[pt] Métodos baseados na cromatografia líquida micelar (MLC) com detecção fluorimétrica foram desenvolvidos para dois estudos de caso. No primeiro deles, seis B-carbolinas (harmol, harmalol, harmane, norharmane, harmine e harmaline) foram plenamente separadas de modo a se aplicar o método para a determinação dos alcaloides em formulações de Passiflora incarnata L., extrato seco de Passiflora alata Dryander e em urina. A separação foi realizada em coluna cromatográfica C18, utilizando fase móvel tamponada (pH 8,0) contendo 220 mmol L-1 de dodecilsulfato de sódio (SDS)/acetonitrila (97/3 por cento v/v). A detecção fluorimétrica permitiu que se atingissem limites de detecção (LOD) abaixo de 3,6 ng g-1 com repetibilidade e precisão intermediária entre 0,1 e 5 por cento. As incertezas combinadas associadas à concentração das B-carbolinas ficaram entre 1 e 9 por cento. O método proposto foi comparado ao método baseado na cromatografia eletrocinética micelar (MEKC) com detecção absorciométrica e os resultados com MLC se mostraram superiores do ponto de vista da capacidade de detecção (por serem os alcaloides naturalmente muito fluorescentes) e de recuperação. Para as amostras de fitoterápicos, o harmol foi quantificado tanto nas amostras de medicamento de Passiflora incarnata quanto em urina de voluntário após a administração de fitoterápico. O harmine foi detectado apenas na amostra de medicamento. Os alcaloides norharmane, harmine e harmaline foram identificados no chá misto de Maracujá, no qual continha em sua composição extrato seco de Passiflora alata Dryander. Na segunda abordagem, um método por MLC foi desenvolvido para a determinação de galantamina e de seus principais metabólitos (N-desmetil galantamina, O-desmetil galantamina, epigalantamina e N-óxido galantamina). A separação da galantamina e dos metabólitos foi feita utilizando coluna cromatográfica C18 com porosidade de 300 angstroms e fase móvel constituída de tampão (pH 5,0) contendo 25 mmol L-1 de SDS/acetonitrila (97/3 por cento v/v). A detecção fluorimétrica permitiu limites de detecção abaixo de 343 ng g-1, repetibilidade e precisão intermediária entre 2,2 e 4 por cento. As incertezas combinadas associadas à concentração de galantamina e dos metabólitos ficaram entre 0,3 e 11 por cento. Ensaios de recuperação foram realizados em amostra de urina fortificada com padrões da galantamina e metabólitos e os resultados obtidos ficaram entre de 91,4 e 114,8 por cento. A comparação entre o método por MLC e o método por cromatografia líquida de alta eficiência com fase reversa foram feitas com amostras de medicamento cujo princípio ativo era o hidrobrometo de galantamina. Os resultados dos teores de galantamina e as variâncias das medidas obtidas por ambos os métodos foram estatisticamente iguais. A sensibilidade da curva analítica obtida por MLC foi duas vezes maior do que a encontrada por cromatografia por fase reversa. Análises de urina de ratos coletadas após a administração do medicamento foram realizadas com o método proposto, sendo que a galantamina mais os metabólitos N-óxido galantamina e N-desmetil galantamina foram identificados nas amostras. Para melhor avaliar o efeito de amplificação de fluorescência da galantamina pelo ambiente organizado, experimentos foram feitos com a separação cromatográfica de fase reversa e com a adição de solução rica em micelas de SDS após a passagem do analito pela coluna cromatográfica. As condições de separação incluíram o uso de coluna cromatográfica C18, fase móvel constituída por tampão (pH 5,0) contendo 2 por cento de propano-2-ol (v/v)/ acetonitrila (80/20 por cento v/v). A solução micelar de SDS (100 mmol L-1), misturada à fase móvel após a coluna cromatográfica, foi preparada com a mesma composição da fase móvel. A sensibilidade da curva analítica de galantamina foi três vezes maior quando o meio micelar foi usado. O resultado prova que o ambiente micelar favorece a medição fluorimétrica de galantam / [en] Methods based on micellar liquid chromatography (MLC) with fluorimetric detection have been developed for two case studies. In the first, six B-carbolines (harmol, harmalol, harmane, norharmane, harmine and harmaline) were fully separated allowing the application of the method for the determination of these alkaloids in Passiflora incarnata L. formulations, in Passiflora alata Dryander dry extract and in urine. The chromatographic separation was performed on a C18 column using a buffered mobile phase consisting of disodium hydrogen phosphate (pH 8.0) containing 220 mmol L-1 of sodium dodecyl sulfate (SDS)/acetonitrile (97/3 percent v/v). The fluorimetric detection allowed limits of detection (LOD) below than 3.6 ng g-1 to be achieved with intermediary precision and repeatability between 0.1 and 5 percent. The calculated combined uncertainties of the concentration of B-carbolines were between 1 and 9 percent. The proposed method was compared with the method based on micellar electrokinetic chromatography (MEKC) with absortionmetric detection. The MLC results were superior from the viewpoint of the detection power (since they are very fluorescent alkaloids) and also in terms of recovery. The harmol was quantified in Passiflora incarnata