Nenhuma medição é realizada com perfeição absoluta, uma vez que todos os valores encontrados são aproximações do valor real e todas as medidas, independente de sua finalidade ou qualidade, possuem uma incerteza. A incerteza de medição é um parâmetro associado ao resultado, que caracteriza a dispersão em torno dos seus valores. O conceito de incerteza de medição já é adotado em laboratórios de calibração e também muito aplicado na área de engenharia; no entanto em análises toxicológicas esta abordagem ainda é recente e há poucos relatos na literatura científica. Portanto, este trabalho teve como objetivo o estudo da incerteza de medição em análises toxicológicas confirmatórias de substâncias psicoativas - anfetaminas (anfetamina e metanfetamina), ácido 11-nor-Δ9-tetraidrocanabinol carboxílico (THC-COOH) e benzoilecgonina - em urina, detectados pela técnica de cromatografia em fase gasosa acoplada à espectrometria de massas (GC-MS). A microextração em fase líquida (LPME) mostrou-se eficaz na determinação de THC-COOH, e após a completa validação, o método desenvolvido foi aplicado na quantificação de amostras de urina de referência provenientes do National Institute of Standards and Technology (NIST) dos Estados Unidos da América (SRM1507b - NIST). As principais contribuições para a incerteza do método foram a concentração do analito, a acurácia, seguidos da precisão e do volume de amostra. A incerteza combinada obtida foi equivalente a 8%. A LPME também apresentou-se eficiente para a extração das anfetaminas e a incerteza combinada obtida por este método foi 2,1%. No método de detecção de benzoilecgonina, a principal fonte de incerteza foi a acurácia do método e o resultado da incerteza combinada da análise de uma urina de referência (SRM1508a - NIST) foi 4,8%. Todos os valores de incerteza de medição encontrados em nosso estudo estão de acordo com as normas e referências internacionais e também são condizentes com os valores estipulados pela NIST nos laudos de análise das amostras de referência. / Neither measurement is made with absolute perfection, once all the values are approximations of the actual value and all measures, independent of its purpose or quality, have an uncertainty. Measurement uncertainty is a parameter associated with the result, which characterizes the dispersion around their values. The concept of measurement uncertainty is already used in calibration laboratories and also widely applied in engineering, however, in toxicological analysis, this approach is recent and there are few reports in the scientific literature. Therefore, this work aimed to study the measurement uncertainty in confirmatory toxicological analysis of psychoactive drugs - amphetamines (amphetamine and methamphetamine), acid 11-nor-Δ9-tetrahydrocannabinol carboxylic acid (THC - COOH) and benzoylecgonine - in urine detected by the technique of gas chromatography-mass spectrometry (GC- MS). The liquid phase microextraction (LPME) was effective in the determination of THC-COOH, and after complete validation, the method was applied to the quantification of urine samples of reference from the National Institute of Standards and Technology (NIST) of United States of America (SRM1507b - NIST). The main contributions to the uncertainty of the method were the analyte concentration, accuracy, followed by the precision and the sample volume. The combined uncertainty obtained was equivalent to 8%. The LPME also presented efficient for the extraction of amphetamine and combined uncertainty obtained by this method was 2.1%. In the method of detection of benzoylecgonine, the main source of uncertainty was the accuracy of the method and the result of the combined uncertainty of the analysis of a urine reference (SRM1508a - NIST) was 4.8%. All values of measurement uncertainty found in our study are in accordance with international standards and references and are also consistent with the values stipulated by certificate of analysis of NIST reference samples.

