Development of a rapid method for the detection of fecal enterococci and pathogenic microorganisms in beach water was attempted utilizing an evanescent wave fiber optic biosensor. Various assay formats including a sandwich immunoassay were tested in the development of a rapid assay. Fluorophore labeled antibodies were used for specific detection of bacteria captured or adsorbed directly to the surface of a polystyrene fiber optic waveguide. Binding of the fluorescent labeled antibody to its specific target or binding of a fluorescent labeled anti-IgG within 100-1000 nm of the waveguide surface caused excitation of the fluorescent conjugate resulting in a quantifiable signal. Enterococcus faecalis, Staphylococcus aureus, Escherichia coli O157:H7, and Vibrio cholerae were used as model organisms for biosensor detection in phosphate buffered saline and seawater. Seawater samples were selectively enriched for the presence of these model organisms, which were later detected on the biosensor. The sensitivity and specificity of the biosensor was examined by testing various assay formats, sample preparations, and molecules for capture and detection. Finally, an enrichment protocol combined with filter concentration was utilized to enhance detection of low levels of enterococci. The fiber optic biosensor has the potential to be a sensitive and specific system for the detection of fecal enterococci. The lower limit of detection in seawater and phosphate buffered saline was 2.8 x 106 CFU/ml. As few as 6 CFU/100ml (0.06CFU/ml) could be detected in seawater following a 14-24 hour enrichment and concentration step. Vibrio alginolyticus was found to grow under the same enrichment conditions as the enterococci. V. alginolyticus crossreacted with the polyclonal anti-Strep group D antibody used in the immunoassay at high cell concentrations. Staphylococcus aureus was the only other organism which showed significant cross-reactivity with this antibody. The biosensor was also able to detect other bacterial pathogens in PBS and seawater. The lower limit for detection of E. coli O157:H7 was 3.6 x 105 CFU/ml. The lower limit for detection of Vibrio cholerae O1 was 1.3 x 108 CFU/ml. The antibodies used in these assays were found to crossreact with other gram negative microorganisms. The biosensor was not able to detect Staphylococcus aureus.

Biosensor development has the potential to meet the need for rapid, sensitive, and specific detection of pathogenic bacteria from natural sources. An antibody-based fiber-optic biosensor assay to detect low levels of Vibrio vulnificus in estuarine waters following an enrichment step was developed. The principle of the sensor is based on an immuno-sandwich assay where an anti-V. vulnificus polyclonal capture antibody preparation was first immobilized on a polystyrene fiber-optic waveguide using a biotin-avidin association. The capture antibody is responsible for binding the target cells to the waveguide. Cyanine-5-conjugated anti-V. vulnificus polyclonal antibodies are subsequently allowed to bind to immobilized cells, and detection occurs when a photodetector collects emitted light (670-710 nm) from the fluorophore, which is excited with 635-nm laser light produced by the Analyte 2000 biosensor. Any detection signal greater than a pre-determined threshold signal is considered to be a positive detection event, while any signal lower than the threshold is considered no detection. This immunosensor assay proved highly specific when tested against whole cells and cell extracts from V. cholerae, V. parahaemolyticus, V. alginolyticus, and E. coli. isolates. Following a four hour enrichment in PNCC broth, and in a total of less than seven hours, the assay was able to detect cell extracts from as few as 100 V. vulnificus colony forming units suspended in sterile water. This method holds promise for detection of low numbers V. vulnificus and other autochthonous pathogens in estuarine waters.

