Although prescribed burning has been used frequently for manipulating habitat, it has been rarely used in grassland areas with the intent to improve habitat quality for wild turkeys (Meleagris gallapavo). I studied breeding season and nest location habitat use of wild turkey hens in response to grassland fire management, and quantified its impact on foraging habitat and insect prey for turkey poults, in a diverse agricultural landscape in southern Illinois. Fire management was conducted on 28 ha of grassland in March and April 2012. Radiolocations from 64 radiomarked hens from 2008-2010 and 44 radiomarked hens from 2012-2013 were used to create turkey habitat and nest location models for both pre-fire and post-fire time periods. Areas used by turkeys throughout all models and time periods were characterized by high proportions of forested habitat. Nest locations during both time periods also included areas with higher percentages of shrubland and edge habitat. Turkey use areas during the post-fire time period were found to be further from burned areas, suggesting that fires on short rotations (1-2 years) were slightly detrimental to habitat needed during the breeding season and for nest locations. Field trials using human-imprinted wild (n=54) and commercial (n=64) strain turkey poults were conducted to assess the foraging efficiency and mobility of poult type within burned and unburned grassland sections. Foraging efficiency of turkey poults did not differ between burned or unburned fields, or between wild and commercial strain poults. All poults showed selection of invertebrate categories Coleoptera, Hymenoptera, and Isopoda throughout all grassland field trials, whereas avoidance of Araneae, Diptera, Entognatha, Hemiptera, Lepidoptera, and Orthoptera was exhibited by all poults in all trials. There was evidence to believe that burned grasslands increased the travel efficiency of poults compared to unburned grasslands. Wild poults were able to travel further distances and along less sinuous paths than commercial poults.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O estado nutricional perinatal tem influência persistente sobre o desenvolvimento neural e a função cognitiva. Em humanos e outros animais, a malnutrição protéica durante o período perinatal acarreta alterações permanentes, incluindo a síndrome metabólica na idade adulta. A alimentação é modulada principalmente por fatores neuronais e hormonais que chegam ao hipotálamo. As reservas de glicogênio hipotalâmicos são uma fonte de glicose em altas demandas energéticas, como durante o desenvolvimento dos circuitos neurais. Como alguns circuitos hipotalâmicos estão sendo formados durante o período de lactação, focamos o estudo nos efeitos da desnutrição protéica, durante os primeiros 10 dias de lactação, sobre as reservas de glicogênio em núcleos hipotalâmicos envolvidos no controle do metabolismo energético. Ratas grávidas foram aleatoriamente separadas individualmente em gaiolas e alimentadas ad libitum com uma dieta normoproteíca (22% proteína). Após o parto, cada mãe ficava com 6 filhotes machos. Durante os 10 primeiros o grupo experimental recebia uma dieta isenta de proteína (D) e o grupo controle uma dieta normoproteíca (C). Em P10 a marcação para as reservas de glicogênio era muito intensa nos animais C no núcleo arqueado (ARC) e eminência média (EM). Em P20 no animais C, as reservas eram menores em comparação com P10. Os animais D apresentaram uma marcação menor do que os controles. Após P45 foi difícil determinar diferença entre os grupos porque o as reservas estavam diminuídas. Nós também mostramos que os tanicitos eram as células que apresentavam reservas de glicogênio. Nossos dados reforçam que o estado nutricional materno durante a lactação é crítico para a maturação cerebral, pois a malnutrição materna resulta em menor marcação nas reservas de glicogênio no hipotálamo, o que pode ser crítico para o estabelecimento da circuitaria neural. / Perinatal nutrition has persistent influences on neural development and cognition. In humans and other animals protein malnutrition during the perinatal period causes permanent changes, inducing to adulthood metabolic syndrome. Feeding is mainly modulated by neural and hormonal inputs to the hypothalamus. Hypothalamic glycogen stores are a source of glucose in high energetic demands, as during development of neural circuits. As some hypothalamic circuits are formed during lactation, we attempt to study the effects of malnutrition, during the first 10 days of lactation, on glycogen stores in hypothalamic nuclei involved in the control of energy metabolism. Female pregnant rats were fed ad libitum with a normorprotein diet (22% protein). After delivery each dam was kept with 6 male pups. During the first 10 days of lactation dams from experimental group received a protein free diet and the control group a normoprotein diet. By P10 glycogen stores were very high in the arcuate nucleus and median eminence of control group. Glycogen stores decreased during development. In P20 control animals, glycogen stores were lower when compared to P10 control animals. Animals submitted to malnutrition presented a staining even lower than control ones. After P45 it was difficult to determine differences between control and diet groups because glycogen stores were reduced. We also showed that tanycytes were the cells presenting glycogen stores. Our data reinforce that maternal nutritional state during lactation may be critical for neurodevelopment since it resulted in a low hypothalamic glycogen store, which may be critical for establishment of neuronal circuitry.