Uma avaliação da concentração dos aminoácidos primários em amostras de urina de crianças com refluxo vésico-ureteral (VUR) em busca de caminhos para o diagnóstico não invasivo desta doença. Dois métodos analíticos por eletroforese capilar com detecção UV/vis foram desenvolvidos para a quantificação dos analitos. No método 1 empregou-se a detecção UV/vis direta em 200 e 214 nm com as condições eletroforéticas eletrólito tampão fosfato 90 mmol L-1 pH 2,1; tensão de +15 kV; injeção de 7 s a 0,5 psi; capilar de 75 µm de diâmetro interno; 40,2 cm de comprimento total e 30,0 cm de comprimento efetivo. No método 2, fez-se uso da detecção indireta em 254 nm, com as condições eletroforéticas eletrólito tampão TEA 20 mmol L-1 e DNB 10 mmol L-1 pH 10,84; modificador de fluxo DDAB a 4 mmol L-1; tensão de -15 kV; injeção de 7 s a 0,5 psi; capilar de 75 µm de diâmetro interno; 50,2 cm de comprimento total e 40,0 cm de comprimento efetivo. O método 1 apresentou parâmetros de validação linearidade, precisão intra-dia e inter-dia, seletividade, robustez e recuperação satisfatórios. A quantificação de creatinina, fenilalanina (Phe), histidina (His), triptofano (Trp), tirosina (Tyr) nas amostras de urina foi possível pelo método 1, porém inviável para quantificação de arginina (Arg). O método 2 apresentou valores de robustez e recuperação satisfatórios para os aminoácidos alanina (Ala), aspartato (Asp), glutamato (Glu) e glicina (Gly) satisfatórios, mas a quantificação dos mesmos na maioria das amostras de urina diluída não foi possível por estarem em nível de concentração abaixo da detecção ou quantificação. Para avaliar a potencialidade dos resultados como ferramenta no diagnóstico do VUR, os aminoácidos His, Phe, Trp e Tyr, quantificados em todas as amostras, foram empregados como variáveis na classificação das amostras em dois grupos distintos (1) grupo de crianças saudáveis e (2) grupo de crianças diagnosticadas com VUR. A classificação realizada pelo método de análise de componente principal (PCA) apresentou valores estatísticos satisfatórios e poder de predição: R2 (capacidade de ajuste) e Q2 (capacidade de predição) foram 0.9993 e 0.65, respectivamente com os dois componentes principais (PC1 e PC2). A separação total com valor de Q2 desejável (acima de 0,8) poderia ser alcançada com uma quantidade maior de informação, sendo neste caso, número maior de aminoácidos quantificados. Assim, este trabalho abre caminho para estudos mais aprofundados na investigação da concentração dos aminoácidos primários em pacientes com VUR, objetivando o desenvolvimento de um potencial biomarcador para VUR. / An assessment of the concentration of primary amino acids in urine samples from children with vesicoureteral reflux (VUR) using capillary electrophoresis separation with UV/vis detection has been proposed to help establishing a means for non invasive diagnosis of the disease. Two analytical methods were developed. Method 1 used direct UV/vis detection at 200 and 214 nm, 90 mmol L-1 phosphate buffer at pH 2.1, high voltage separation at +15 kV, injection of 0.5 psi during 7 s, and a fused-silica capillary of 75 µm inner diameter, 40.2 cm total length, and 30.0 cm effective length. Method 2 used indirect UV/vis detection at 254 nm, TEA at 20 mmol L-1 and DNB at 10 mmol L-1 electrolyte at pH 10.84, 4 mmol L-1 DDAB as flow modifier, separation voltage at -15 kV, injection of 0,5 psi during 7 s, fused-silica capillary of 75 µm inner diameter, 50.2 cm total length, and 40,0 cm effective length. Method 1 presented satisfactory results for linearity, intra-day and inter-day precision, selectivity, robustness, and recovery. By method 1 it was possible to quantify creatinine, phenylalanine (Phe), histidine (His), tryptophan (Trp), tyrosine (Tyr) but not arginine (Arg) in the urine samples under investigation. Method 2 presented satisfactory robustness and recovery for alanine (Ala), aspartate (Asp), glutamate (Glu) and glycine (Gly), but the contents of these metabolites in the urine samples were not established because they lay below the limits of detection and quantitation. To assess the potentiality of the results as diagnostic tool for VUR condition, the concentrations of the amino acids His, Phe, Trp and Tyr, quantified in all samples, were used as variables in a classification procedure where samples were divided in two distinct groups: (a) a group of healthy children and (b) a group of children diagnosed with VUR. The classification by principal component analysis (PCA) showed a partial separation with good statistics and prediction power: R2 (goodness of fit) and Q2 (goodness of prediction) were 0.9993 and 0.65, respectively with two components analysis (PC1 and PC2). Values of Q2 greater than 0.8 are usually desired and it could be provided if more information was available, such as a greater number of amino acids being quantified. Thus, this research opens the way for further investigative studies of amino acids concentration in patients with primary VUR, aimed at developing a potential biomarker for VUR.