phitotherapic samples and in urine collected from a voluntary after of the administration of herbal medicine. The harmine was detected only in the phitotherapic sample. The norharmane, harmine and harmaline alkaloids were identified in Passion Fruit tea, which contained dry extract of Passiflora alata Dryander. In the second approach, a method for MLC was developed for the determination of galantamine and its main metabolites (N-demethyl galantamine, O-demethyl galantamine, epigalantamine and N-oxide galantamine). The separation was performed using a C18 chromatography column with porosity of 300 angstroms and with mobile phase consisting of buffer (pH 5.0) containing SDS (25 mmol L-1)/acetonitrile (97/3 percent v/v). The fluorimetric detection enabled LOD below 343 ng g-1 with intermediate precision and repeatability between 2.2 and 4 percent. Combined uncertainties for concentration of galantamine and metabolites were between 0.3 and 11 percent. Recovery experiments were performed on urine samples fortified with galantamine standards and metabolites with results between 91.4 and 114.8 percent. A comparison between the proposed MLC method and reverse phase high performance liquid chromatography was made using medicine samples containing galantamine hydrobromide as the active principle. Galantamine levels and variances achieved with these methods were statistically equal. The analytical curve sensitivity by MLC was two times higher than that found with reverse phase high performance liquid chromatography. Analysis of rat urine, collected after the administration of the medicine, were performed with the proposed method enabling the identification of galantamine, N-oxide galantamine and N-demethyl galantamine. To better evaluate the fluorescence amplification effect of galantamine in the organized environment, experiments were made with conventional chromatographic separation and post-column addition of the aqueous solution rich in micelles. The separation conditions included the use of C18 chromatographic column, mobile phase consisting of buffer (pH 5.0) containing 2 percent of propan-2-ol (v/v)/acetonitrile (80/20 percent v/v). The micellar solution of SDS (100 mmol L-1), added after the chromatographic column, was prepared with the same composition of the mobile phase. The column temperature was 25 degrees C and the sample volume was 20 uL. The sensitivity of the analytical curve of galantamine was three times higher when the micellar solution was mixed, proving that the organized environment favors the galantamine fluorescence even at flow regime. Recoveries with or without post-column mixing with the micelle rich solution were between 97.5 and 102.2 percent.

Drug facilitated assault (DFA) is an increasing problem in the UK. The crime often occurs through the surreptitious administration of a drug into a victims drink, rendering the victim unable to resist the assault. The detection of these drugs in a biological specimen from the victim is one of the most challenging facets of forensic chemistry. Drug concentrations can be very low, as often only a single dose is administered, and the pharmacodynamics of commonly employed drugs further hinders the testing process. The research presented in this work shows the development of several new assays for the detection of flunitrazepam, gamma-hydroxybutyrate (GHB) and ethyl glucuronide (EtG) in a variety of biological matrices. New methods of drug testing in blood and urine are demonstrated, as well as interesting developments in the field of hair testing. Using hair to detect drug exposure allows a much wider window of detection than the more traditional matrices of blood and urine. New methods are presented in this work using gas chromatography-tandem mass spectrometry (GCMS/MS) to detect drugs in hair. Validation data is presented along with the results of authentic DFA testing. All aspects of the drug testing procedure have been evaluated, from new extraction techniques utilising water instead of solvents, to novel clean up stages involving the unique combination of SFE and SPME. Several confirmation techniques are explored including single quadrupole, triple quadrupole and ion trap mass spectrometry. In addition to developing assays for DFA cases, the versatility of this type of analytical chemistry is explored in two population studies. The first study evaluates alcohol consumption between two groups; drugs users and non drug users in medico-legal cases. There is an anecdotal belief amongst drug clinic staff that alcohol use is lower in drugs users than it is in non drug users. This study presents the first scientific confirmation of this belief through EtG (an alcohol metabolite) testing in hair of the two groups. The second study investigates whether there is a correlation between EtG and cocaethylene (a metabolite of cocaine only produced in the presence of alcohol) in cocaine users. Results f this study suggest that there is no positive correlation between the two compounds. The research presented in this thesis aims to further the analytical science surrounding FA investigation and provide accurate, sensitive and reliable methodology for drug esting in blood, urine and hair.