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O estado nutricional perinatal tem influência persistente sobre o desenvolvimento neural e a função cognitiva. Em humanos e outros animais, a malnutrição protéica durante o período perinatal acarreta alterações permanentes, incluindo a síndrome metabólica na idade adulta. A alimentação é modulada principalmente por fatores neuronais e hormonais que chegam ao hipotálamo. As reservas de glicogênio hipotalâmicos são uma fonte de glicose em altas demandas energéticas, como durante o desenvolvimento dos circuitos neurais. Como alguns circuitos hipotalâmicos estão sendo formados durante o período de lactação, focamos o estudo nos efeitos da desnutrição protéica, durante os primeiros 10 dias de lactação, sobre as reservas de glicogênio em núcleos hipotalâmicos envolvidos no controle do metabolismo energético. Ratas grávidas foram aleatoriamente separadas individualmente em gaiolas e alimentadas ad libitum com uma dieta normoproteíca (22% proteína). Após o parto, cada mãe ficava com 6 filhotes machos. Durante os 10 primeiros o grupo experimental recebia uma dieta isenta de proteína (D) e o grupo controle uma dieta normoproteíca (C). Em P10 a marcação para as reservas de glicogênio era muito intensa nos animais C no núcleo arqueado (ARC) e eminência média (EM). Em P20 no animais C, as reservas eram menores em comparação com P10. Os animais D apresentaram uma marcação menor do que os controles. Após P45 foi difícil determinar diferença entre os grupos porque o as reservas estavam diminuídas. Nós também mostramos que os tanicitos eram as células que apresentavam reservas de glicogênio. Nossos dados reforçam que o estado nutricional materno durante a lactação é crítico para a maturação cerebral, pois a malnutrição materna resulta em menor marcação nas reservas de glicogênio no hipotálamo, o que pode ser crítico para o estabelecimento da circuitaria neural. / Perinatal nutrition has persistent influences on neural development and cognition. In humans and other animals protein malnutrition during the perinatal period causes permanent changes, inducing to adulthood metabolic syndrome. Feeding is mainly modulated by neural and hormonal inputs to the hypothalamus. Hypothalamic glycogen stores are a source of glucose in high energetic demands, as during development of neural circuits. As some hypothalamic circuits are formed during lactation, we attempt to study the effects of malnutrition, during the first 10 days of lactation, on glycogen stores in hypothalamic nuclei involved in the control of energy metabolism. Female pregnant rats were fed ad libitum with a normorprotein diet (22% protein). After delivery each dam was kept with 6 male pups. During the first 10 days of lactation dams from experimental group received a protein free diet and the control group a normoprotein diet. By P10 glycogen stores were very high in the arcuate nucleus and median eminence of control group. Glycogen stores decreased during development. In P20 control animals, glycogen stores were lower when compared to P10 control animals. Animals submitted to malnutrition presented a staining even lower than control ones. After P45 it was difficult to determine differences between control and diet groups because glycogen stores were reduced. We also showed that tanycytes were the cells presenting glycogen stores. Our data reinforce that maternal nutritional state during lactation may be critical for neurodevelopment since it resulted in a low hypothalamic glycogen store, which may be critical for establishment of neuronal circuitry.

O processamento de peles para a fabricação de couro utiliza uma mistura complexa de substâncias - dentre elas, os taninos - que torna o tratamento de águas residuais difícil e oneroso. Vários estudos estão sendo conduzidos na tentativa de melhorar o tratamento e até mesmo reutilizar o efluente tratado. Neste trabalho foi avaliado o desenvolvimento de adsorventes funcionais sintetizados pelo método solgel e dotados de impressão molecular, para adsorção de compostos de taninos. Por serem de fácil preparação, os materiais híbridos obtidos viabilizam a sua produção para o uso como sólidos adsorventes. A caracterização dos materiais híbridos orgânico-inorgânico obtidos foi realizada por análise de Porosimetria de nitrogênio (BET), Espalhamento de raios-X a baixo ângulo (SAXS), Espectroscopia de refletância difusa no UV-vis (DRS), Espectroscopia de infravermelho com transformada de Fourier (FT-IR) e Potencial Zeta (PZ). As características estruturais e texturais dos materiais híbridos gerados variaram para cada rota sol-gel e processo de extração de template empregado, resultando em materiais com diferentes capacidades de adsorção. As sílicas sem funcionalização não foram capazes de adsorver os compostos de tanino em solução aquosa, enquanto que as sílicas funcionalizadas com APTES demonstraram sua potencialidade como adsorvente para os compostos testados, atingindo remoções superiores a 80%. Diferenças nas capacidades de adsorção entre as sílicas funcionalizadas com e sem impressão molecular também foram