Diagnostiquer précocement les maladies est un défi à relever pour améliorer la prise en charge des patients concernés et leur offrir une meilleure qualité de vie. Les analyses 'omiques', qui quantifient globalement, simultanément et sans a priori l'abondance de milliers de molécules dans les liquides biologiques, s'avèrent très prometteuses pour l'identification de biomarqueurs précoces des maladies complexes. Dans ce contexte, mon travail de thèse avait pour objectif de développer des outils de diagnostics, à partir d'analyses du peptidome et métabolome urinaire, pour détecter précocement la présence d'une maladie rénale chronique (MRC) et la survenue de ses complications cardiovasculaires. La première étude, insérée dans le projet européen 4C (Cardiovascular Complications in Children with Chronic kidney disease), s'est centrée sur les complications cardiovasculaires associées à la MRC en pédiatrie. Ces complications constituent la principale cause de mortalité des enfants en insuffisance rénale, et leur diagnostic précoce est impossible à ce jour. En analysant par électrophorèse capillaire couplée à la spectrométrie de masse (CE-MS) le peptidome urinaire de 86 enfants souffrant, ou non, de complications cardiovasculaires secondaires à la MRC, nous avons identifié des peptides qui permettent de prédire à l'avance les patients à haut risque cardiovasculaire : 190 peptides étaient associés à l'épaississement de la paroi carotidienne (AUC 0.87, sensibilité 80%, spécificité 100%) et 22 peptides prédisaient l'augmentation de la rigidité artérielle (AUC 0.83, sensibilité 83%, spécificité 70%). Le second projet relevait de la médecine vétérinaire. Dans cette étude menée sur 50 chiens avec et sans MRC, nous avons caractérisé pour la première fois le peptidome urinaire canin via la technologie CE-MS et nous avons découvert 133 peptides urinaires associés à la MRC. Ces derniers ont permis de diagnostiquer la présence d'une MRC dans 80% des chiens. Les métabolites sont mieux corrélés au phénotype que les autres strates moléculaires. Cependant l'apport de la métabolomique en clinique est encore limité, dû au manque de technologies analytiques performantes. Le troisième objectif de ma thèse était donc de mettre au point une procédure de dosage par CE-MS des métabolites urinaires. Grâce à une méthode unique de normalisation interne, basée sur l'utilisation de métabolites endogènes stables, il est maintenant possible d'analyser le contenu en métabolites d'un même échantillon urinaire avec une très haute reproductibilité sur le long terme (4 ans). Comme preuve de concept, nous avons mis en évidence, via cette procédure, la présence d'une combinaison de 32 métabolites dans l'urine qui permet de repérer avec une sensibilité de 76% et une spécificité de 86% les nouveau-nés porteurs d'une malformation rénale obstructive. Enfin, la quatrième problématique s'inscrivait dans une démarche translationnelle. Son but était de développer des aptasenseurs capables de détecter avec de hautes affinités et spécificités les biomarqueurs d'origine omique, pour un diagnostic simple, rapide et à moindre coût. La cible choisie était un fragment urinaire de l'alpha-1-antitrypsine, qui est ~1000 fois plus abondant chez les adultes atteints de MRC que les chez les sains. La sélection de l'aptasenseur s'est faite par le Systematic Evolution of Ligands by EXponential enrichment (SELEX). Nous présentons ici les travaux préliminaires de la mise au point du SELEX sur cette cible. En conclusion, cette thèse démontre le potentiel de l'analyse du contenu urinaire en peptides et métabolites pour le diagnostic précoce des pathologies complexes telles que la MRC et les complications cardiovasculaires associées. De plus l'obtention d'aptasenseurs dirigés contre ces biomarqueurs précoces et utilisables au chevet du patient devrait révolutionner dans le futur les méthodes diagnostiques. / Early diagnosis of diseases is a big challenge to improve patients' health and quality of life. 'Omics' analyses, which allow the global and simultaneous quantification of the relative abundance of thousands of molecules in biological fluids are promising for the identification of early biomarkers of complex diseases. In this context, the objective of my thesis was to develop diagnostic tools, based on urinary peptidome and metabolome analyses, for the early detection of chronic kidney disease (CKD) and associated cardiovascular complications. The first study, as part of the 4C European project (Cardiovascular Complications in Children with Chronic kidney disease), focused on analyzing the cardiovascular complications associated to CKD in children. These complications are the main cause of mortality in children with CKD and their early diagnosis is impossible for now. Analysis of the urinary peptidome of 86 children with or without cardiovascular complications associated to CKD by capillary electrophoresis coupled to mass spectrometry (CE-MS), led to the identification of two sets of peptides for the early prediction of high cardiovascular risk in pediatric patients: 190 peptides were associated to an increase of the carotid intima-media thickness (AUC 0.87, sensitivity 80%, specificity 100%) and 22 peptides were associated to an increase in arterial stiffness (AUC 0.83, sensitivity 83%, specificity 70%). The second study falls in the field of veterinary medicine. In this study, carried out on 50 dogs with or without CKD, we analyzed for the first time the canine urinary peptidome using the CE-MS technology. We identified 133 urinary peptides associated to CKD allowing an accurate diagnosis of CKD in 80% of the dogs. Metabolites correlate best to phenotype compared to other molecular traits. However, the use of metabolomics for identification of clinically relevant biomarkers is very limited due to the lack of high-performance analytical technologies. The third part of my thesis was to develop a procedure for the quantification of urinary metabolites by CE-MS. Using a unique method of internal normalization based on endogenous and stable metabolites, we can now analyze the metabolite content of the same urine sample with a high reproducibility over the long-term (4 years). As a proof-of-concept, we demonstrated that this developed procedure led to the identification of a set of 32 urinary metabolites that allow the early identification of newborns with an obstructive kidney anomaly with a sensitivity of 76% and a specificity of 86%. Finally, the fourth study was dedicated to improving translational research. The aim was to develop aptasensors able to detect 'omics'-identified biomarkers with a high affinity and specificity to obtain a simple, rapid and low-cost diagnostic test. The biomarker chosen as target is a urinary fragment of alpha-1-antitrypsin, which is ~1000 more abundant in adults with CKD compared to healthy subjects. Aptasensors were selected by the Systematic Evolution of Ligands by EXponential enrichment (SELEX). Here we present preliminary work on the development of the SELEX for our target. In conclusion, this thesis shows the strength of the urinary content, in terms of peptides and metabolites, for the early diagnosis of complex pathologies like CKD and the associated cardiovascular complications. Moreover, the selection of aptasensors targeting these early biomarkers and that can be used at bedside, will revolutionize future diagnostic methods.