The thesis reports on 6 studies (2 of which were part of a multi-centre trial) examining hydration and fluid balance. The first study described in this thesis investigated the impact of hydration status on Dual energy x-ray absorptiometry (DXA) and other methods that are popular tools to determine body composition in athletes. We observed that it is important to ensure a euhydration when assessing body composition, particularly when considering changes associated with nutritional or exercise interventions. The second and third studies reported identified beverages that promote longer term fluid retention and maintenance of fluid balance in adults. We investigated the effects of 13 different commonly consumed drinks on urine output and fluid balance when ingested in a euhydrated state, with a view to establishing a beverage hydration index (BHI), i.e., the volume of urine produced after drinking expressed relative to a standard treatment (still water) for each beverage. The beverages with the highest BHI were oral rehydration solution, full fat milk and skimmed milk. BHI may be a useful measure to identify the short term hydration potential of different beverages when ingested in a euhydrated state. The fourth study aimed to systematically examine the influence of carbohydrate, sodium and caffeine content of beverages on the BHI. The BHI was greater in beverages with higher carbohydrate or higher sodium content, but not influenced by caffeine. The carbohydrate content of beverages has no effect on BHI at concentration up to 10% carbohydrate. Sodium content of beverages in concentrations of 27mmol/L and higher can improve the hydration potential of beverages. Caffeine doses in beverages up to 400mg/L do not have an impact upon diuresis when ingested in a euhydrated state. The fifth study compared net fluid balance (NFB) responses to the ingestion of commonly consumed drinks in young and older men. We observed that in young adults milk helps to maintain positive net fluid balance for longer than other drinks. In older adults this effect of milk is not observed despite similar net electrolyte balance responses. Future work should more fully explore these potential differences in fluid balance responses to drink ingestion between young and older adults. The final study investigated the hydration habits of Scottish young and older adults (+50 years old), identifying their fluid choices, volume, and preferences in relation to time of day. The results showed that 26.1% of the young females, 30.3% of the young males, 25.8% of the older females and 50.4% of the older males did not meet the European (EU) Food Safety Authority (EFSA) fluid intake recommendations. We also observed that the difference between those who met and those who did not meet the EFSA adequate intake could be attributed to differences in water ingestion, mainly during the mid-morning (after breakfast until 11 am) and during the early-afternoon (after lunch time up to 5 pm). It was concluded that these moments might be key when implementing interventions to improve hydration status especially in the older population.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-08T08:39:40Z No. of bitstreams: 2 Dissertação - kamila Pereira de Amorim - 2015.pdf: 1653482 bytes, checksum: 57f33de27efd496be7b4807548727455 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-08T08:41:11Z (GMT) No. of bitstreams: 2 Dissertação - kamila Pereira de Amorim - 2015.pdf: 1653482 bytes, checksum: 57f33de27efd496be7b4807548727455 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-08T08:41:11Z (GMT). No. of bitstreams: 2 Dissertação - kamila Pereira de Amorim - 2015.pdf: 1653482 bytes, checksum: 57f33de27efd496be7b4807548727455 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-08-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cloud point extraction method (CPE) was used for the determination of estrone (E1) and 17β-estradiol (E2) hormones in human urine. The combination of the electrochemical detection techniques with high-performance liquid chromatography (HPLC-EC) was used for the detection and quantification of these hormones. A boron doped