La cryoconservation de cortex ovarien est la seule technique envisageable pour les patientes pré-pubères et les femmes dont la pathologie nécessite l’administration d’un traitement hautement gonadotoxique dont l’initiation ne peut être différée. L’autotransplantation est jusqu’à présent la seule méthode disponible de réutilisation du tissu ovarien cryoconservé, et a permis d’obtenir plus de 130 naissances dans le monde, dont trois au CHRU de Besançon. Toutefois, en cas de pathologie néoplasique à risque de localisation métastatique ovarienne, cette technique peut présenter un risque de réintroduction de cellules malignes susceptibles d’être présentes dans le greffon. Des méthodes alternatives à l’autogreffe de tissu ovarien cryopréservé fondées sur l’utilisation de follicules ovariens isolés sont actuellement en développement. L’objectif de cette thèse a été de développer un protocole permettant l’isolement et la qualification de follicules ovariens qui pourront être utilisés en thérapie cellulaire à usage humain. Dans un premier temps, une validation de la technique d’isolement de follicules ovariens a été réalisée à partir de la dissociation de fragments de cortex ovarien, issus de patientes ayant subit une résection percœlioscopique, à l’aide d’une collagénase NB6 produite selon les bonnes pratiques de fabrication. Les follicules ainsi obtenus ont été analysés en termes de viabilité (immédiate et après culture in vitro), rendement, morphologie et état prolifératif. Dans un deuxième temps, la sécurité carcinologique des suspensions folliculaires obtenues après isolement a été évaluée par cytométrie en flux multicouleurs à l’aide d’une modélisation ayant consisté en la contamination de suspensions folliculaires avec des cellules leucémiques issues de patients souffrant de leucémies aigües myéloïdes (LAM) ou lymphoblastiques (LAL) d’immunophénotype connu. La collagénase NB6 a permis l’isolement d’un grand nombre de follicules vivants, principalement au stade primordial, et dont la majorité était non activée, même après trois jours de culture in vitro dans un gel de fibrine. La technique d’isolement suivie de trois lavages a permis d’éliminer les cellules leucémiques préalablement ajoutées aux suspensions folliculaires dans 23 cas sur 24, sans endommager les follicules isolés. La cytométrie en flux multicouleurs est une technique d’analyse efficace pour évaluer la contamination, par des cellules leucémiques, de suspensions contenant des follicules ovariens isolés. Le protocole d’isolement de follicules ovariens humains, ayant été réalisé avec la collagénase NB6 de grade clinique, peut être envisagé pour une reconstruction ovarienne à visée thérapeutique humaine. / The cryopreservation of ovarian cortex is the only technique available for prepubertal girls and women when their pathology requires the administration of a highly gonadotoxic treatment whose initiation cannot be delayed. Autotransplantation has so far been the only available method to re-use cryopreserved ovarian tissue, and has resulted in more than 130 births worldwide, including three at the Besançon Hospital. However, in cases of neoplastic disease with a risk of ovarian metastatic localization, this technique may present a risk of reintroducing malignant cells likely to be present in the graft. Alternative methods to cryopreserved ovarian tissue autograft based on the use of isolated ovarian follicles are currently under development. The aim of this thesis was to develop a protocol allowing the isolation and the qualification of ovarian follicles that can be used in cell therapy for human purposes. In a first step, a validation of the technique to isolate ovarian follicles was carried out from the dissociation of cortical ovarian fragments, taken from patients undergoing a laparoscopic ovarian drilling, using collagenase NB6 which is produced according to good manufacturing practices. Follicles thus obtained were analyzed in terms of viability (before and after in vitro culture), yield, morphology and proliferative state. In a second step, the carcinologic safety of follicular suspensions obtained after isolation was evaluated by multicolor flow cytometry using a model involving the contamination of follicular suspensions with leukemic cells from patients suffering from acute myeloid leukemia (AML) or acute lymphoblastic leukemia (LAL) with known immunophenotype. Collagenase NB6 has allowed the isolation of a large number of viable follicles, mostly at the primordial stage and the majority of which were unactivated even after three days of in vitro culture in a fibrin matrice. Our isolation technique followed by three washes has allowed the elimination of leukemic cells previously added to follicular suspensions, in 23 out of 24 cases, without damaging isolated follicles. Multicolor flow cytometry is an effective analytical technique for assessing leukemic cell contamination of suspensions containing isolated ovarian follicles. The protocol to isolate human ovarian follicles, performed with the clinical grade collagenase NB6, may be considered for an ovarian reconstruction to human therapeutic purposes.