Engineers are exploring a new paradigm in wastewater treatment; focus is shifting to the recovery and reuse of energy, water, and nutrients. Ecological sanitation (EcoSan) technologies, which allow for this recovery and reuse, are an environmentally sound option for the future of sanitation. While the technology to achieve this goal of recovery and reuse exists, a limiting factor is user attitudes and perceptions. Social sciences, especially anthropology, can and should inform engineering projects to ensure socio-cultural sustainability. Since 2003, rural indigenous Ngäbe communities in Panama have been implementing ecological sanitation projects, mainly double vault urine diverting (DVUD) latrines known as composting latrines. With the help of governmental agencies and the Peace Corps, over 200 of these latrines have been built across the province of Bocas del Toro and the ñÖ Kribu region of the Comarca Ngäbe-Buglé. To this point, little monitoring and evaluation has taken place in these communities. Interviews and observations in 23 communities throughout this coastal region revealed that 70.6% of composting latrines constructed (n = 201) were completed and 71.8 % of the completed composting latrines (n = 142) are still in use. Based on observations, 65% of the latrines in use were determined to be used properly, which translates to the proper use of 45.8% of the completed latrines. To promote composting latrine adoption, social marketing and pilot latrine projects can be employed, and to improve the percentage of properly used composting latrines, education campaigns can be deployed as follow up. Utilizing suggestions made in recent literature as guidelines for the proper application of compost, analysis showed that new training messages have not reached the communities with older composting latrines. Informal interviews in 18 communities identified compost production, the lack of mosquitoes and flies, and the lack of odor as the most frequently mentioned advantages. With respect to the disadvantages, the inability to use water for anal cleansing was the most frequently mentioned disadvantage. In three communities, informal interviews and 124 surveys were used to characterize the perceptions of Ngäbes regarding feces and their use of composted human excrement as a soil amendment in agriculture. In general, the responses reflected perceptions that show no strong barrier to the operation and maintenance of composting latrines. Utilizing the Fisher's exact test and Kruskal-Wallis test, the community, sanitation classification, gender, primary occupation, and age all showed some level of association with the perceptions expressed in the survey responses. Filo Verde was more likely to respond with perceptions accepting of composting latrine use, while San San Puente was more likely to respond with "don't know" or with perceptions objecting to composting latrine use. At times, up to 37.9% of the respondents responded with negative perceptions; thus, evaluations of perceptions prior to the implementation stage are still beneficial. One discrepancy existed between the overall majority and the composting latrine user majority; 56.5% of the 124 respondents perceived the handling of human excrement as a great health risk, whereas 59.1% of the 22 composting latrine users did not. As expected, the composting latrine users responses represent the positive perceptions of feces and their reuse, but pit latrine owners were most likely to respond with perceptions contrary to those indicative of proper composting latrine behavior. Overall, males were more likely to agree with the perceptions related to composting latrine use. Regarding primary occupations, farmers consistently replied with more favorable perceptions of feces and their use as a soil amendment, while banana company workers showed more dissidence. Additionally, older participants gave responses reflecting favorable perceptions of composting latrines more than younger participants. Finally, education and household size do not have any statistically significant associations with the perceptions reflected in the survey responses.

