Studies were conducted to investigate the tissue distribution of stearoyl-CoA desaturase-1 (SCD) and the regulation of SCD1 protein expression by dietary fat, insulin, polyunsaturated fatty acids (PUFA), and linoleic acid (cis-9, cis-12 18:2). The first study examined tissue distribution of SCD1 protein in Holstein calves (n=6/diet) fed one of four milk replacer diets for a nine wk period after which they were sacrificed. Milk replacer diets varied in fat content and were formulated and administered as follows: 0.4 kg/d 20% protein, 20% fat (20:20; CON), 0.97 kg/d (28:20; HPLF), 0.97 kg/d (28:28; HPHF), or 1.46 kg/d (28:28; HPHF+). Samples of subcutaneous adipose tissue (AT), perirenal AT, omental AT, duodenum, proximal jejunum, distal jejunum, ileum, and liver were collected from calves fed the HPHF+ diet to determine SCD1 tissue distribution. Tissue homogenates were prepared and used for Western blotting. Additionally, dietary effects were analyzed on tissues expressing SCD1 protein for all 24 calves. The second study investigated the regulation of SCD1 protein expression by insulin, fatty acids increasing in degree of unsaturation, and increasing concentrations of linoleic (18:2) acid. Subcutaneous AT was collected from Smith Valley Meats in Rich Creek, VA and used to prepare explants cultured in treatment media for 24 h. Treatments consisted of insulin at 0, 7, 14, and 21 nM; stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids at 100 μM; and linoleic (18:2) acid at concentrations of 0, 25, 50, 75, and 100 μM. Tissue explant homogenates were used for Western blotting to detect SCD1. In the first study, we found that SCD1 protein was detectable in subcutaneous AT, perirenal AT, and omental AT; however, it was not detectable in liver or small intestine samples. Also, the HPHF+ diet increased SCD1 protein expression in subcutaneous AT and perireanl AT. In the second study, SCD1 protein expression increased linearly with insulin concentration. There was no fatty acid treatment effect, but there was a negative linear effect with increase in degree of unsaturation. Finally, there was no effect on SCD1 protein expression with linoleic acid increasing in concentration. In conclusion, results indicate that SCD1 protein expression was detected in bovine AT depots, regulated by dietary fat, insulin, and by PUFA . / Master of Science

L'objectif de cette thèse était de faire le point sur la place de l'analyse de la moelle osseuse (MO) en tant que matrice alternative au sang en toxicologie médicolégale. Une méthode analytique a été développée et validée pour la quantification du citalopram, du diazepam et ses métabolites (nordazepam, temazepam, oxazepam) dans la MO et 10 autres matrices d'intérêt médicolégal. Cette procédure a été appliquée avec succès dans des cas réels pour l'analyse de matrices dégradées et a permis l'établissement d'une cinétique tissulaire chez l'animal au cours d'une étude pharmacocinétique. Cette cinétique animale a été intégrée dans une modélisation PBPK afin de prédire chez l'homme la distribution tissulaire du citalopram, du diazepam et son métabolite principal, le nordazepam, après administration orale thérapeutique. Ces simulations donnent des clefs intéressantes pour l'interprétation quantitative des concentrations tissulaires en toxicologie médicolégale. Une étude a été conduite pour déterminer l'influence du site de prélèvement sur la détermination des concentrations médullaires de caféine et sur la corrélation de ces concentrations avec les dosages sanguins. Elle montre que le site de prélèvement de MO est un paramètre important à prendre en considération dans l'interprétation quantitative des analyses de MO. L'ensemble de ce travail confirme l'intérêt de la MO en toxicologie médicolégale. Des études expérimentales ont permis d'approfondir les connaissances de cette matrice autour des problématiques du prélèvement, de l'analyse et de la distribution ante mortem afin de contribuer à l'interprétation qualitative et quantitative des analyses réalisées sur la MO / The aim of this work was to evaluate the interest of bone marrow (BM) analysis in forensic toxicology, as an alternative matrix to blood. An analytical method was developed and validated for the quantification of citalopram, diazepam, and its main metabolites (nordazepam, temazepam, oxazepam) in BM and 10 others matrices of forensic interest. This procedure was successfully applied to real cases for putrified sample analyses and to establish a tissue kinetic in rabbit samples for a pharmacokinetic study. These animals kinetics were implemented in PBPK modeling to predict in human tissue distribution of citalopram, diazepam, and its metabolite, nordazepam, after oral therapeutic administration. These predictions gave some clues to interpret quantitatively tissue concentrations in forensic toxicology. A study was also performed to examine whether BM sample location may influence post mortem BM quantification and correlation between BM and blood concentrations. Caffeine was used as test compound. Sample location was found to be an important parameter to consider in quantitative interpretation of BM analyses. This work confirmed the interest of BM in forensic toxicology. Experimental studies improved our knowledge on this matrix about the problematic of sample location, analytical procedure and ante mortem distribution to contribute to qualitative and quantitative interpretation of BM analyses

