fungi. Woodworkers showed significant positive correlations between percentage cross-shift change (decrease) in lung function and respiratory symptoms. Significant inverse correlations were also found among percentage predicted lung function and respiratory symptoms. The elevated inhalable dust exposures observed in this study can be explained by a combination of factors, including: lack of awareness of potential health effects of wood dust exposure among both management and workers, aging equipment, inadequate and ineffective dust extraction systems or usually none especially for hand held tools, poor maintenance of the ventilation system in some, non-segregation of dusty processes, dry sweeping, and the use of compressed air jets. The determinant-of-exposure analysis confirmed the field observations. The significant determinants of personal inhalable dust exposures (n=163) were found to be: local exhaust ventilation, job title, use of hand-held tools, cleaning method used, use of compressed air, and green or dry wood processed. Type of wood processed was not found to be statistically significant. A majority of workers (~90%) did not wear appropriate respirators approved for wood dust, while the workers who did wear them, used them on average less than 50% of the time. Workers should be protected by controlling dust at its source. When exposure to wood dust cannot be avoided, engineering controls should be supplemented with the use of appropriate personal protective equipment.

SUMMARY Respiratory mucosal surfaces are constantly exposed to a broad range of non-pathogenic environmental antigens. In the absence of proinflammatory signals, inhalation of harmless antigens results in immunological tolerance. Indeed, lung dendritic cells stimulate the development of antigen-specific regulatory T cells. Nevertheless, epidemiological studies have shown that ambient air contains not only inert antigens but also immunostimulatory molecules of microbial origin. Of particular interest are endotoxins, a cell wall component of gram-negative bacteria that is ubiquitous in the environment. In spite of the fact that high levels of endotoxin exposure in early life protect against allergic sensitization, most evidence indicates that exposure to house-dust endotoxin is a significant risk factor for increased asthma prevalence and severity. When the respiratory tract is stimulated with airborne endotoxins, lung dendritic cells lose their tolerogenic properties and rather promote the development of an allergic response directed against concomitant aeroantigens. Although endotoxins are omnipresent in the environment and favour airway allergy, only a minority of people develops asthma. A unifying model reconciling these conflicting observations is still lacking. We report here that LPS-triggered airway allergy is tightly controlled by lung interstitial macrophages, a cell population that remains largely uncharacterized. Interstitial macrophages could be distinguished from alveolar macrophages by their unique capacity to inhibit lung dendritic cell maturation and migration upon LPS stimulation, thereby preventing sensitization to concomitant inhaled antigens. We furthermore demonstrated that functional paralysis of LPS-stimulated dendritic cells involves interleukin-10 production by interstitial macrophages. Finally, we demonstrate that specific in vivo elimination of interstitial macrophages leads to overt asthmatic reactions to innocuous airborne antigens inhaled along with low LPS doses. Our study thus reveals a crucial role for interstitial macrophages in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that airborne LPS has the ability to promote the induction of Th2 responses by lung dendritic cells but does not provoke airway allergy under normal conditions. In the presence of LPS, interstitial macrophages, but not alveolar macrophages, break the link between innate and adaptive immunity, allowing harmless inhaled antigens to escape from T cell-dependent responses. RÉSUMÉ Le système respiratoire est continuellement exposé à de nombreux antigènes environnementaux non pathogéniques. En labsence de signal proinflammatoire, linhalation dantigènes inoffensifs aboutit au développement dune tolérance immunologique. Dans ces conditions, les cellules dendritiques pulmonaires tolérogènes stimulent le développement de lymphocytes T régulateurs. Cependant, les études épidémiologiques montrent que lair ambiant ne contient pas que des antigènes inertes mais également des molécules immunostimulatrices dorigine microbienne dont les endotoxines (LPS, lipopolysaccharide). La présence dans lenvironnement de ce composant de la paroi des bactéries Gram négatives est ubiquiste. Malgré le fait que lexposition à de hauts niveaux de LPS durant lenfance semble protéger contre la sensibilisation allergique, la plupart des études montrent que les endotoxines contenues dans la poussière domestique constituent un facteur de risque significatif pour la prévalence et la sévérité de lasthme. Quand le système respiratoire est stimulé par le LPS aérogène, les cellules dendritiques perdent leurs propriétés tolérogènes et deviennent capables dinduire le développement dune réponse allergique. Bien que les endotoxines soient omniprésentes dans lenvironnement et favorisent lallergie des voies respiratoires, seulement une minorité de personnes est asthmatique. Ces observations contradictoires impliquent lexistence de mécanismes protecteurs non encore décrits capables de prévenir les réponses allergiques induites par les endotoxines. Nous montrons dans ce travail que lallergie des voies respiratoires induite par le LPS est étroitement contrôlée par les macrophages interstitiels, une sous-population de macrophages pulmonaires dont la fonction in vivo navait jamais été caractérisée. Les macrophages interstitiels peuvent être distingués des macrophages alvéolaires par leur capacité unique à inhiber la maturation et la migration des cellules dendritiques induites par lexposition du système respiratoire au LPS, prévenant ainsi la sensibilisation aux aéroantigènes inhalés concomitamment. De plus, nous démontrons que linhibition fonctionnelle des cellules dendritiques implique la sécrétion dIL-10 par les macrophages interstitiels. Finalement, nous montrons que lélimination spécifique des macrophages interstitiels in vivo aboutit au développement dune réponse asthmatique dirigée contre les aéroantigènes inoffensifs inhalés avec de faibles doses de LPS. Notre travail révèle un rôle crucial des macrophages interstitiels dans le maintien de lhoméostasie immunitaire du tractus respiratoire et fournit une explication au paradoxe que le LPS aérogène a la capacité de favoriser linduction de réponses Th2 par les cellules dendritiques mais ne provoque pas dallergie des voies respiratoires dans les conditions normales. En présence de LPS, les macrophages interstitiels, mais pas les macrophages alvéolaires, brisent le lien entre limmunité innée et limmunité adaptative, permettant aux antigènes inhalés déchapper aux réponses dépendantes des lymphocytes T.