New Zealand is dominated by its agricultural industry with one of the most intensive farming practices being that of intensive dairying. New Zealand currently has approximately 5.3 million dairy cows that excrete up to 2.2 L of urine, per urination event, up to 12 times per day. This equates to 5.1 x10¹⁰ L per year or enough urine to fill over 1.2 million milk tankers. This sheer volume of urine and its associated N content has implications for the cycling of N within the pasture soils utilised, and New Zealand’s greenhouse gas budget due to the emission of N₂O from urine affected areas. The emission of nitric oxide (NO) from agricultural systems is also receiving increasing attention due to concerns about alterations in the balance of atmospheric trace gases and sinks. Worldwide there is a dearth of information with respect to the emissions of NO from urine-N deposition onto soils with only two in situ studies and no studies on the effects of soil pH, environmental variables or urine-N rate on NO fluxes. This present study has provided some fundamental information on the factors and processes affecting the emission of NO from bovine urine applied to pasture soils. Five experiments were performed in total; three laboratory experiments and two field experiments. The first laboratory experiment (chapter 4) examined the effect of the initial soil pH on NOx emissions from urine-N applied at 500 kg N ha⁻¹. Soil was treated to alter the initial soil pH over the range of 4.4 to 7.6. Initial soil pH affected rates of nitrification which in turn affected the decline in soil pH. Emissions of NO increased with increasing soil pH. However, a strong positive linear relationship was established between the NO-N flux, expressed as a percentage of the net NH4⁺-N depletion rate, and the level of soil acidity. The NO-N fluxes were higher under the more acidic soil conditions where N turnover was lower. The fluxes of N₂O did not follow the same pattern and were attributed to biological mechanisms. In experiment two (chapter 5) the objectives were to concurrently examine the effects of varying the soil temperature and the water-filled pore space (WFPS) on NOx emissions from urine-N. In this experiment increasing the soil temperature enhanced both the rate of nitrification and the rate of decrease in soil pH. The relationship between the net NO-N flux, expressed as a percentage of the net NH4⁺-N depletion rate, and the level of soil acidity was again demonstrated at the warmest soil temperature (22°C) where soil acidification had progressed sufficiently to enable abiotic NO formation. The NO-N fluxes increased with decreasing soil moisture and increasing soil acidity indicating abiotic factors were responsible for NO production. The Q10 response of the NO flux between 5 to 15°C decreased from 4.3 to 1.5 as WFPS increased from 11% to 87% respectively. Fluxes of N₂O increased with increasing WFPS and temperature indicating that denitrification was the dominant process. Results from experiments 2 and 3 indicated that the rate of nitrification had a direct bearing on the ensuing soil acidity and that it was this in conjunction with the available inorganic-N pools that affected NOx production. Therefore the third experiment examined the effect of urine-N rate on NOx emissions, with urine-N rate varied over 5 levels from 0 to 1000 kg N ha⁻¹, the highest rate being that found under maximal urine-N inputs to pasture. Rates of nitrification were diminished at the highest rates of urine-N applied and decreases in soil acidity were not as rapid due to this. Again significant but separate linear relationships were developed, for each urine-N rate used, between the NO-N flux, expressed as a percentage of the net NH4⁺-N depletion rate, and the level of soil acidity. The slope of these relationships increased with increasing urine-N rate. The NO-N flux, expressed as a percentage of the net NH4⁺-N depletion rate, versus soil acidity was higher under 1000 kg N ha⁻¹, despite the lower soil acidity in this treatment. This indicated that the enhanced inorganic-N pool was also playing a role in increasing the NO flux. The N₂O fluxes were of limited duration in this experiment possibly due to conditions being disadvantageous for denitrification. In the field experiments two urine-N rates were examined under both summer and winter conditions at two urine-N rates. The emission factors after 71 days for NO-N in the summer were 0.15 and 0.20% of the urine-N applied for the 500 and 1000 kg N ha⁻¹ rates respectively while the respective N₂O-N fluxes were 0.14 and 0.16%. Under winter conditions the emission factors after 42 days for NO-N were <0.001% of the urine-N applied regardless of urine-N rate while the N₂O-N fluxes were 0.05 and 0.09% for the 500 and 1000 kg N ha⁻¹ urine-N rates respectively. The relationships and predictors of NO-N flux determined in the laboratory studies did not serve as strong indicators of the NO-N flux under summer conditions. Low emissions from urine-N over winter were due to the low soil temperatures and high WFPS. These studies have demonstrated that soil chemical and environmental variables influence the production of NOx and N₂O emissions from urine-N applied to soil and that seasonal effects have a significant impact on the relative amounts of NO-N and N₂O-N emitted from urine patches. Suggestions for future work are also made.