observadas, onde a sílica sem impressão apresentou uma adsorção superior de taninos, indicando que mais estudos são necessários para encontrar um processo de extração eficiente do template. É importante salientar que as sílicas que obtiveram a maior capacidade de adsorção, puderam adsorver os compostos de taninos por vários ciclos consecutivos. / The processing of hides for leather manufacturing uses a complex mix of chemicals that makes the wastewater treatment difficult and costly. Several studies are being conducted in attempt to improve the treatment and even reuse the treated effluent. This study evaluated the development of functional adsorbent synthesized by the molecular imprinting method in a sol-gel matrix, for adsorbing tannin compounds. Easy to prepare, hybrid materials obtained enable their production for use as solid adsorbents. The characterization of organic-inorganic hybrid materials obtained was performed by nitrogen porosimetry analysis (BET), small-angle X-ray scattering (SAXS), diffuse reflectance spectroscopy (DRS), Fourier-Transform Infrared Spectroscopy (FT-IR) and Zeta Potential (PZ). The structural and textural characteristics of hybrid materials ranged for each sol-gel route and template employed extraction process resulting in materials with different adsorption capacities. The silicas without functionalization were unable to adsorb the tannin compounds in aqueous solution, whereas the silica functionalized with APTES demonstrated the potential of the adsorbent for the tested compounds, with removals above of 80%. Differences in adsorption capacities between functionalized silica with and without molecular imprinting were observed where the unprinted silica had a higher adsorption of tannins, indicating that more studies are needed to find a process of efficient template extraction to improve adsorption capacity to the imprinted materials. Further, the silicas with improved adsorption capacity could adsorb the tannin compounds for several consecutive cycles of adsorption.

Le domaine Dlk1-Dio3 est l’un des rares domaines imprimés contrôlés par une région de contrôle d'impression méthylée sur le chromosome paternel, nommée IG-DMR. Dans l’embryon, au niveau du domaine Dlk1, Rtl1 et Dio3 les gènes codant pour des protéines sont exprimés à partir du chromosome paternel, tandis que les ARNs non-codants dont Meg3, les snoRNAs à boite C/D et les micro-ARNs sont exprimés à partir du chromosome maternel.Il a été montré que la copie maternelle de l'IG-DMR est nécessaire pour l'expression des gènes imprimés de ce domaine et que les ARNs de types enhancer (de la même région) activent la transcription des ARNs non-codants. Cependant, les mécanismes qui régulent l'expression imprimée de gènes codant pour des protéines restent indéterminés. Dans ce projet, nous avons cherché à élucider les mécanismes qui contrôlent l'expression spécifiquement paternelle des gènes codant pour des protéines ainsi que le rôle possible des ARNs non-codants dans ce processus.Pour nos études alléliques, nous avons utilisé des cellules ES hybrides qui ont été obtenues en croisant des lignées de M. musculus domesticus et M. musculus molossinus. Ces cellules ont été différenciées in vitro dans des lignées neurales. Dans les cellules ES, l'expression Dlk1 est détectée à partir des deux chromosomes parentaux à des niveaux très bas. Lors de la différenciation, l'allèle paternel de Dlk1 devient actif tandis que le niveau d'expression de l'allèle maternel reste faible. Nos études de la chromatine ont montré que cette surexpression est due à l’activation de la chromatine sur l'allèle paternel de Dlk1.L'un de nos objectifs était d'explorer le rôle de Meg3 (un long ARN non-codant) dans la régulation de l’empreinte de Dlk1. A cet effet, nous avons généré des cellules souches embryonnaires déficientes en Meg3. Dans toutes les lignées déficientes, de suppressions maternelles ou bi-alléliques, nous avons constaté une perte d’expression de tous les ANRs non-codants. De plus, l’expression de Dlk1 devient bi-allélique dans ces cellules. Pour élucider le mécanisme de l'empreinte de ce gène, nous avons décidé d'étudier les caractéristiques de la chromatine au niveau du promoteur Dlk1 dans les cellules déficientes en Meg3. Nous avons examiné les modifications activatrices et répressives des histones ainsi que l'occupation de l'ARN Pol II. Nous avons observé l'acquisition des marques d’une chromatine active sur les deux chromosomes ainsi que le recrutement bi-allélique de l'ARN Pol II.Bien que nous n’ayons pas pu détecter une perte de la marque répressive H3K27me3 suite à la surexpression de Dlk1, nous avons observé un gain d'acétylation sur ce résidu lysine. Afin de comprendre davantage le rôle de la marque H3K27me3 sur l’empreinte de Dlk1, nous avons généré des cellules ES dépourvues de EZH2, la méthyltransférase de H3K27. L’expression de Dlk1 dans les cellules différenciées dépourvues de H3K27me3 est bi-allélique.Enfin, ces données suggèrent que l'expression des ARNs non-codant empêche l'activation de Dlk1 sur le chromosome maternel via l’activité de EZH2 au cours du développement. / The Dlk1-Dio3 imprinted domain is one of the few imprinted domains that are controlled by a paternally methylated imprinting control region, IG-DMR. Protein-coding genes of the domain, Dlk1, Rtl1 and Dio3 are expressed from the paternal chromosome, and