It has been suggested that the rising amounts of chemicals in the environment may affect host resistance and increase susceptibility to infections. Studies have also shown that infections can change the toxicity of pollutants. The aim of this thesis was to study interactions between environmental pollutant exposure in terms of polybrominated diphenyl ethers (PBDE) and a common human coxsackievirus B3 (CVB3) infection adapted to Balb/c mice. The studies focused on virus levels, cytokines, metabolising cytochrome P450 (CYP) enzymes and tissue distribution of PBDE. A novel finding was an organ-specific effect of CVB3 infection on the metabolising capacity of PBDE. The PBDE metabolising enzyme CYP2B10 was down-regulated by the CVB3 infection in the liver, up-regulated in the lungs, but not affected in the pancreas. Accordingly, CVB3 infection increased the concentration of PBDE in the livers of infected mice. However, serum levels of PBDE were not affected by the infection, indicating that serum does not reflect the actual organ exposure of PBDE in infected individuals. The change in metabolising capacity was likely mediated by infection-induced cytokines and associated effects on the nuclear factor-κB (NF-κB) pathway. PBDE drastically decreased serum levels of several cytokines and chemokines, an event that may create a slot for viruses to replicate. Accordingly, some results show that infected mice exposed to a high dose of PBDE had higher virus levels than mice exposed to a low dose. In conclusion, infected individuals showed organ-specific changes in metabolism and tissue levels of PBDE, which potentially could change the toxicity of PBDE. PBDE also seems to affect the fate of the infection. NF-κB activated pathways may mediate one possible mechanism underlying these effects. Thus, further investigations of this pathway are warranted. In addition, future studies should address how PBDE exposure affects viral replication.

The blood-brain barrier is formed by tightly joined capillary cells with transporter proteins and acts as to regulate the brain concentration of nutrients as well as many drugs. When developing central nervous system drugs it is necessary to measure the unbound drug concentration in the brain, i.e. the unbound brain exposure. This is to ensure that the drug reaches the site of action. Furthermore, when designing new drugs it is extremely valuable to be able to predict brain exposure from a tentative drug structure. Established methods to measure total drug concentrations are of limited (if any) utility since the pharmacologically active, unbound, concentration is not obtained. The aim of the conducted research was to develop an efficient methodology to measure unbound drug in the brain and to generate a dataset for developing computational prediction models describing the relationship between drug structure and unbound brain exposure. First it was demonstrated that unbound brain exposure can be efficiently assessed using a combination of total drug concentrations in the brain and separate measurements of drug binding in the brain slices. The in vitro brain slice method was refined and made high-throughput. Improvements were also made to the in vivo measurements of total concentrations by introducing an appropriate correction for drug in residual blood. Modeling of a 43-drug dataset in the rat showed that unbound brain exposure is related to the drug hydrogen bonding potential and not to lipid solubility, which contrasts the common understanding. Further, the drug concentrations in cerebrospinal fluid approximated unbound concentrations in the brain (r2=0.80) and were also correlated with corresponding measurements in humans (r2=0.56). Therefore, rat-derived prediction models can be used when designing drugs for humans. This thesis work has provided drug industry and academia with efficient tools to obtain and to use relevant estimates of drug exposure in the brain for evaluating drugs candidates.