Sepsis is a common diagnose in the intensive care population, burdened with a high mortality. The systemic inflammatory reaction underlying the development of septic organ dysfunction can be modeled using Gram-negative bacterial lipopolysaccharide, endotoxin. This thesis used a porcine endotoxemic experimental sepsis model to address clinical questions difficult to answer in clinical trials; furthermore a model of secondary sepsis was developed. No additional effect on the development of renal dysfunction by tobramycin was found, indicating that a single dose of tobramycin does not further compromise renal function in inflammatory-induced acute kidney injury. Antiendotoxin treatment had no measurable effect on TNF-α-mediated toxicity once the inflammatory cascade was activated. There was an effect on the leukocyte response that was associated with improvements in respiratory function and microcirculation, making it impossible to rule out fully the beneficial effect of this strategy. However, the effects were limited in relation to the magnitude of the endotoxin concentration reduction and the very early application of the antiendotoxin measure. The lungs stood out compared to the other organ systems as having a threshold endotoxin dose for the protective effect of endotoxin tolerance. As to the development of circulatory and renal dysfunction, tolerance to endotoxin was evident regardless of the endotoxin pre-exposure and challenge dose. There was a temporal variation of endotoxin tolerance that did not follow changes in plasma TNF-α concentrations and maximal tolerance was seen very early in the course. More pronounced endotoxin tolerance at the time of maximum tolerance was associated with a more marked hyperdynamic circulation, reduced oxygen consumption and thrombocytopenia eighteen hours later. It might be of interest to use the experimental model of long-term endotoxemia followed by a second hit, which has been designed to resemble an intensive care setting, for the study of treatment effects of immunomodulating therapies in secondary sepsis. / Paper 3, previous title as submitted: "Compartmentalization of organ endotoxin tolerance in a porcine model of secondary sepsis"

The objective of this study was to investigate the physiology and productivity in Escherichia colicultures with emphasis on the temperature-limited fed-batch (TLFB) culture. The TLFB techniquecontrols the oxygen consumption rate of the growing culture by a gradually declining temperaturefrom 37-35 °C down to 20-18 °C. The temperature regulated the DOT around a set-point (30 % airsat.), and all nutrients were in excess. Glucose was fed into the culture continuously, however, highacetate formation was avoided by keeping the glucose at a low, yet excessive, concentration. Thebiomass productivity was approximately the same in TLFB as in glucose-limited fed-batch (GLFB)cultures, since the specific growth rate and the oxygen consumption rate are limited by the oxygentransfer capacity of the reactor in both techniques.High concentrations of endotoxins were found in the medium of E. coli fed-batch cultures at lowspecific growth rates (below 0.1 h-1) and severe glucose limitation. In this thesis the TLFB techniquewas found to suppress the endotoxin release even at low specific growth rates. The repressed release of endotoxins in TLFB cultures was due to the high availability of glucose and not to the low growthrate or the lower temperature. The conclusion was drawn from comparing with the GLFB technique performed at 20 °C, which resulted in high endotoxin release.Extensive release of endotoxin, accompanied with high concentrations of soluble proteins was foundin a TLFB culture exposed to a higher energy dissipation rate, 16 kW m-3, instead of 2 kW m-3, due toa higher stirrer speed (1000 instead of 500 rpm). The hypothesis that this is a result of mechanicalstress is discussed in context with the common view that cells like E. coli, which are smaller than the Kolmogoroff’s microscale of turbulence, should not be influenced by the turbulence.TLFB cultured cells exhibited more stable cytoplasmic membranes when treated with osmotic shockas compared to the GLFB cultured cells. The concentrations of DNA and soluble proteins in the periplasmic extracts from the TLFB cultured cells were lower than from GLFB cultured cells. Inaddition, the specific productivity of periplasmic β-lactamase was higher in the TLFB cultures,suggesting that this technique could be an alternative for protein production. The solubility of apartially aggregated recombinant protein increased in the TLFB compared to the GLFB cultures.However some time after induction, in spite of the gradually declining temperature, the solublefraction decreased.For obtaining better understanding of the performance of the process and for identifying criticalparameters, a mathematical model was developed based on the growth, energy and overflowmetabolism at non-limiting nutrient conditions. The temperature-dependent maximum specific glucoseand oxygen uptake rates were determined in pH-auxostat cultures for temperatures ranging from 18 to37 °C. A dynamic simulation model of the TLFB technique was developed and the results were compared to experimental data. The simulation program was also used for sensitivity analysis of some physiological and process parameters to study the impact on biomass concentration and temperatureprofiles. An effect on the biomass concentration profile but not on the temperature profile wasobserved when changing the oxygen transfer coefficient. If the maximum specific glucose uptake ratewas altered, or if the glucose concentration was permitted to assume other values, the temperatureprofile but not the biomass concentration profile was influenced. Cell death affected both the biomassconcentration profile and the temperature profile. / QC 20100922