In response to LH surge, the selected dominant follicle undergoes a series of complex events viz., meiotic maturation of the oocyte, terminal differentiation of follicular cells, cumulus expansion and follicular rupture leading to ovulation (release of fertilizable oocyte) and formation of corpus luteum. These complex set of events are mediated by dramatic changes in the temporo-spatial transcriptional regulation of a large number of genes in the preovularory follicle. In the present thesis, attempts have been made to delineate the transcriptional changes occurring in the periovulatory follicle in response to gonadotropin surge in monovulatory species (bonnet monkeys and buffalo cows). Further, attempts have also been made to investigate effects of increased circulating IGF-I and glucose on the periovulatory events. Chapter I provides a review of available literature on regulation and factors influencing various aspects of growth and development of ovarian follicles. Chapter II describes initial studies carried out in the bonnet monkeys to examine expression patterns of various genes considered as markers of ovulation and luteinization in granulosa cells of multiple preovulatory-like follicles before and after exposure to luteinizing dose of hCG. Chapter III describes the standardization and validation of an induced ovulation model system in buffalo cows involving determination of the presence of a large follicle by ultrasonography before administering exogenous PGF2αon day 7 of the cycle to induce luteolysis and administration of hCG 36 h post PGF2αto mimic LH surge-like stimulus. Attempts made to characterize the responsiveness of buffalo cows to a range of exogenous doses of GnRH in terms of LH secretion during summer and winter seasons and determination of the exogenous dose of GnRH necessary to elicit a surge-like increase in circulating LH levels during breeding season in buffalo cows are described in Chapter IV. Chapter V describes the global changes in the gene expression pattern in the periovulatory follicle of buffalo cows before and at various time points after the onset of gonadotropin surges. IGF-I is known to regulate the proliferation and survival of follicular cells and play an important role in the selection of dominant follicle however, its role during the periovulatory events remains to be delineated. Chapter VI describes the effects of increased circulating and intrafollicular IGF-I levels on the periovulatory events. An ongoing study to delineate the effects of transient increase in circulating glucose levels on the periovulatory events are described in Appendix I. Thus, this thesis aims at delineating (i) changes in the gene expression patterns regulating periovulatory events and (ii) effects of increased circulating IGF-I and glucose on the periovulatory events.

The role oxygen plays in the developing ovarian follicle is of interest not only to the field of developmental biology but also to in-vitro fertilisation (IVF) technologists, as oxygenation of the oocyte is considered to be a potential determinant of oocyte competence. Oxygen transport through the developing ovarian follicle, and practical aspects of the analysis of oxygen in human follicular fluid were investigated in this work. Mathematical modelling of oxygen transport in the pre-antral, and antrallpreovulatory follicle revealed a number of interesting findings, Contrary to previous conclusions (Gosden & Byatt-Smith, 1986), oxygen can reach the oocyte in the small pre-antral follicle. Improved estimates of diffusion coefficients through the granulosa cell layer and the inclusion of fluid voidage in this layer showed that oxygen can also reach the oocyte in large pre-antral follicles. The amount of oxygen that reaches the oocyte in the pre-antral follicle is a function of its size and degree of vascularisation. Symmetrically distributed vascularisation is superior in achieving a well oxygenated follicle. However, the large pre-antral follicle will eventually reach a size beyond which it cannot grow without anoxic regions developing. The size at which this occurs is consistent with the size at which antrum formation is observed in human follicles. The model predicts that the follicle can avoid an anoxic state through antrum formation, and shows that the follicle develops in a way that is