non-coding RNAs (ncRNAs) including Meg3, C/D box snoRNAs and microRNAs are expressed from the maternal chromosome exclusively in the embryo. Maternal copy of the IG-DMR is required for the imprinted gene expression at this domain. Enhancer RNAs transcribed from this region are involved in activation of ncRNA expression on the maternal chromosome. However, the regulation of imprinted expression of protein-coding genes remains unknown. In this project, we aimed to elucidate the mechanisms controlling the paternal specific expression of protein-coding genes and a possible role of ncRNAs in this process.For our allelic studies, we made use of hybrid ES cells that were obtained by crossing M. musculus domesticus and M. musculus molossinus strains. These cells were differentiated in vitro into neural lineages. In ES cells, Dlk1 expression is detected from both parental chromosomes at very low levels. Upon differentiation, paternal allele of Dlk1 gets activated while low level of expression is detected from maternal allele. Our chromatin studies showed that this upregulation is through the acquisition of active chromatin on the paternal allele of Dlk1.One of our aims was to explore the role of Meg3 long non-coding RNA (lncRNA) in the regulation of Dlk1 imprinting. For this purpose, we generated ES cells deficient in Meg3. In all maternal or biallelic deletion lines, we observed complete loss of all ncRNA expression. Interestingly, in these cells Dlk1 expression becomes biallelic. To elucidate the mechanism of imprinting of this gene, we set out to study the chromatin features at the Dlk1 promoter in Meg3 deficient cells. We looked into active and repressive histone modifications and RNA Pol II occupancy. We observed acquisition of active chromatin marks on both chromosomes as well as biallelic recruitment of RNA Pol II.Although we could not detect a loss of repressive mark H3K27me3 upon Dlk1 upregulation on the paternal allele, we observed gain of acetylation on this lysine residue. To further investigate the role of H3K27me3 mark on Dlk1 imprinting, we generated ES cells that lack functional EZH2, the H3K27 methyltransferase. Dlk1 is biallelically expressed in the differentiated cells that are devoid of H3K27me3.Combined, these data suggest a model in which non-coding RNA expression prevents the developmental activation of Dlk1 on the maternal chromosome by a process that also requires the activity of EZH2.

Chez les mammifères, l'empreinte génomique parentale est un mécanisme épigénétique restreignant l'expression d'une centaine de gènes à un seul allèle, déterminé selon son origine parentale. Les gènes affectés et les mécanismes sous-jacents à leur expression mono-allélique sont essentiellement déterminés par une marque épigénétique différentielle portés par les allèles maternel et paternel. D'un point de vue fonctionnel et au niveau physiologique, l'empreinte est actuellement comprise comme un mécanisme contrôlant la quantité de ressources attribuées par la mère à sa progéniture. Les gènes soumis à empreinte s'inscrivent dans un même réseau transcriptionnel (IGN), et plusieurs études indiquent qu'ils contrôleraient l'équilibre entre prolifération et quiescence de cellules souches adultes. A travers cette étude, nous montrons une induction coordonnée de la plupart des gènes soumis à empreinte lors de la sortie du cycle cellulaire, que celle-ci soit réversible (quiescence) ou non (différenciation). De plus, dans un modèle de pré-adipocytes 3T3-L1, la perturbation de la dynamique d'expression de plusieurs de ces gènes semble conforter l'hypothèse d'un contrôle des transitions entre différents états cellulaires (prolifération, quiescence et différenciation) par l'IGN. Outre l'identification d'une fonction cellulaire commune aux gènes soumis à empreinte, nos résultats ouvrent la voie d'une meilleure compréhension des mécanismes de régulation de la quiescence. De plus, nos conclusions permettent de suggérer un nouveau scénario pour la sélection de l'empreinte parentale au cours de l'évolution des mammifères. / Mammalian genomic imprinting is an epigenetic mechanism that restrains the expression of about a hundred genes to a single allele, in a parent-of-origin specific manner. The identity of imprinted genes and the molecular basis of their monoallelic expression mostly rely on a differential epigenetic marking of the parental alleles. Presently, imprinting is understood as a mechanism aimed at controlling the amount of maternal resources allocated to the offspring. Imprinted genes belong to the same transcriptional network (IGN) and, according to different reports, they seem to control the balance between proliferation and quiescence of adult stem cells. In this study, we show that most imprinted genes are induced upon cell cycle exit, whether reversible (quiescence) or not (differentiation). In addition, within the 3T3-L1 preadipocytes cell line, impairing the dynamics of expression of several imprinted genes impairs the transitions between different cellular states, namely proliferation, quiescence and differentiation. Our results highlight the existence of a common cellular function of imprinted genes, and provide a new frame to understand cellular quiescence, at a molecular level. Furthermore, they suggest a new plausible scenario for the implementation of genomic imprinting during mammalian evolution.