The neosporosis is a disease of great economic importance, is distributed worldwide and is considered today one of the major causes of abortion and neonatal deaths in cattle. The pathogenesis of Neospora caninum infection is not fully understood and some issues in this regard are important in determining the most efficient measures of control and prophylaxis of a flock. The exclusive use of animals in these studies are expensive and time consuming so, some species of animals, such as, the rodents, can be used to make it possible. The gerbils (Meriones unguiculatus) mimic infection in cattle and are considered highly sensitive to replication of the protozoan. This study evaluated the responses of these animal models, at different moments of infection, especially in the amount and severity of acute and chronic/persistent infections by N. caninum. In addition, the research sheds light the tissues preferred location/replication the protozoan in these rodent at different conditions depending on disease progresses. The results are presented in two articles. In chapter one, the aim was to characterize the acute infection by the protozoan for to determine the major tissue sites of replication in experimental models of M. unguiculatus. Thus we have demonstrated that, initially the protozoan made a replication, but there is a distribution in virtually all tissues of the host. In addition, we observed that without an effective action of the immune system to combat agent, the infection progresses to death of animals with characteristic signs rapidity detected between 3 and 5 days after initiation of infection. The second chapter, sought to establish in rodents (M. unguiculatus) chronically infected, which places the N. caninum prefers to develops chronic evolutive form bradyzoites that are characteristic of a phase of persistent disease, especially in regions of the CNS. Furthermore, we note that the dose inoculated interfere significantly in the induction of acute and chronic infection and this is reflected in the amount of detection of nucleic acids in different tissues, above all in the CNS. In this context, it was observed that the emergence of clinical signs was milder without the induction of severe and lethal infections in most chronically infected animals. Thus, through this study we can conclude that highly sensitive techniques are necessary to detect nucleic acids from N. caninum, especially in the CNS tissues in animals chronically infected. / A neosporose é uma enfermidade de grande importância econômica, encontra-se distribuída em todo o mundo e pode ser considerada atualmente, umas das maiores causas de aborto e perdas neonatais em bovinos. A patogenia da infecção pelo Neospora caninum não está totalmente esclarecida e algumas questões a este respeito são importantes para determinar medidas mais eficientes de controle e profilaxia de um rebanho. A utilização exclusiva de bovinos nestes estudos é dispendiosa e demorada, com isso, algumas espécies de animais, como os roedores, podem ser utilizadas para torna-la possível. Os gerbils (Meriones unguiculatus) mimetizam a infecção em bovinos e são considerados altamente sensíveis a replicação do protozoário. Neste estudo foram avaliados as respostas destes modelos animais, em momentos de infecção diferentes, principalmente em relação a quantidade e severidade da infecção aguda e crônica/persistente pelo N. caninum. Além disso, a pesquisa esclarece os tecidos preferenciais de localização/replicação do protozoário nestes roedores em diferentes condições conforme a evolução da doença. Os resultados estão apresentados em dois artigos. No capítulo um, o objetivo foi caracterizar a infecção aguda pelo protozoário, a fim de determinar os principais sítios teciduais de replicação em modelos experimentais de M. unguiculatus. Desta forma conseguimos demonstrar que, inicialmente o protozoário fez uma replicação, mas que há uma distribuição em praticamente todos os tecidos do hospedeiro. Além disso, foi possível observar que sem uma atuação efetiva do sistema imune no combate ao agente, esta infecção evolui para a morte dos animais com sinais característicos rapidamente detectados, entre 3 a 5 dias após o inicio da infecção. Já o segundo capítulo, buscou estabelecer em roedores (M. unguiculatus) cronicamente infectados, quais os locais onde preferencialmente o N. caninum desenvolve a sua forma evolutiva crônica de bradizoitos que são características de uma etapa persistente da doença, principalmente em regiões do SNC. Além disso, observamos que a dose inoculada interfere significativamente na indução de infecção aguda e crônica e isso se reflete na quantidade de detecção de acidos nucléicos nos diferentes tecidos, sobretudo no SNC. Neste contexto, foi possível observar que o aparecimento de sinais clínicos foram mais brandos sem a indução de infecções letais e severas na maioria dos animais cronicamente infectados. Assim, através desse estudo podemos concluir que são necessárias técnicas altamente sensíveis para detectar ácidos nucléicos do N. caninum, especialmente em tecidos do SNC em animais infectados crônicamente.

Depuis quelques décennies une diminution de la qualité du sperme a été constatée dans la majorité des pays occidentaux. Si les raisons de cette diminution sont encore incomplètement comprises, des facteurs environnementaux pourraient y contribuer grandement. Parmi eux, l’exposition à certains matériaux, comme les nanomatériaux et l’aluminium, a grandement augmenté durant cette même période. Notre objectif a donc été d’évaluer l’impact de ces expositions sur la fonction de reproduction masculine. Pour ce faire, nous avons réalisé deux études de la biodistribution testiculaire des particules après injection par voie intramusculaire sur un modèle de souris, une étude clinique visant à évaluer la charge du plasma séminal en nanoparticules et à la corréler avec le nombre de spermatozoïdes dans l’éjaculat et une étude clinique visant à évaluer l’impact de la charge spermatique en aluminium sur les paramètres spermatiques. Les résultats de ces travaux couplés à l’analyse de la littérature nous rassurent sur la reprotoxicité masculine des nanoparticules mais montrent l’existence d’un impact potentiellement négatif de l’aluminium. Dans les deux cas, de nouvelles études devront poursuivre l’exploration de ces questions / In recent decades a decrease in sperm quality was observed in most Western countries. While the reasons for this decline are not yet fully understood, environmental factors could greatly contribute. Among them, exposure to materials such as nanomaterials and aluminium, has greatly increased during this period. Therefore, our goal was to assess these exposures impact on male reproductive function. To do so, we conducted two studies to determine testis biodistribution of particles after intramuscular injection in a mouse model. We also conducted two clinical studies, first one to assess the burden of nanoparticles in seminal plasma and to correlate it with sperm count and the second one to evaluate the impact of the aluminum load in semen on sperm parameters. The results of these works and the analysis of literature reassure us about male reproductive toxicity of nanoparticles but revealed a potentially negative impact of aluminum. In both cases, new studies will be needed to further explore these issues