La barrière entre l'aspect bénéfique du sélénium (Se) et son aspect toxique est étroite. Afin de mieux contrôler les apports de cet élément dans l'alimentation, de nombreux travaux s'intéressent à la compréhension de son métabolisme. Cette thèse présente le développement et l'optimisation de méthodes d'analyse des espèces séléniées dissoutes et volatiles présentes dans l'urine de sujets non supplémentés. Le couplage entre la chromatographie liquide et la spectrométrie de masse atomique précédé d'un prétraitement de l'échantillon par extraction sur phase solide a permis non seulement de confirmer la présence de métabolites séléniés précédemment détectés dans l'urine de sujets supplémentés mais également, de mettre en évidence des composés inconnus. Un de ces composés a été identifié par spectrométrie de masse moléculaire. Les couplages entre la chromatographie gazeuse et les spectrométries de masse atomique et moléculaire, précédés d'une micro-extraction sur phase solide ont été utilisés pour l'analyse des formes volatiles de Se. Ces méthodes ont permis de confirmer la présence de composés séléniés et mixtes Se/S dans les urines de sujets non supplémentés et, de définir des conditions de stockage permettant de préserver la spéciation originelle dans l'échantillon.

<p>En metod för detektering och kvantifiering av <em>gamma</em>-hydroxysmörsyra (GHB) i urin med gaskromatografi (GC) är framtagen på Sahlgrenska universitetssjukhuset. Metoden är relativt unik då den inte kräver upparbetning i form av derivatisering, indunstning eller extraktion. Urinen surgörs med koncentrerad saltsyra och internstandard, <em>gamma</em>-valerolakton, tillsätts. GHB övergår då till laktonformen, <em>gamma</em>-butyrolakton (GBL). Därefter injiceras provet direkt på en GC-FID med en kapillärkolonn för glykoler och alkoholer. Detektion ner till 100 μmol/L är möjligt med en variationskoefficient mellan 6 och 12 %. Provsvar erhålls efter 6,5 minuter. Metoden är dock inte fullständig då en del frågetecken kvarstår. Bland annat bör det undersökas om andra föreningar, som kan förekomma i urin, kan eluera samtidigt som GHB. Om ja så bör vidare analyser genomföras för att separera GHB och den andra föreningen. Metoden kan däremot användas i nuläget som en screeninganalys för att snabbt få ett svar på om GHB finns närvarande eller inte. Verifiering kan sedan ske med GC-MS.</p> / <p>A method for determination and quantification of <em>gamma</em>-hydroxyburyric acid (GHB) in urine samples is developed at Sahlgrenska universitetssjukhus. No time consuming procedures as derivatization and exctration is required, which makes the method fairly unique. Hydrochloric acid and internal standard, <em>gamma</em>-valerolakton, is added to the urine sample before the sample is injected to a gas chromatograph with a flame ionization detector and a column for glycols and alcohols. The hydrochloric acid makes the GHB convert into <em>gamma</em>-butyrolactone (GBL) which is easier to separate in the gas chromatograph. Limit of detection was found to be 100 μmol/L and test result is received after 6,5 minutes. There are still some question marks around the method, for example, there is a possibility that another substance elute at the same time as GHB. More tests are required to determine whether or not it is so. For now the method can be used as a screening analysis to hastily detect GHB presence. Verification can be done with GC-MS.</p>

Neuweltkameliden, Trächtigkeitsdiagnose, Hormone, Speichel, Milch, Urin Ziel der vorliegenden Arbeit war es, die trächtigkeitsassoziierten Hormone Progesteron (P4), Pregnanediol-Glucuronid (PdG), Östronsulfat (E1S) und Relaxin (RLN) in Spei-chel, Milch und Urin von tragenden und nicht tragenden Alpakas im Vergleich zur je-weiligen Blutkonzentration zu bestimmen, um ihre Eignung zur nicht invasiven Träch-tigkeitsdiagnostik zu untersuchen. Beprobt wurden, über einen Zeitraum von zwei Jahren, 36 Alpakastuten von sechs pri-vaten Züchtern in Sachsen jeweils vor der Bedeckung und in verschiedenen Stadien der Trächtigkeit (verifiziert durch eine transabdominale Ultraschalluntersuchung). Es wurden jeweils Serum-, Plasma-, Speichel-, Urin- und Milchproben gewonnen und die Hormonkonzentrationen mittels Enzymimmunoassay (EIA) bestimmt. Weiterhin wurden einige Milchproben in einem semiquantitativen Progesteron-Schnelltest für Rinder ein-gesetzt. P4-Konzentrationen steigen signifikant von Basalwerten beim nicht tragenden Tier von 0,35 ± 0,04 ng/ml auf 2,94 ± 0,11 ng/ml Plasma (bzw. von 