diamond electrode (BDD) pretreated cathodically was used as electrode material for all electrochemical measurements. The optimized chromatographic parameters resulted in a mobile phase composition of KH2PO4 (0.01 mol L-1; pH 5.0) / ACN (72:28 V/V), flow 1.2 ml min-1. An applied potential for electrochemical detection of 1.0 V x Ag/AgCl (3.0 mol L-1) was selected from hydrodynamic voltammograms constructed for each hormone changing the potential between 0.3 V and 1.2 V x Ag/AgCl (3.0 mol L-1). Limits of detection (S/N = 3) of 500 ng mL-1 and limits of quantification of 800 ng mL-1 were obtained for both E1 and E2 hormones without any extraction process. Urine samples at pH 5.0 and 7.0 were investigated aiming the influence of pH on the efficiency of the CPE process, and the optimum results for the most current signal of the hormones was obtained at pH 7.0. Extractor solvent volumes were changed in the 0.5-2.5 mL range, and the optimum results were obtained when using 1.0 mL of Tergitol TMN-6 surfactant (10% aqueous solution). From the equation of the calibration curves obtained with and without the CPE procedure it was possible to determine the pre-concentration factor (FC) and all the other parameters involving the efficiency of CPE method. A comparison of the efficiency of CPE method with direct liquid-liquid extraction with the organic solvent CCl4 was carried out and the results showed that the CPE method was quite superior to liquid-liquid extraction. The validation of the method was carried out from intra-day recovery experiments and inter-day and evaluated the accuracy, precision and repeatability. The proposed method was applied to individual samples of urine of 1 man, 1 pregnant woman, 1 woman in fertile age, and 1woman in lactating stage. The values of the variation coefficients of the recovery percentages were lower than 15%. / Método de extração por ponto nuvem (EPN) foi usado para a determinação dos hormônios estrona (E1) e 17β-estradiol (E2) em urina humana. A combinação entre as técnicas de detecção eletroquímica e cromatografia líquida de alta eficiência (CLAE-EC) foi usada para a detecção e quantificação desses hormônios. Um eletrodo de diamante dopado com boro (DDB) pré-tratado catodicamente foi usado como material de eletrodo para todas as determinações eletroquímicas. Os parâmetros cromatográficos otimizados resultaram em uma composição de fase móvel de KH2PO4 (0,01 mol L-1; pH 5,0) / ACN (72:28 V/V) vazão de 1,2 mL min-1. Um potencial aplicado para a detecção eletroquímica de 1,0 V x Ag/AgCl (3 mol L-1) foi selecionado a partir de voltamogramas hidrodinâmicos construídos para cada hormônio variando-se o potencial entre 0,3 V e 1,2 V x Ag/AgCl (3,0 mol L-1). Limites de detecção (S/R = 3) de 500 ng mL-1 e limites de quantificação de 800 ng mL-1 foram obtidos para ambos os padrões dos hormônios E2 e E1 sem qualquer processo de extração. Amostras de urina em pH 5,0 e 7,0 foram investigadas quanto a influência do pH na eficiência do processo de extração, e o melhor resultado referente ao maior sinal de corrente dos hormônios foi obtido em pH 7,0. Os volumes de solvente extrator foram variados na faixa de 0,5-2,5 mL e o melhor resultado referente ao sinal de corrente dos hormônios foi obtido pelo uso de 1,0 mL de solução aquosa 10% do surfactante Tergitol TMN-6. A partir da equação da reta obtida das curvas analíticas, com e sem o procedimento de EPN, foi possível determinar o fator de pré-concentração (FC) e todos os demais parâmetros envolvendo a eficiência do método de EPN. Uma comparação sobre a eficiência dos métodos de EPN com extração direta líquido-líquido com o solvente orgânico CCl4 foi realizada e os resultados mostraram que método de EPN mostrou-se bastante superior a extração líquido-líquido. A validação do método foi feita a partir de ensaios de recuperação intra-dia e inter-dia, sendo avaliadas a exatidão, precisão e repetitividade. O método proposto foi aplicado em amostras individuais de urina de 1 homem, 1 mulher gestante, 1 mulher em idade fértil e 1 mulher lactante. Os valores dos coeficientes de variação das porcentagens de recuperação foram menores que 15%.