This thesis dissertation assessed workers who work in poultry barns and their occupational environment in relation to the type of bird housing in which they were exposed (cage-housed birds (CH) or floor-housed birds (FH)) and examined the environmental variables including dust and endotoxin and potential relationships to respiratory symptoms of workers. <p>A cross sectional study was undertaken to assess the environmental exposure levels and respiratory health effects of workers who worked in CH and FH poultry operations. The respiratory results suggested an asthma-like syndrome in these workers. Workers who worked in CH facilities reported greater current and chronic respiratory symptoms and significantly greater current and chronic phlegm as compared to workers from FH facilities. Workers from CH poultry facilities were exposed to greater endotoxin load than workers from FH facilities, but workers from FH operations were exposed to greater levels of total dust. It was found that endotoxin load (EU/mg) was a significant predictor of chronic phlegm for all poultry workers.<p>The effects on dust and endotoxin measurements when utilizing a Marple impactor with greased or ungreased impaction surfaces when sampling in an agricultural environment were unknown, and the potential for effects was tested. There were no significant differences in the aerosol mass median aerodynamic diameters between the greased and ungreased Marple impactors. Endotoxin analysis results appeared to be influenced by impaction grease particularly when very low amounts of endotoxin were present. <p>Size fractioning the dust and endotoxin using Marple impactors in CH and FH poultry operations showed that endotoxin load (EU/mg) was significantly higher in the respirable fraction of area samples in CH poultry operations as compared to FH operations. There were no differences in endotoxin load in the non-respirable size fractions for area samples between CH and FH operations. FH poultry operations had significantly greater dust mass and dust concentration in both respirable and non-respirable fractions for FH operations. There was significantly greater endotoxin load (EU/mg) in the 3.5-6.0 micron size fraction for the CH poultry operations as compared to the FH operations.

This thesis dissertation assessed workers who work in poultry barns and their occupational environment in relation to the type of bird housing in which they were exposed (cage-housed birds (CH) or floor-housed birds (FH)) and examined the environmental variables including dust and endotoxin and potential relationships to respiratory symptoms of workers. <p>A cross sectional study was undertaken to assess the environmental exposure levels and respiratory health effects of workers who worked in CH and FH poultry operations. The respiratory results suggested an asthma-like syndrome in these workers. Workers who worked in CH facilities reported greater current and chronic respiratory symptoms and significantly greater current and chronic phlegm as compared to workers from FH facilities. Workers from CH poultry facilities were exposed to greater endotoxin load than workers from FH facilities, but workers from FH operations were exposed to greater levels of total dust. It was found that endotoxin load (EU/mg) was a significant predictor of chronic phlegm for all poultry workers.<p>The effects on dust and endotoxin measurements when utilizing a Marple impactor with greased or ungreased impaction surfaces when sampling in an agricultural environment were unknown, and the potential for effects was tested. There were no significant differences in the aerosol mass median aerodynamic diameters between the greased and ungreased Marple impactors. Endotoxin analysis results appeared to be influenced by impaction grease particularly when very low amounts of endotoxin were present. <p>Size fractioning the dust and endotoxin using Marple impactors in CH and FH poultry operations showed that endotoxin load (EU/mg) was significantly higher in the respirable fraction of area samples in CH poultry operations as compared to FH operations. There were no differences in endotoxin load in the non-respirable size fractions for area samples between CH and FH operations. FH poultry operations had significantly greater dust mass and dust concentration in both respirable and non-respirable fractions for FH operations. There was significantly greater endotoxin load (EU/mg) in the 3.5-6.0 micron size fraction for the CH poultry operations as compared to the FH operations.