0,26 ± 0,03 auf 2,87 ± 0,10 ng/ml Serum) bei tragenden Tieren an. Auch in Milch und im Urin tragender Alpakas sind signifikant höhere P4-Konzentrationen messbar: Sie steigen von basal 0,83 ± 0,06 ng/ml auf 4,09 ± 0,38 ng/ml Milch bzw. von 0,29 ± 0,04 ng P4/mg Krea auf 0,60 ± 0,06 ng P4/mg Krea im Urin. Die Urin-Konzentrationen von PdG sind signifikant höher bei graviden (152,73 ± 17,37 ng PdG/mg Krea) als bei ingraviden Alpakas (26,70 ± 2,80 ng PdG/mg Krea). Im Speichel sind weder von P4 noch von PdG Konzentrationsunterschiede zwischen den beiden Gruppen nachweisbar. Der P4-Schnelltest erkannte 28 von 31 Milchproben tragender Tiere richtig als tragend, was einem Prozentsatz von 90 % entspricht. Dage-gen wurden 22 von 32 Proben nicht tragender Tiere als nicht tragend identifiziert (69 %), wobei von den falsch positiven Milchproben jedoch 70% auch mit dem labor-gebundenen EIA falsch positive Ergebnisse lieferten. Während Blutkonzentrationen von RLN signifikant nach dem zweiten Trächtigkeitsmo-nat von basal 1,65 ± 0,56 ng/ml auf 11,69 ± 2,31 ng/ml (Plasma) bzw. von 0,95 ± 0,30 ng/ml auf 16,23 ± 3,05 ng/ml (Serum) ansteigen, sind keine Unterschiede in Milch, Speichel und Urin zwischen tragenden und nicht tragenden Tieren nachweisbar. Konzentrationen von E1S steigen erst im letzten Trächtigkeitsmonat signifikant an: Blutwerte steigen von basal 0,59 ± 0,07 ng/ml auf 3,43 ±0,55 ng/ml (Plasma) bzw. 0,32 ± 0,02 ng/ml auf 2,16 ± 0,43 ng/ml (Serum) und Urinwerte von basal 6,14 ± 0,53 ng E1S/mg Krea auf 104,03 ± 24,09 ng E1S/mg Krea. Speichel und Milchkonzentrationen unterscheiden sich nicht signifikant zwischen den beiden Gruppen. Die gemessenen Konzentrationen von P4, E1S und RLN im Blut bzw. PdG und E1S im Urin stimmen mit den Ergebnissen früherer Untersuchungen überein und können somit als Trächtigkeitsmarker bestätigt werden. Dies ist die erste Arbeit, die trächtigkeitsassoziierte Hormone in Speichel und Milch von Alpakas untersucht. Während die P4 Bestimmung in Milch sowie die Bestimmung von PdG und E1S in Urin geeignete Alternativen darstellen, ist Speichel für eine Trächtig-keitsdiagnostik beim Alpaka ungeeignet. Die Nutzung von Milch und Urin zur Trächtigkeitsdiagnose stellt insofern eine Vereinfa-chung der derzeitig gängigen Methoden (u. a. Blutprogesteron) dar, als dass der Besit-zer das Probenmaterial selbst gewinnen kann und dies mit erheblich weniger Stress für die Stuten verbunden ist. Die Bestimmung von P4 in Milch und PdG in Urin stellen so-mit geeignete Alternativen zur Frühdiagnostik im ersten Trächtigkeitsmonat dar, da zu diesem Zeitpunkt eine transabdominale Ultraschalluntersuchung noch nicht aussage-kräftig ist. Die vorliegende Arbeit leistet einen Beitrag, um die noch vergleichsweise kleine vor-handene Datenbank zur Endokrinologie der Reproduktion bei NWK zu erweitern. / Aims of the present study were the measurement of pregnancy-associated hormones progesterone (P4), pregnanediol-glucuronide (PdG), relaxin (RLN) and oestrone sul-phate (E1S) in saliva, milk and urine of pregnant and non-pregnant alpacas, to compare to their respective blood concentrations and to assess their potential use for pregnancy diagnosis. Samples were obtained over a course of two years from 36 female alpacas of 6 private alpaca breeders in Saxony (Germany) before mating and at different stages throughout pregnancy (confirmed by ultrasonography). Hormone concentrations in serum, plasma, saliva, urine and milk samples were determined using enzyme immunoassays (EIA). Some milk samples were also tested using a commercial on-farm P4 kit which is de-signed for dairy cattle. Concentrations of P4 increased significantly from basal values in non-pregnant alpacas of 0.35 ± 0.04 ng/ml to 2.94 ± 0.11 ng/ml in plasma (and from 0.26 ± 0.03 to 2.87 ± 0.10 ng/ml in serum) in pregnant animals. Milk and urine concentrations of P4 were sig-nificantly higher in pregnant alpacas: Values increased from basal 0.83 ± 0.06 ng/ml to 4.09 ± 0.38 ng/ml in milk and from 0.29 ± 0.04 ng P4/mg Cr to 0.60 ± 0.06 ng P4/mg Cr in urine. While PdG concentrations in urine were significantly higher in pregnant (152.73 ± 17.37 ng PdG/mg Cr) than in non-pregnant animals (26.70 ± 2.80 ng PdG/mg Cr), there were no differences in concentrations of P4 or PdG in saliva. The on-farm milk P4 test kit showed a sensitivity of 90% for diagnosis of pregnancy and a specificity of 69% for non-pregnancy. RLN concentrations in blood increased significantly after the 2nd month from basal 1.65 ± 0.56 ng/ml to 11.69 ± 2.31 ng/ml