A Cannabis sativa L., conhecida popularmente no Brasil como maconha, tem sido apontada como a droga de uso ilícito de maior consumo no mundo contemporâneo. As análises toxicológicas utilizadas para verificar o seu uso vem ganhando cada vez maior importância em diversos ambientes de trabalho, desportivos, prisões, clínicas para tratamento de farmacodependência, centros de emergência toxicológica, enfermarias de psiquiatria, entre outros. A presença de canabinóides na urina indica o uso de produtos derivados da planta Cannabis. A maioria dos estudos encontrados na literatura especializada se refere ao perfil de eliminação do 11-nor-&#916 9-THC-COOH, principal produto de biotransformação do &#916 9-THC, que, dependendo da sensibilidade e especificidade da técnica analítica utilizada, poderá ser detectado a longo prazo na urina. Uma constante preocupação é determinar o período de detecção, ou seja, o tempo transcorrido desde a interrupção do uso da droga até a obtenção do primeiro resultado negativo na urina. Este trabalho teve como objetivo investigar o período de detecção de canabinóides urinários em uma população usuária de Cannabis, residentes em uma clínica de tratamento, pela técnica de imunofluorescência polarizada, considerando 50 ng/mL como valor de referência. Foi avaliada a influência do padrão de uso da droga no período de detecção de canabinóides. Entre os pacientes estudados, o período de detecção foi de grande variação: de 33 a 498 horas. Não houve correlação estatística relevante entre o período de detecção e as variáveis freqüência de uso, tempo total de utilização e quantidade de cigarros utilizada na última exposição. / The marijuana, one of the products originated from the Cannabis sativa L., is the illicit used drug of major consume in the world. The toxicological analysis to verify the use of the drug are increasing importance in several workplaces drug-testing programs, sportive, prisions, drug treatment and rehabilitation clinics, emergency toxicology departments and other. The cannabinoids in urine indicates the exposition of products originated from the Cannabis. An important concern is to determine the detection times of cannabinoids in urine i.e. the time from the use of the drug until the first negative result. Most of the studies in the speciallized literature report the elimination profile of the 11-nor-9-carboxy- &#916 9-tetrahydrocannabinol (THCCOOH), which the primary cannabinoid metabolite. According to those studies, the urinary cannabinoids detection times can be made for a long time depending on the sensibility and accuracy of the methods used. The purpose of this work was estudy the detection time of the urinary cannabinoids in the marijuana users population after drug abstaining, by FPIA, using a 50ng/mL cutoff. The pattern in drug use influence on the urinary cannabinoids detection times was evaluated. The detection time in the studied population ranged from 33 to 498 hours. The statistic correlation between the detection time and the drug use pattern was not significant (p&#8804 0,05).

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors. Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any preconcentration scheme beyond sample work up by solid phase extraction (SPE). Using this hyphenated approach, submicromolar levels of catecholamines and metanephrines in normal human urine and indolamines in human plasma were detected after the removal of the analytes from their biological matrices and after preconcentration by SPE on mixed mode cation-exchange sorbents. The CE-TOFMS and SPE methods were individualized for each group of compounds. While catecholamines and metanephrines in urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard, deuterated isotopes, considered ideal internal standards, were used for the quantitation of indolamines. Because the angiotensin peptides are present in biological fluids at much lower concentrations than the previous two families of analytes, their analysis required the application of additional preconcentration techniques. In this work, the coupling of either of two types of electrophoretic preconcentration methods - field amplified injection (FAI) and isotachophoresis (ITP) - to capillary zone electrophoresis with both UV and MS detection was evaluated. Using FAI-CE-UV, angiotensins were detected at ~1 nM concentrations. Using similar conditions but TOFMS detection, the detection limits were below 10 nM. ITP was evaluated in both single-column and two-column comprehensive arrangements. The detection limits achieved for the ITP-based techniques were approximately one order of magnitude higher than for the FAI-based preconcentration. While the potential usefulness of these techniques was demonstrated using angiotensins standards, substantial additional research would be required to allow these approaches to be applied to plasma as part of clinical assays.

No description available.

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...