consistent with overcoming mass transport limitations. The oxygen status of the follicle during the antrallpre-ovulatory phase of growth requires that the volume of granulosa cells be balanced by the volume of follicular fluid. Further predictions suggest that oocyte respiration becomes sub-maximal at follicular fluid volumes below approximately 4m1, vascularisation levels below 38%, or fluid i dissolved oxygen levels below 5.1 ~01%. These values are consistent with observations in the literature. It was also shown that the measurement of follicular fluid dissolved oxygen levels could provide a simple measure of the respiratory status of the oocyte, and this may be superior to the measurement of follicular vascularisation which requires knowledge of more parameters. Methodology for the analysis of follicular fluid oxygen solubility and diffusivity was developed using a Clark oxygen electrode. Analysis of these parameters showed that they are similar to human plasma, and allowed the predictive uncertainty of the model to be reduced. Experimental studies into the effects of IVF aspiration on follicular fluid were carried out. Aspiration results in significant changes in the properties of follicular fluid. Dissolved oxygen levels rose 5 * 2 vol%, pH increased by 0.04 * 0.01 pH units, and temperature dropped by 7.7 * 1.3 "C. Mathematical modelling of blood contaminated follicular fluid also showed that contamination results in significant changes in the dissolved oxygen of the fluid. This suggests that if the composition of follicular fluid is to be determined (particularly dissolved oxygen), sampling andlor measurement of fluid must take place before the collection vial of the aspiration kit, and blood contamination must be eliminated. Based on this result, the design and testing of devices capable of reliable sampling andlor rneasurement of oxygen levels of follicular fluid was considered. This presents a continuing challenge, including the integration of routine follicular fluid oxygen measurement into clinical practice.

Made available in DSpace on 2016-08-15T20:27:59Z (GMT). No. of bitstreams: 1 AntonioCLC_TESE.pdf: 1302444 bytes, checksum: e465e8b76dbdec077ba3f1534f5d9898 (MD5) Previous issue date: 2015-04-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Gossypol is a polyphenol compound produced by the pigment glands of cotton plants (Gossypium spp.) and presents proven deleterious action on spermatogenesis, rodent´s estrous cycle, granulosa cells steroidogenesis in pigs, early termination of pregnancy and embryonic development. However, the effects on sheep ovaries were not yet studied. This study was divided into two stages and aimed to determine in vitro and in vivo effect of gossypol on sheep ovarian follicles. In the first stage, eight ovarian fragments from each ewe were cultured in &#945;-MEM supplemented culture and subjected to three concentrations of gossypol-acetic acid (5, 10 and 20 µg/mL) for periods of 24 hours and seven days. In vitro action of gossypol resulted in a significant reduction in the number of viable ovarian follicles of all development stages, and a significant increase in the number of atretic follicles after 24 hours and seven days of cultivation (p < 0,01). In the latter it was remarkable the absence of viable antral follicles and the primordial follicles presented less than 50% of viability. In the second stage, we used twelve crossbred ewes, which were randomly divided into two groups and fed diets containing cottonseed cake or not (control group). Concentrate feed was offered at a rate of 1.5% of body weight of the animal for 63 days. Concentrations of total and free gossypol in cottonseed cake was 3.28 mg/g and 0.11 mg/g, respectively. Throughout the trial period no animal showed clinical signs of toxicosis. There was no interference on progesterone concentrations, weight, weight gain and ovaries measurements. Only occasional changes on vaginal cytology, hematological and biochemical parameters were noted. However, treatment with gossypol was responsible for significant reduction in the number of viable ovarian follicles (20.6% in treated sheep and 65.1% the control group) and significant increase in the number of atretic follicles (79.4% in treated sheep and 34.9% in control group), interfering at all stages of follicular development (p < 0,01). In conclusion, the gossypol promotes direct damage to the ovarian follicles of sheep, resulting in an increase on the proportion of atretic follicles and reduction of viable follicles both in vitro and in vivo / O gossipol é um composto polifenólico produzido pelas glândulas de pigmento do algodoeiro (Gossypium spp.); apresentando ação deletéria comprovada sobre a espermatogênese, ciclo estral de roedores, esteroidogênese nas células da granulosa em suínos, interrupção precoce da gestação e desenvolvimento embrionário inicial. No entanto, os efeitos sobre os ovários de ovelhas ainda não havia sido estudado. O presente estudo foi dividido em duas etapas: determinar o efeito in vitro e in vivo do gossipol sobre folículos ovarianos de ovelhas. Na primeira etapa, oito fragmentos ovarianos de cada animal foram cultivados em Meio &#945;-MEM suplementado e submetidas a cultivo em três concentrações de gossipol-ácido acético (5, 10 e 20 &#956;g/mL) por períodos de 24h e sete dias. A ação do gossipol in vitro proporcionou redução significativa no número de folículos viáveis em todos os estágios de desenvolvimento folicular; e aumento significativo no número de folículos atrésicos após 24h e sete dias de cultivo (p < 0,01). Neste último também foi notório a ausência de folículos antrais viáveis, e, os folículos primordiais apresentaram viabilidade inferior a 50%. Na segunda etapa, foram utilizadas doze ovelhas mestiças, que foram aleatoriamente separadas em dois grupos e alimentadas com rações contendo torta de algodão ou não (grupo controle). A ração foi oferecida na proporção de 1,5% do peso vivo do animal por 63 dias. As concentrações de gossipol total e livre na torta de algodão foram 3,28 mg/g e 0,11 mg/g, respectivamente. Durante todo o período experimental, nenhum animal apresentou qualquer sinal clínico de intoxicação. Não houve interferência sobre as concentrações de progesterona, peso, ganho em peso e medidas ovarianas. Apenas alterações ocasionais sobre a citologia vaginal, parâmetros hematológicos e bioquímicos foram evidenciadas. Entretanto, o tratamento com gossipol foi responsável por redução significante no número de folículos ovarianos viáveis (20,6% nas ovelhas tratadas e 65,1% no grupo controle) e aumento significante no número de folículos atrésicos (79,4% nas ovelhas tratadas e 34,9% no grupo controle), com interferência em todas as fases de desenvolvimento folicular (p < 0,01). Em conclusão, o gossipol promove danos diretos aos folículos ovarianos de ovelhas, resultando em aumento na proporção de folículos atrésicos e redução dos folículos viáveis tanto in vitro como in vivo

Made available in DSpace on 2016-08-15T20:30:58Z (GMT). No. of bitstreams: 1 GabrielaLL_DISSERT.pdf: 3154281 bytes, checksum: e51e3f107ffe738761818a96584d12a8 (MD5) Previous issue date: 2011-01-10 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / It were done the estimative of the follicular population of the collared peccaries ovaries and the short term preservation of it using PBS or ACP. Pair of ovaries from 6 females were collected. A half of one ovary was designated to the estimative and characterization of the follicle population, and it was immediately submitted to histology and transmission electron microscopy (TEM). The other ovary was divided into 9 fragments and one of it was immediately submitted to histology procedures, the other fragments were stored either in phosphate-buffered saline (PBS; control group) or ACP medium in isothermal Styrofoam boxes containing biological ice packs. The boxes were sealed and opened only after 12, 24, or 36 h. After opening each box, the ovarian fragments were submitted to histological analysis and TEM. The temperature into the boxes and pH of the medium were checked. A total of 33273.45 ± 3019.30 preantral follicles (PF) were estimated for each ovary. Most of them was morphologically normal (94.4%) and just a few was atretic (5.6%). At histological analysis, a lot of lipids droplets was observed at oocyte cytoplasm, which was confirmed by TEM. About short term storage, it was observed that the follicular integrity was kept until 4 h, using ACP medium. The medium pH was kept constant. This work shows, for the first time, the estimative of the PF population of the ovary, totally and in each follicle category, and it´s characterization