Un polymère à empreintes moléculaires (MIP) est un récepteur synthétique supramoléculaire, un matériau possédant des cavités pouvant reconnaître spécifiquement une molécule cible. Il est synthétisé en mettant en contact la molécule cible, avec un mélange de monomères fonctionnels et réticulants qui permettent d'obtenir un réseau polymérique tridimensionnel rigide. L'élimination de la molécule empreinte laissera des sites vides complémentaires de cette dernière. Ces cavités sont maintenant capables de la recapturer spécifiquement. Ces polymères sont utilisés dans les domaines tels que l’extraction en phase solide, la chromatographie d’affinité, la catalyse enzymatique, les biocapteurs et la vectorisation des médicaments. Bien que le concept des MIPs a pour origine les travaux réalisés sur des matériaux sol-gel imprimés dans les années 1930, ces derniers sont restés dans l’ombre jusqu’à l'introduction de polymères organiques imprimés plus versatiles. Par rapport aux MIPs organiques, les MIPs sol-gel présentent quelques avantages comme une plus grande stabilité thermique, une meilleure compatibilité avec l'eau et une plus grande porosité. Dans cette thèse, nous avons développé des MIPs organiques et des MIPs sol-gel pour leur application en cosmétologie et pour la vectorisation de médicaments. Dans la première partie, nous présentons des MIPs pouvant adsorber d’une façon spécifique l’acide oléique (OA), un biomarqueur de l’état pelliculaire sur le cuir chevelu. Pour la préparation des MIPs organiques, nous avons employé plusieurs monomères basiques dont l’acryloylaminobenzamidine (AB), que nous avons tout spécialement synthétisé. Tous les MIPs pouvaient lier l’OA mais beaucoup d’interaction non-spécifique était observé. D’autre part, les MIPs sol-gel présentaient une bonne reconnaissance spécifique et une capacité élevée pour OA; par exemple, un MIP de composition OA:APTES:TEOS = 1:1.6:1.7 pouvait adsorber 625 μmol.g-1 de OA dans le sébum artificiel. Des tests pour capturer l’OA sur le stratum corneum et la peau reconstruite (Episkin) ont également été effectués. La pénétration de l’OA sur les deux types de peau était plus faible en présence de MIP que de NIP. Les MIPs comme matériaux désodorisants font l’objet de la deuxième partie de cette thèse. Des MIPs pouvant adsorber les précurseurs de molécules malodorantes comme les conjugués glutamine des acides (E)-3-méthyl-2-hexénoïque (3M2H) et 3-hydroxy-3-méthyl-hexanoïque (3H3MH) ont été préparés. Le N-hexanoyl glutamine et le N-hexanoyl glutamate ont été utilisés comme template. Nous observons que le MIP synthétisé avec AB comme monomère fonctionnel possède la plus grande capacité d'adsorption pour le N-hexanoyl glutamine, ainsi que pour les précurseurs glutamines des molécules malodorantes. Des résultats préliminaires et très prometteurs ont également été obtenus dans la sueur. La dernière partie de cette thèse concerne des MIPs pour la vectorisation de médicaments. L'acide salicylique (SA) est un médicament efficace utilisé dans le traitement de l’acné. Des MIPs organiques et sol-gel contre SA ont été synthétisés. Les MIPs sol-gel ont une plus grande capacité d’adsorption, 180 μmol.g-1, que les MIPs organiques et ils lient le SA sept fois plus que le NIP. Les tests de relargage du SA ont été effectués dans plusieurs milieux, avec la plus grande efficacité dans l’eau pure. En conclusion, les applications de MIPs en cosmétologie et en vectorisation de médicaments ont étés étudiés. Nos résultats montrent que les MIPs sol-gel sont les plus appropriés pour ce type de travail. / Molecularly imprinted polymers (MIPs) are tailor-made synthetic receptors possessing specific cavities for a given target molecule. They are produced by introducing, into the polymer precursors, guest molecules that act as templates at the molecular level. Interacting and cross-linking monomers are then copolymerized to form a cast-like shell. After removal of the template, cavities complementary to the template in size, shape and position of functional groups are revealed in the polymer, which can now specifically bind the template. Thanks to these specific molecular recognition properties, MIPs have found applications in areas like bio sensors, solid phase extraction, affinity chromatography, catalysis, and drug delivery. Although the MIP concept originated from imprinted silica in the 1930s, imprinted sol-gel materials received little attention afterwards due to the introduction of the more versatile organic polymers as imprinting matrix. However, compared to organic polymers, sol-gels possess higher thermal stability, better water compatibility and larger inner surface area. There have been many applications to biomolecules in aqueous conditions with sol-gel imprinting materials. In this thesis, we have developed organic and silica sol-gel