Av samtliga globala processer som reglerar jordsystemet är de biogeokemiska flödena av kväve (N) och fosfor (P) mest påverkade av mänskliga aktiviteter. Inerta former av N och P omvandlas till reaktiva former som sprids i miljön, där de orsakar eutrofiering och påverkar marina ekosystem negativt. Majoriteten av de reaktiva N- och P-formerna används för framställningen av mineralgödsel. Ett alternativt sätt att producera gödsel är att återvinna näringsämnena i avloppet. En teknik som återvinner näringsämnen i urin är basisk urintorkning. Teknologin stabiliserar urea med ett basiskt torkningsmedium och koncentrerar näringsämnena genom att evaporera vattnet i urinen. Slutprodukten är ett torrt gödsel i pulverform. I det här projektet testades urintorkningsteknologin för första gången i ett fältförsök. Ett system för urintorkning med kapacitet att förånga 40 kg urin dygn-1 m-2 konstruerades och integrerades i ett befintligt torrt sanitetssystem för användning under en period på tre månader. I projektet utvärderades 13 dygn av de 3 månaderna. Resultaten visade att 24 kg urin tillfördes systemet och att systemet kunde upprätthålla en kontinuerlig torkning av urinen. Efter torkningen återvanns majoriteten (97 %) av N i slutprodukten. På grund av att den tillförda mängden urin var liten blev växtnäringshalterna i slutprodukten och i torrsubstansen (TS) av slutprodukten låga. Systemet hade emellertid potential att torka mycket större kvantiteter urin. Om systemets fulla potential hade använts, det vill säga att torka 40 kg urin dygn-1 m-2, så hade särskilt N- och P-halterna ökat avsevärt. N-halterna hade även ökat ytterligare om torkningen hade utförts vid en lägre temperatur. Systemets energiförbrukning var hög, eftersom systemet hade en kontinuerlig energikonsumtion och även komponenter med hög effekt. I jämförelse med den konventionella avloppsvattenreningen och produktionen av mineralgödsel har systemet en hög energikonsumtion, men i jämförelse med en förbränningstoalett är systemets energiförbrukning likvärdig. För att minska energiförbrukningen kunde reglertekniska åtgärder utföras så att systemets energitillförsel upphör när systemet inte används. Systemets energiförbrukning får även ställas i relation till de problem som dagens system för livsmedelsproduktion och sanitet medför. Till skillnad från nämnda system möjliggör urintorkningsteknologin besparing av dricksvattenresurser, ett slutet kretslopp av näringsämnen och en minskad påverkan på miljön. / Of all global processes that regulate the earth system, the biogeochemical flows ofnitrogen (N) and phosphorus (P) are the most affected by human activities. Inert forms of N and P are converted into reactive forms that are dispersed in the environment, causing eutrophication and affecting marine ecosystems. The majority of the reactive N and P are used for the production of mineral fertilizers. An alternative way of producing fertilizers is to recycle nutrients from waste water. A technology that reuses nutrients in urine is alkaline urine drying. The technology stabilizes urea with an alkaline drying medium and concentrates the nutrients by evaporating the water in urine. The end-product is a dry fertilizer in powder form. In this master project, the alkaline urine drying technology was tested for the first time in field conditions. A system for urine drying with the capacity to evaporate 40 kg of urine day-1 m-2 was constructed and integrated into an existing dry sanitation system for use over a period of three months. The master project evaluated the system for 13 days of the 3 months. The results showed that 24 kg of urine was collected in the system, significantly less than what the system had been designed to dry. Furthermore, the results showed that the system functioned smoothly recovering 97 % of the urine-N in the end-product. The nutrient content in the end-product and the dry matter of the end-product was low due to the low amount of urine that was collected. However, the system had the potential to dry much larger quantities of urine. If the system would have been operated to function at full potential (drying 40 kg of urine day-1 m-2) the N- and P-content in the end-product would be much higher than that observed during the 13 days. Furthermore, the system if operated at lower temperatures has the potential to recover more N. The system’s energy consumption was high, as the system had a continuous energy consumption. In comparison with the conventional waste water treatment and the production of mineral fertilizers, the system has a high energy consumption, but compared to an incineration toilet, the system’s energy consumption is equivalent. In order to reduce the energy consumption, automatic control could be implemented so that the energy is switched off when the system is not used. The system’s energy consumption may also be set in relation to the problems that today’s systems for food production and sanitation entail. Unlike the aforementioned systems, the urine dehydration technology does not consume drinking water, it enables recycling of nutrients as well as a reduced impact on aquatic life.