The human metabolic syndrome is characterized by a heterogenic complex of symptoms, including central obesity. Obesity itself is linked to major features of the metabolic syndrome such as insulin resistance, dyslipidemia or type 2 diabetes mellitus. It has been shown that obesity risk and resulting metabolic alterations are associated with adipose tissue distribution, adipocyte size and secretion of adipocytokines, which are in turn influenced by environmental factors and genetic susceptibility. It might be assumed that currently known genetic variants associated with obesity and/or BMI (body mass index) as well as fat distribution explain up to 20 % of the variability in BMI and so, studies employing novel strategies are inevitable. In addition to the role of genetic variation, mRNA levels of several genes have been shown to be differentially expressed in subcutaneous (SC) and visceral (Vis) adipose tissue and to be correlated with obesity-related traits. It is scarcely investigated whether the obesity risk variants also might account for the variability in mRNA expression. The present thesis deals with novel obesity candidate genes, characterized by a differential mRNA expression in various fat depots. The association of genetic variants in these genes with obesity as part of the metabolic syndrome, and related traits was investigated in well characterized German cohorts. The main method used for genotyping was described in detail in a comprehensive review providing explicit troubleshooting and description of modified protocols for specific experimental needs. Further, the influence of genotypes on the gene expression levels was examined. While the differential expression for FTO could be described for the first time, the variant rs8050136 was shown to be significantly associated with obesity but not with the expression. Genetic variants in FASN were shown to be significantly associated with obesity and related traits in a cohort of European ancestry for the very first time. Moreover, one polymorphism showed effects on the ratio of Vis/SC FASN mRNA expression. While CNR1 is controversially discussed in the literature, the present work showed rather moderate effects of genetic variants on obesity. BMPR2 could be described as a novel obesity candidate gene. Amongst others, one variant was associated with obesity in a case-control design and with BMPR2 mRNA expression in Vis adipose tissue. In conclusion, the present study revealed novel genetic variants promoting obesity, and therefore a metabolic risk, which might be partly explicable through an influence of these variants on the mRNA expression levels of the genes in the adipose tissue depots. These findings contribute to better understanding of the genetic background of obesity which is essential in order to translate experimental data into diagnostic, preventive and treatment strategies.

Mitochondrial uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carriers. The longest known is UCP1, predominantly expressed in brown adipose tissue, where it takes part in nonshivering thermogenesis. In the late 1990s were discovered other sequence homologs of UCP1 with tissue specific distribution. The Function of these "new" uncoupling proteins is still uncertain. It is assumed that each of the isoforms has a specific function depending on the type of tissue. This thesis showed differences in tissue transcription pattern between rat and mice using RT-PCR absolute quantification. Significant differences in pattern were found in lungs, brain and muscle. In each case UCP expression was higher in mice tissues. Mice lungs express mainly UCP2. The difference in mice brain is caused by ucp4 and ucp5 genes transcription and finally in muscle is highest content of UCP3 mRNA. We investigated whether any of ucp transcript can complement ucp2 transcripton in spleen or lungs of ucp2 -/- mice. We did not find any difference which can explain, that in isolated lung mitochondria of fasted ucp2-/- mice were uncoupled in state 4. In the last project, we found relationship between ucp2 transcription in insulinoma INS-1E cells and oxygen levels of the cultivation atmosphere.

No description available.

BACKGROUND AND PURPOSE: Pharmacokinetic assessment of drug tissue permeation following iontophoresis is limited. The depth of ketoprofen tissue permeation following cathodic iontophoresis (4 mA, 40 minutes) and the stereoselectivity of drug delivery were examined in this study. SUBJECTS: Ketoprofen (750 mg) was iontophoresed onto one porcine medial thigh, with passive drug permeation conducted on the other thigh. METHODS: Skin, subcutaneous fascia, and muscle biopsies from the drug delivery sites were harvested and stored separately, and the "R" and "S" ketoprofen enantiomers were determined. Results. Iontophoretic and passive applications yielded equivalent total ketoprofen concentrations in the skin and fascia. In contrast, multivariate analysis demonstrated that the ketoprofen concentration in the first centimeter of muscle following iontophoresis was greater than the drug concentration in the deeper underlying muscle layers and greater than that delivered to any muscle layer following passive delivery. No transcutaneous stereoselective delivery) of ketoprofen was detected. CONCLUSION AND DISCUSSION: Compared with passive delivery, iontophoresis enhances nonstereoselective ketoprofen permeation into the fascia-muscle interface. With delivery to deeper tissue sites, however, there is no apparent enhancement over passive application.