MIPs for applications in cosmetics and drug delivery. MIPs able to adsorb the dandruff-inducing molecule oleic acid (OA) were produced via both the organic and inorganic routes. In the organic MIPs synthesis, different positively charged monomers were used, one of which, acryloyl aminobenzamidine, was specifically synthesized. Although some binding of oleic acid was obtained, specificity and capacity of these polymers were not satisfying. Sol-gel MIPs, on the other hand, exhibited good specific recognition and high binding capacity for OA. A MIP of the composition OA:APTES:TEOS= 1:1.6:1.7 yielded a capacity of 625 μmol.g-1 in artificial sebum. Furthermore, tests were carried out to capture OA on stratum corneum and reconstructed skin (Episkin). Less penetration of OA was observed in the presence of a MIP than with a non-imprinted control polymer. Deodorant materials are another topic of this thesis. MIPs that are able to adsorb certain precursors of odorant molecules, the glutamine conjugates of (E)-3-methyl-2-hexenoic acid (3M2H) and 3-hydroxy-3-methyl-hexanoic acid (3H3MH) were prepared. N-hexanoyl glutamine and N-hexanoyl glutamate were used as templates. After optimization of the MIP composition, we found that MIPs synthesized with acryloyl aminobenzamidine as functional monomer had the highest adsorption capacity for N-hexanoyl glutamine, and also recognised the glutamine targets of 3M2H and 3H3MH. Some preliminary promising binding results were obtained in artificial sweat. The third part of this work concerns a drug delivery MIP. Salicylic acid (SA) is a drug used to treat acne. SA-imprinted polymers were prepared via both organic imprinting and the sol-gel process.Compared to organic MIPs, sol-gel MIPs have a higher capacity, 180 μmol.g-1, and 7 times higher binding than to a non-imprinted control polymer was observed. Release tests were carried out in different aqueous media, the most efficient drug release was observed in pure water. In conclusion, applications of molecularly imprinted polymers for cosmetics and drug delivery have been investigated. Our results demonstrate the great potential of in particular sol-gel MIPs for these purposes.

Epigenetic differences between maternally inherited and paternally inherited chromosomes, such as CpG methylation, render the maternal and paternal genome functionally inequivalent, a phenomenon called genomic imprinting. This functional inequivalence is exemplified with imprinted genes, whose expression is parent-of-origin specific. The dosage of imprinted gene expression is disrupted in cells with uniparental disomy (UPD), which is an unequal parental contribution to the genome. I have derived mouse embryonic stem (ES) cell sub-lines with maternal UPD (mUPD) for mouse chromosome 6 (MMU6) to characterize regulation and maintenance of imprinted gene expression. The main finding from this study is that maintenance of imprinting in mitotic UPD is extremely variable. Imprint maintenance was shown to vary from gene to gene, and to vary between ES cell lines depending on the mechanism of loss of heterozygosity (LOH) in that cell line. Certain genes analyzed, such as Peg10, Sgce, Peg1, and Mit1 showed abnormal expression in ES cell lines for which they were mUPD. These abnormal expression levels are similar to that observed in ES cells with meiotically-derived full genome mUPD (parthenogenetic ES cells). Imprinted CpG methylation at the Peg1 promoter was found to be abnormal in all sub-lines with mUPD for Peg1. Two cell sub-lines which incurred LOH through mitotic recombination showed hypermethylation of Peg1, consistent with the presence of two maternal alleles. Surprisingly, a cell sub-line which incurred LOH through full chromosome duplication/loss showed hypomethylation of Peg1. The levels of methylation observed in these sub-lines correlates with expression, as the first two sub-lines showed a near-consistent reduction of Peg1, while the latter showed Peg1 levels close to wild-type. Altogether these results suggest that certain imprinted genes, like Peg1 and Peg10, have stricter imprinting maintenance, and as a result show abnormal expression in UPD. This strict imprint maintenance is disrupted, however, in UPD incurred through full chromosome duplication/loss, possibly because of the trisomic intermediate stage which occurs in this mechanism. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Orientador: Fabio Augusto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T13:57:50Z (GMT). No. of bitstreams: 1 Silva_RaquelGomesdaCosta_D.pdf: 2096184 bytes, checksum: a4a32e68eebe713c6f80bc5415484d51 (MD5) Previous issue date: 2009 / Resumo: Esse trabalho apresenta o desenvolvimento de materiais sorventes com impressão molecular, para utilização na Extração em Fase Sólida (SPE) a partir da tecnologia sol-gel. No capítulo 1 foi preparado um material com impressão molecular seletivo para metilxantinas, utilizando-se o processo sol-gel. Posteriormente, esse material foi aplicado na extração de cafeína em águas e urina humana, seguido de análise cromatográfica. No capítulo 2, foi sintetizado um ormosil (sílica organicamente modificada) seletivo para fenobarbital. Nessa etapa, como molécula molde foi utilizado um análogo estrutural (ácido barbitúrico) ao analito alvo. O material foi aplicado na determinação de fenobarbital em plasma humano, demonstrando ser seletivo para esse composto. No capítulo 3 foi relatado o preparo de um ormosil impresso molecularmente seletivo para compostos triazínicos. Esse material foi aplicado com sucesso na determinação de simazina, propazina e atrazina em amostras de caldo de cana. Paralelamente, o material preparado em laboratório foi comparado ao material comercial seletivo para triazinas. Ambos materiais demonstraram ser seletivo para esses compostos. Os materiais preparados a partir do processo sol-gel apresentaram potencialidade na determinação de diferentes analitos alvo. / Abstract: This work presents the development of sorbent materials with molecular imprinting for use in Solid Phase Extraction (SPE) through sol-gel technology. In chapter 1, a molecularly imprinted material selective for methylxanthines was prepared, using the sol-gel process. Subsequently, this material was used in the extraction of caffeine from water and human urine, followed by chromatographic analysis. In Chapter 2, an ormosil (organically modified silica) selective for phenobarbital was synthesized. For this, a analogue structural was used as template (barbituric acid) for the target analyte. The material was applied to the determination of phenobarbital in human plasma, proving to be selective for this compound. In Chapter 3 the preparation of a imprinted ormosil selective for triazine is reported. This material has been successfully applied for the determination of simazine, atrazine and propazine in samples of sugar cane juice. In addition, the material prepared in the laboratory was compared to commercial material selective for triazines. Both materials were shown to be selective for these compounds. The materials prepared by the sol-gel process showed potential for the determination of different target analytes. / Doutorado / Quimica Analitica / Doutor em Ciências

GNAS est un locus soumis à empreinte parentale. Ce locus produit différents transcrits et protéines en utilisant des promoteurs distincts. Le transcrit bialléique Gsα, les transcrits soumis à empreinte maternelle GNAS-AS1, XLαs et A/B et un transcrit soumis à empreinte paternelle NESP55. L’expression de ces transcrits est contrôlée par des régions différentiellement méthylées (DMRs). Le locus GNAS est impliqué dans plusieurs maladies. La PseudoHypoParathyroïdie (PHP) également appelée inactivating PTH/PTHrP Signaling Disorder (iPPSD, pour la description de la nouvelle classification) englobe un groupe de maladies rares, très hétérogène caractérisé par la résistance des organes à l'action de la PTH. Les iPPSD2 sont dues à des mutations perte de fonctions de Gsα, les iPPSD3 à des anomalies de méthylation d’une ou plusieurs DMRs. Les tumeurs intracanalaires papillaires et mucineuses du pancréas (TIPMPs) sont des tumeurs kystiques caractérisées par une prolifération papillaire intracanalaire de l’épithélium des canaux pancréatiques. Les TIPMPs sont des précurseurs de l’adénocarcinome pancréatique comprenant trois degrés de dysplasie selon le phénotype histologique, légère, moyenne et haut grade.L’objectif de mon travail était d’étudier l’implication des transcrits non codants et des GNAS-miRNAs à l’aide de deux modèles pathologiques les iPPSD2 et 3 et les TIPMPs.Projet TIPMP :Nous avons identifié que la perte d’empreinte du locus GNAS est un événement fréquent dans les TIPMPs et semble associée à des marques d’activation de la transcription du locus. De plus, d’autres gènes soumis à l’empreinte parentale, comme MEG3 et DIRAS3, présentent également des modifications de leurs marques épigénétiques.Projet iPPSD2 et 3 :Résultat 1 : Croissance longitudinale, taille finale et IMC des patients iPPSD2 et 3. Les patients iPPSD2 naissent hypotrophiques, ont une petite taille finale et développe un surpoids. Les patients iPPSD3 naissent macrosomiques mais atteignent une taille finale normale et un IMC moyen normal. Mes résultats suggèrent un rôle majeur de Gsα et de Xlαs dans la croissance fœtale, postnatale, pubertaire et l’accumulation de la masse grasse.Résultat 2 : Cibles des GNAS-miRNAs et phénotype des patients iPPSD3. Mes données d’expression des GNAS-miRNAs dans les lymphocytes et les fibroblastes de patients matUPD20/patUPD20 montrent qu’ils sont soumis à l’empreinte parentale. In silico, les cibles