Einleitung: Die Leptospirose ist eine der weltweit bedeutendsten Zoonosen. Durch den häufig asymptomatischen oder unspezifischen Krankheitsverlauf, wird von einer hohen Dunkelziffer ausgegangen. Menschen können sich über direkten Tierkontakt oder indirekten Kontakt mit dem Urin infizierter Tiere, zum Beispiel über kontaminierte Gewässer oder Erde, anstecken. Die Infektion erfolgt dabei über den Eintritt des Erregers in Schleimhäute oder Hautwunden. Ziel der Untersuchungen: Obwohl die Überlebenszeit von Leptospiren in der Umwelt einen entscheidenden Einfluss auf das Infektionsrisiko des Menschen hat, wurden bisher nur sehr wenige Untersuchungen dazu durchgeführt. Diese konzentrierten sich dabei vor allem auf das Überleben der Erreger in Erde oder Wasser. Daher war es Ziel dieser Arbeit die Umweltstabilität eines häufig in Deutschland gefundenen Leptospirenserovars unter verschiedenen Umweltbedingungen zu untersuchen. Zusätzlich zu diesen Untersuchungen war es Ziel dieser Arbeit, durch die Veröffentlichung eines Übersichtsartikels über die Leptospirose Ärzte in Deutschland für diese häufig unerkannt bleibende Erkrankung zu sensibilisieren. Material und Methoden: Ein jahrelang an Kulturmedium adaptierter Labor- und ein erst vor 3 Jahren isolierter Feldstamm von Leptospira kirschneri Serovar Grippotyphosa wurden auf ihr Überleben in der Umwelt untersucht. Es wurde ihre Widerstandsfähigkeit gegenüber verschiedenen Umwelteinflüssen, wie z.B. Tierurin als umgebendes Medium bei verschiedenen Temperaturen oder auch der Einfluss einer Trocknung, untersucht. Nachdem die Leptospiren den Umwelteinflüssen für unterschiedliche Zeitspannen ausgesetzt waren, wurde versucht sie in EMJH-Medium wieder zu kultivieren. Während einer Inkubationszeit von mindestens 28 Tagen bei 29 °C wurden diese Kulturen wöchentlich unter dem Dunkelfeldmikroskop auf das Vorhandensein motiler Leptospiren untersucht. Zusätzlich wurde durch Kultivierungsversuche in EMJH-Medium das Überleben der Leptospiren in einem Wasserstrom mit einer definierten Fließgeschwindigkeit und ihre Verbreitung in diesem Strom mittels real-time PCR untersucht. Alle Versuche wurden im Dreifachansatz durchgeführt. Statistische Untersuchungen wurden mittels eines zweiseitigen Mann-Whitney-U Tests (Fehler 1. Art α = 0,05) durchgeführt. Ergebnisse: Die beiden untersuchten Stämme von L. grippotyphosa überlebten nicht in unverdünntem Tierurin. In verdünntem Tierurin überlebten die Stämme, je nach Temperatur und Verdünnungsmedium, zwischen 1 - 72 Stunden (Laborstamm) und 4 - 24 Stunden (Feldstamm). Beide Stämme überlebten signifikant länger bei 15 °C als bei 37 °C (p < 0,001, bzw. p = 0,041). Der Laborstamm überlebte signifikant länger in verdünntem Rindereurin (max. 72 h bei 15 °C) als in verdünntem Hundeurin (max. 4 h; p = 0,027). Im Gegensatz dazu, überlebte der Feldstamm signifikant länger in Hundeurin (max. 24 h bei 15 °C) als in Rinderurin (max. 4 h; p = 0,028). Das vollständige Trocknen auf einer festen Oberfläche war bei Temperaturen zwischen 15 °C und 37 °C für beide Stämme letal. Jedoch war, unabhängig von der untersuchten Temperatur, eine halbe Stunde vor der vollständigen Trocknung eine Kultivierung der Leptospira spp. noch möglich. In einem Wasserstrom konnten sich die Leptospiren aufgrund ihrer Eigenbewegung schneller und langsamer als die Durchschnittsgeschwindigkeit (0,01 m / s) des Wassers bewegen, überlebten jedoch die mechanischen Schäden während des Schlauchdurchflusses nicht. Schlussfolgerungen: Das Überleben von Leptospira spp. ist offensichtlich von vielen Faktoren abhängig. Eine schnelle Verdünnung nach der Ausscheidung mit dem Urin scheint dabei essentiell zu sein. Niedrigere Temperaturen sowie eine feuchte Umgebung verbessern ihre Widerstandsfähigkeit gegen schädliche Einflüsse, während Trockenheit oder mechanische Schädigung ihr Überleben nicht ermöglichen. Wegen der großen Bedeutung der leptospiralen Überlebenszeit in der Umwelt für das Infektionsrisiko von Mensch und Tier sind weitere Untersuchungen auf diesem Forschungsgebiet in der Zukunft nötig.:1. Einleitung 2. Literaturübersicht 2.1 Die Geschichte der Leptospirose 2.2 Morphologie und Übertragungswege der Erreger 2.3 Taxonomie 2.4 Haupt- und Nebenwirte 2.5 Die Erkrankung beim Menschen 2.5.1 Vorkommen 2.5.2 Klinik und Therapie 2.6 Die Erkrankung beim Tier 2.6.1 Bei Nutztieren 2.6.2 Bei Haustieren 2.6.3 Bei Wildtieren 2.7 Diagnostik 2.8 Die Umweltstabilität der Erreger 2.8.1 In Erde 2.8.2 In Wasser 2.8.3 In Urin 3. Publikationen 