in plasma and from 0.95 ± 0.30 ng/ml to 16.23 ± 3.05 ng/ml in serum, whereas there were no differences in milk, saliva and urine between pregnant and non-pregnant animals. Hormone concentrations of E1S increase during the last month of pregnancy: Blood concentrations rise from basal values of 0.59 ± 0.07 ng/ml to 3.43 ± 0.55 ng/ml in plasma and from 0.32 ± 0.02 ng/ml to 2.16 ± 0.43 ng/ml in serum; urine concentrations from 6.14 ± 0.53 ng E1S/mg Cr to 104.03 ± 24.09 ng E1S/mg Cr. There were no sig-nificant differences in E1S concentrations in saliva and milk between pregnant and non-pregnant alpacas. Values of P4, E1S and RLN in blood as well as PdG and E1S in urine are comparable to previous reports in alpacas and therefore can be confirmed as an indicator for preg-nancy. This is the first study to include determination of pregnancy associated hormones in saliva and milk of alpacas. However, saliva seems to be unsuitable for pregnancy di-agnosis in alpacas, whereas P4 in milk, as well as PdG and E1S in urine seem to be adequate tools. The use of milk and urine would simplify pregnancy diagnosis in alpacas since, in con-trast to the current methods (e.g. blood P4 concentration and ultrasonography), the owners themselves can take the samples. The avoidance of blood sampling results in a considerable stress reduction for the animals and therefore reduces the risk for potential loss of pregnancies. The measurements of P4 in milk and PdG in urine are useful alternatives to pregnancy diagnosis, especially during the first month of pregnancy, when transcutaneous ultrasonography is not yet reliable. This work adds information to the comparatively small database for camelid reproduc-tive endocrinology.

<p>Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type II diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not&nbsp / reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes. Twenty Wistar rats were used. The rats were initially fed a CHOW diet, which is the standard balanced diet for rats, for 4 weeks. The rats were then divided into 2 groups of 10 where 1 group was fed CHOW and another was fed a high fat (HF) diet in order to induce obesity. The two groups were fed their respective diets for 18 weeks. Rats were weighed. Rats were placed in metabolic chambers and 24 hour urine samples were collected. Ketone levels were measured by Ketostix. Urine proteins were precipitated by acetone, quantified and separated on both the 1D SDS-PAGE and the 2D SDS-PAGE. Protein expression changes between CHOW and HF fed rats were determined and identified using MALDI-TOF mass spectrometry. Protein spots intensities increased and decreased between the CHOW and HF fed rats. Transducin (beta)-like 3 was identified as the only differentially expressed protein, which might serve as a potential biomarker for prediabetes.</p>

The ability of prey to detect and respond appropriately to predator risk is important to overall prey fitness. Many aquatic organisms assess risk through the use of chemical cues that can change with predator diet. Two variable characteristics of diet are: 1. prey type and 2. prey mass. To assess the effect of these two characteristics on the assessment of risk by the mud crab Panopeus herbstii, I exposed mud crabs to the urine of the blue crab Callinectes sapidus fed one of 5 diet treatments: 10g of oyster shell free wet mass, 5g of oyster shell free wet mass, 10g crushed mud crabs, 5g crushed mud crabs, and a mix of 5g of oyster shell free wet mass and 5g crushed mud crab. Effects on P. herbstii foraging were tested in a previously developed bioassay by measuring shrimp consumption over a 4 hour period. I hypothesized that P. herbstii would have a larger magnitude response to urine from C. sapidus fed a diet of crushed mud crabs than to urine from C. sapidus fed a diet of oysters. I further hypothesized that P. herbstii would have a larger magnitude response to urine from C. sapidus fed a high mass diet relative to a lower mass diet. Contrary to expectations there was no observed effect of urine on P. herbstii foraging in any of the treatments. Results suggest that bioassay protocol may be unreliable suggesting further replication to determine the difference between this study and previous results. Future studies examining how P. herbstii varies with urine concentration will aid in understanding the ecological scale of this predator cue system. Determining the role of other potential cue sources will improve the predictive abilities of these studies.