A presente dissertação tem por objetivo solucionar duas questões acerca do enriquecimento sem causa. Primeiramente, busca definir se há utilidade na aplicação da figura jurídica, ou seria caso de uma regra descartável no ordenamento jurídico privado brasileiro. A partir da resposta à primeira indagação, questiona-se qual o campo de incidência da regra de vedação ao enriquecimento sem causa. Destarte, será possível formular um critério rígido, que evite a arbitrariedade do julgador na aplicação de normas abertas e cláusulas gerais. Porém, cabível um critério abrangente, vez que inclui no conceito de enriquecimento sem causa novo parâmetro, para além da posição tradicional. Para tanto, imprescindível analisar os elementos e fundamentos do enriquecimento sem causa e da subsidiariedade de maneira a evitar sua aplicação desmedida e sem critérios, ao bel-prazer do julgador. A hipótese, derradeiramente, repousa na necessidade de estabelecimento do campo de atuação da figura jurídica que fundamente o critério de aplicação no caso concreto. / The present dissertation has as a goal to solve two questions regarding the unjustified enrichment. First, it aims to define if there is any use in the application of the juridical figure or if it is the case of a disposable rule on the Brazilian private juridical order. Then, based on the answer to the first inquiry, it is questioned which is the application field for the prohibition to the unjustified enrichment. Therefore, it will be possible to formulate a rigid criterion that prevents the arbitrarity of the ruler in the applications of open norms and general terms. However, been appropriate a comprehensive criterion once it includes in the unjustified enrichment concept a new parameter, beyond the traditional understandment. For that matter, it is indispensable to analyze the elements and foundations of the unjustified enrichment and the subsidiarity in a way to prevent its unmeasured application, without any criteria and at the will of the ruler. Finally, the hypothesis lays on the necessity for the establishment of the action field of the juridical figure that justify the application criteria in the actual case.

Individuals exposed to life-threatening trauma are at increased risk for developing post-traumatic stress disorder (PTSD). Not all people exposed to trauma, however, go on to develop PTSD. Some evidence suggests that individuals who receive social stimulation, such being involved in supportive social networks, are less likely to develop PTSD compared to those lacking social interactions. Although human research has been effective in demonstrating associations between higher levels of social stimulation and lower incidences of PTSD, there has been a lack of experimental evidence suggesting that social stimulation protects against the onset of the disorder after trauma. Here, we tested the hypothesis that providing animals with daily social stimulation (DSS) would ameliorate psychosocial stress-induced changes in behavior and physiology produced by our previously developed animal model of PTSD which generates responses comparable to patients with the disorder. The major findings of this study revealed that providing animals with DSS initiated shortly after an acute stress experience blocked the development of PTSD-like responses in adult rats exposed to chronic psychosocial stress, such as heightened anxiety, exaggerated startle, and contextual fear. These results are consistent with human research suggesting that social stimulation may confer resistance of a subset of the traumatized population to develop PTSD. This level of analysis in an animal model of PTSD underlies the importance of continuing clinical research examining social phenomena in identifying risk factors for PTSD, as well as non-pharmacological treatments (e.g. social support systems) for the disorder.

Doctor of Philosophy (PhD) / The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

<p>Due to the expansion of the human population we are an increasing threat to all wild animals. They are driven to exist in smaller areas and in the worst case scenario extinction. Zoos are being encouraged to improve the animal’s physical and social surroundings. Environmental enrichment is a term used for such improvements. In this behavioural study, the enrichment introduced to two Pallas cats (<em>Felis manul</em>) at Parken Zoo in Eskilstuna, Sweden, consisted of a fishing rod and a clothes peg with a dead mouse or chick attached. The intention was to see if the feeding enrichment could increase activity levels and how the cats were using their enclosure. The enclosure was divided into seven zones which have a varying degree of opportunities for the animals to climb, hide and rest above ground level. The environmental enrichment effects on the Pallas cats´ behaviour, activity levels and use of the enclosure were measured and recorded using instantaneous scan sampling, and the mean value was calculated for each individual. The behavioural and enclosure results proved that enrichment benefits the cats activity levels. The behaviours that showed a variation when presented with enrichment were: walking, climbing, grooming, aggressive behaviour, out of sight behaviour, crouching position, sitting and standing still.  The results showed that the cats preferred the zones in the enclosure that offered a larger view of the nearby enclosures. This study shows that providing captive felids with inexpensive, easy to administer enrichment objects can have profound effects on activity behaviours and their enclosure.</p><p> </p>

<p>Two propositions connected by and have the same truth-value, irrespective of the order of the conjuncts. However, in a sentence like “I put my socks and shoes on” it becomes obvious that the order of the conjuncts affects the meaning of the sentence. This study concerns the contribution of pragmatics to and by implicit enrichment to and then or and thus. It includes three experiments that investigate and-enrichment in adults and children. Nine five-line stories concerning everyday events were used. After each story the participants were to respond “yes” or “no” to a statement which referred to two events that occurred in the story, conjoined with and. In the critical statement, the two events were presented in the inverse order to which they had occurred. The results show no general developmental effect but awareness of the task plays a critical role for and-enrichment production. Ten-year-olds enrich and to the same extent as adults when no efforts are made to mask the intention behind the task. However, when a more spontaneous response is captured by masking the purpose of the task children respond more logically. There are no clear evidence that and-enrichment is affected by the cognitive demands of the task.</p>