3.1 Publikation Nr. 1 3.2 Publikation Nr. 2 4. Diskussion und Schlussfolgerung 5. Zusammenfassung 6. Summary 7. Literaturverzeichnis 8. Danksagung / Introduction: Leptospirosis is one of the most important zoonosis worldwide. Due to the often asymptomatic or non-specific course of the disease, a high number of unreported cases is assumed. Humans can get infected through direct contact with animals or indirect contact with the urine of infected animals, for example through contaminated water or soil. The infection occurs via entry of the pathogen through mucous membranes or skin wounds. Aims of the study: Although the survival time of Leptospira spp. in the environment has a crucial influence on the human infection risk, very few studies have been carried out so far. These studies focused primarily on the survival of the pathogens in soil or water. It was therefore the aim of this study to investigate the environmental stability of a leptospiral serovar that is frequently found in Germany under different environmental conditions. In addition to these investigations, the aim of this work was to raise the awareness of physicians in Germany for this often undetected and neglected disease by publishing a review article on leptospirosis. Material and Methods: The survival in the environment of both a laboratory strain, which has been adapted to culture medium for years and a field strain, which was isolated only 3 years ago, of Leptospira kirschneri Serovar Grippotyphosa was studied. Their resistance to various environmental influences, such as animal urine as surrounding medium at different temperatures or the influence of drying, were examined. After the leptospires were exposed to these influences for various time periods, an attempt was made to cultivate them in EMJH medium. During an incubation period of at least 28 days at 29 °C, the cultures were examined weekly for the presence of motile leptospires under the darkfield microscope. In addition, the survival of the leptospires in a water stream with a defined velocity and their distribution in this stream were examined by real-time PCR and cultivation experiments in EMJH medium. All experiments were carried out in triplicate. The statistical analysis was done using a two-tailed Mann-Whitney U test (type-1-error α = 0.05). Results: Both examined strains of L. grippotyphosa did not survive in undiluted animal urine. In diluted animal urine, the strains survived between 1-72 hours (laboratory strain) and 4-24 hours (field strain), depending on the temperature and dilution medium. Both strains survived significantly longer at 15 °C than at 37 °C (p < 0.001 or p = 0.041). The laboratory strain survived significantly longer in diluted cattle urine (max. 72 h at 15 °C) than in diluted dog urine (max. 4 h) (p = 0.027) while the field strain survived significantly longer in dog urine (max. 24 h at 15 °C) than in cattle urine (max. 4 h) (p = 0.028). Complete drying on a solid surface at temperatures between 15 °C and 37 °C was lethal for both strains. However, regardless of the temperature examined, Leptospira spp. were still cultivatable half an hour before the time point of complete drying. In a water stream, leptospires were able to move faster or slower than the average velocity of the water (0.01 m / s) due to their intrinsic mobility but were not able to survive the mechanical damage caused by running water in the hose system. Conclusions: Overall, it can be concluded that the survival of Leptospira spp. depends on many factors. Rapid dilution after urine excretion appears to be essential. Lower temperatures and a humid environment improve their survival time, while drought or mechanical damage is lethal to them. Because of the great importance of leptospiral survival in the environment for the infection risk of humans and animals, further studies in this research area will be necessary in the future.:1. Einleitung 2. Literaturübersicht 2.1 Die Geschichte der Leptospirose 2.2 Morphologie und Übertragungswege der Erreger 2.3 Taxonomie 2.4 Haupt- und Nebenwirte 2.5 Die Erkrankung beim Menschen 2.5.1 Vorkommen 2.5.2 Klinik und Therapie 2.6 Die Erkrankung beim Tier 2.6.1 Bei Nutztieren 2.6.2 Bei Haustieren 2.6.3 Bei Wildtieren 2.7 Diagnostik 2.8 Die Umweltstabilität der Erreger 2.8.1 In Erde 2.8.2 In Wasser 2.8.3 In Urin 3. Publikationen 3.1 Publikation Nr. 1 3.2 Publikation Nr. 2 4. Diskussion und Schlussfolgerung 5. Zusammenfassung 6. Summary 7. Literaturverzeichnis 8. Danksagung