de ces miRNAs sont classées en 3 groupes : signalisation de l’AMPc, signalisation du calcium et croissance. La transfection dans les cellules HEK des GNAS-miRNAs m’a permis de confirmer certaines cibles moléculaires comme la cible du miR-296-3p, PRKAG1 et la cible des miRs 296-5p et 296-3p, GNB2 tous les deux impliqués dans la voie de l’AMPc.Résultat 3 : étude d’une nouvelle DMR : GNAS-AS2. Cette DMR récemment identifiée présente une perte de méthylation chez la plupart des patients iPPSD3 comparés aux contrôles. Nous avons identifié pour la première fois, un sous-groupe de patients iPPSD3 qui présente une perte de méthylation de la DMR GNAS-AS1:TSS-DMR, mais pas de GNAS-AS2. Ces patients ont un profil de iPPSD3 autosomique dominant, sans délétion du centre d’empreinte STX16. Mes résultats montrent : 1-absence de délétion ou remaniement génomique en whole genome sequencing dans une famille atteinte, 2-la DMR GNAS-AS2 possède deux sous domaines distincts et 3-la transmission de cette anomalie est maternelle.Les transcrits de GNAS jouent un rôle important dans des phénomènes fondamentaux comme la croissance ante et post natale, la tumorigenèse, la signalisation endocrine AMPc dépendante et l’acquisition de la masse grasse. Afin de mieux comprendre les pathologies de l’empreinte il est important d’identifier des biomarqueurs comme les miRNAs et de les corréler aux phénotypes des patients iPPSD. / GNAS is a locus subjected to parental imprinting. This locus produces different transcripts and proteins using distinct promoters: the Gsα biallelic transcript, the maternally imprinted transcripts GNAS-AS1, XLαs and A/B and a paternal imprinted transcript NESP55. The expression of these transcripts is controlled by differentially methylated regions (DMRs). The GNAS locus is involved in several diseases. PseudoHypoParathyroidism (PHP) also called inactivating PTH/PTHrP Signaling Disorder (iPPSD, for the description of the new classification) encompasses a group of heterogeneous rare diseases, characterized by the resistance of organs to the action of PTH. IPPSD2 are due to Gsα loss of function mutations, iPPSD3 to methylation abnormalities at one or several GNAS DMRs. Intraductal Papillary Mucinous Neoplasm (IPMN) are cystic tumors characterized by Intraductal papillary proliferation of the pancreatic duct epithelium. IPMNs are precursors of pancreatic adenocarcinoma comprising three degrees of dysplasia according to the histological phenotype, low, medium and high grade.The aim of my work was to study the implication of non-coding transcripts and GNAS-miRNAs using two pathological models, iPPSD2 and 3 and IPMN.IPMN project:We have identified that loss of imprinting at the GNAS locus is a common trait in IPMN and appears to be associated with transcriptional activation events of the GNAS transcripts. In addition, other genes subjected to parental imprinting, such as MEG3 and DIRAS3, also show changes in their epigenetic marks.Project iPPSD2 and 3:Result 1: Longitudinal growth, final height and BMI of iPPSD2 and 3 patients. iPPSD2 patients are born hypotrophic, display a final short stature and are overweight. IPPSD3 patients are born macrosomic but reach a normal final height and normal mean BMI. My results suggest a major role of Gsα and Xlαs in fetal, postnatal, pubertal growth and fat mass accumulation.Result 2: Targets of GNAS-miRNAs and phenotype of iPPSD3 patients. Expression of GNAS-miRNAs in lymphocytes and fibroblasts of matUPD20/patUPD20 patients suggested that GNAS-miRNAs are subjected to parental imprinting. In silico, the targets of these miRNAs are classified in 3 groups: cAMP signaling, calcium signaling and growth. Transfection of the GNAS-miRNAs into HEK cells confirmed certain molecular targets such as the miR-296-3p target, PRKAG1 and the miRs 296-5p and 296 -3p target, GNB2, both transcripts being involved in cAMP pathway.Result 3: characterization of a new DMR: GNAS-AS2. GNAS-AS2 is a recent characterized DMR with loss of methylation in most iPPSD3 patients compared to controls. We have identified for the first time a subset of iPPSD3 patients with loss of methylation at the GNAS-AS1:TSS-DMR but not at the GNAS-AS2 DMR. These patients have an autosomal dominant form of iPPSD3, without deletion of the STX16 imprinting control element. My results show: 1- lack of deletion or genomic rearrangement through whole genome sequencing in an affected family, 2- GNAS-AS2 DMR has two distinct subdomains and 3- this anomaly is maternally inherited.GNAS transcripts play an important role in fundamental biological processes such as ante and postnatal growth, tumorigenesis, endocrine cAMP dependent signaling pathways and fat mass acquisition. It is important to identify new biomarkers such as miRNAs and correlate them with phenotypical factors of iPPSD patients.