<p><em>Clostridium difficile</em> is a pathogen for both humans and animals and is often associated with antibiotic-associated diarrhea. Recently, several human cases of <em>C. difficile</em>-infection with increased mortality and morbidity have been reported. In studies performed in different countries <em>C. difficile</em> has been found in meat. Therefore the question whether <em>C. difficile</em> can be a zoonotic agent has been raised. The aim of this study was to optimize a method for isolation of <em>C. difficile</em> from faeces. When <em>C. difficile</em> is isolated from animals that do not have diarrhea the sample must be cultivated in an enrichment broth. Parameters influencing the enrichment were tested such as enrichment before and after spore selection, enrichment time, alcohol and heat chock for spore selection and if the samples had to be centrifuged or not before cultivation on agar plates. Enrichment in broth before spore selection was better than after. Heat and alcohol chock showed similar results, therefore you can chose which method you want. Cultivation from the pellet after centrifugation of the sample was better than cultivating directly from the inoculated broth. When the sample had low concentration of bacteria long enrichment time, 7 days or more, was best. The next step will be isolation of <em>C. difficile</em> from food-producing animals and humans and the strains will then be compared to se if the same strain is found in humans and in animals, to se if <em>C. difficile</em>-infection can be a zoonoz.</p>

Unconventional natural gas extraction from the Marcellus Shale requires millions of gallons of water to fracture shale and release natural gas from the formation. This process produces water with high levels of total dissolved solids (TDS); and, efforts to recycle these fluids has stimulated microbial growth in produced water. The objective of this study was to analyze the ionic composition of and characterize microorganisms from Marcellus produced water samples. A semi-synthetic culture medium was designed with high TDS to enrich for halophilic microbes, which yielded robust cultures that were able to grow over a wide range of salinities. DNA extracted from aerobic cultures was used for 16s rDNA clone libraries and Automated Ribosomal Intergenic Spacer Analysis (ARISA). ARISA and 16S gene sequencing revealed differences in bacterial composition between Marcellus and freshwater samples. Sequencing of 16S gene indicated the presence of Halomonas, Thalassospira and other genera related to halophilic and petroleum degrading species. / Bayer School of Natural and Environmental Sciences / Environmental Science and Management (ESM) / MS / Thesis

Prymnesium parvum Carter, a haptophyte species capable of forming harmful algal blooms (HABs), has been identified in fresh and brackish water habitats worldwide. In Texas, P. parvum blooms have diminished local community revenues from losses to tourism, fishing, and hatchery production. In this thesis, P. parvum dynamics were studied using in-situ microcosm experiments at Lake Possum Kingdom, Texas during three seasons (fall, winter, spring) in 2004-2005. Specifically, nutrient additions were used to test the hypothesis that increased nutrient levels would not enhance P. parvum's ability to invade phytoplankton communities. In addition to full nutrient additions to levels of f/2 media, other treatments included nutrient additions deficient in either nitrogen (N) or phosphorus (P). Additionally, barley straw extract was tested as a growth inhibitor to prevent P. parvum blooms. Furthermore, P. parvum initial population density was examined to test the hypothesis that increased initial populations could promote an increase in P. parvum population densities. Findings indicated that P. parvum populations in Lake Possum Kingdom would not likely gain a selective advantage over other species when inorganic nutrients (nitrogen and phosphorus) were not limiting. P. parvum did, however, gain an advantage during both N- and P-limited conditions as indicated by toxicity, cell concentrations, and bulk phytoplankton community shifts. Furthermore, P. parvum blooms in Lake Possum Kingdom would likely not be inhibited by barley straw extract application. Initial population densities affected the final population density, but only when initial populations were low. A method to quickly and accurately detect the presence of P. parvum is needed due to P. parvum's potential to cause toxic and lethal blooms. This thesis tested whether P. parvum photopigments are conservative regardless of growth conditions and could be used to quantify the relative abundance of P. parvum in mixed community samples. If biomarker pigments are conservative, then an optimized version of CHEMTAX could be employed as an alternative diagnostic tool to microscopy for enumeration of P. parvum. However, P. parvum pigments in the Texas strain were not conservative throughout the growth cycle and therefore may not be a reliable indicator of cell abundance.