in collared peccaries (Tayassu tajacu). It could be concluded that the ACP medium may be used as an alternative medium for the short term preservation of PF in that specie. / Foram realizados a estimativa da população folicular ovariana de catetos, e ainda a refrigeração dos mesmos utilizando PBS ou ACP por curtos períodos. Os pares de ovários de 6 catetos foram coletados. Metade de um dos ovários foi destinada a estimativa e caracterização da população folicular, sendo imediatamente submetida ao processamento para histologia clássica e microscopia eletrônica de transmissão (MET). O outro ovário foi dividido em 9 fragmentos e um deles foi imediatamente destinado à histologia, os demais foram colocados em tubos com solução salina fosfatada tamponada (PBS) ou em solução à base de água de coco em pó (ACP) e acondicionados em caixas isotérmicas de 5L. As caixas foram lacradas e abertas 4h,12h, 24h e 36h após, e os fragmentos ovarianos foram submetidos à histologia e processamento para MET. A temperatura das caixas e o pH do meio foram aferidos. Um total de 33273.45 ± 3019.30 folículos pré antrais (FOPA) foram estimados para a população em cada ovário. A maioria dos FOPA apresentava-se como morfologicamente normal (94.4%) e apenas poucos deles eram atrésicos (5.6%). Na análise histológica, uma grande quantidade de gotas de lipídios foi observada no citoplasma oocitário, os quais foram confirmados através da análise ultraestrutural. Quanto à refrigeração, verificou-se que a integridade folicular foi mantida até as 4 h utilizando-se ACP. O pH dos meios de conservação permaneceu constante. Este trabalho mostra pela primeira vez a estimativa da população de FOPA do ovário, total e por categoria, e também a caracterização destes folículos em catetos (Tayassu tajacu). E pôde-se concluir que a ACP® pode ser utilizada como um meio alternativo para conservação de folículos pré-antrais.

Made available in DSpace on 2014-07-29T15:07:30Z (GMT). No. of bitstreams: 1 Ubirajara_Bilego.pdf: 525711 bytes, checksum: 92b1baad77eca0e7758c3f7bfe166098 (MD5) Previous issue date: 2009-02-26 / The aim of this study was to describe the physiological responses to the Ovsynch protocol in Girolando heifers breeding in extensive model on Centro-Oeste of Brazil region and to determine relationships with environments factors in two stations, dry and rain. This responses were describe through the characterization of ovarian structures how: (NTL) total number of follicles; (DFOLIPR) mean of follicular diameter on protocol s begin; (DFOLOV) ovulatory diameter follicles; (DCL) corpus luteum ovulatory diameter; (SCL) ovulatory corpus luteum brightness score; pregnancy rates and ovulation rate on day 9 obtained in two year stations. Were utilized 40 heifers 18-24 months age, split in two groups of 20 animals being G1 group of dry station and G2 group of rain station. During the evaluations were found differences for the total number of follicles 8,07±0,25 e 8,83±0,26 in two stations applied. The mean of follicular diameter on protocol s begin was not show differences, but the measure of the ovulatory follicle diameter, have differences for the year stations (P<0,01), being the diameter for the dry station 11,88 ± 0,4mm whereas the rain station was 10,13 ± 0,36mm. Have in this study, higher frequency of follicles that ovulate with 11 to 12 mm on dry station and 10 to 11mm on rain station. The mean of the corpus luteum diameter on the present study not differ among the stations (P>0,05), with10,46±0,41mm for the dry station and 10,49±0,34mm for the rain station, being the higher frequency of CL 11 to 12mm in both stages. The CL brightness score not have differ(P>0,05), being 2,6 ± 0,16 for the dry and 2,31 ± 0,11 for the rain. The pregnancy rate have differences on two stages (P<0,01) with values 45% and 11% on station dry and rain respectively. The THI values differ among the stations dry and rain (P<0,01). The ovulation rate on D9 do not differ (P>0,05) although to have numeric differences. It was possible to conclude that in two stations evaluated, dry and rain, have differences on follicular morfometry and pregnancy rates. / Objetivou-se com este estudo descrever a resposta fisiológica do protocolo Ovsynch em novilhas Girolando, criadas extensivamente na região Centro-Oeste do Brasil em duas estações do ano, seca e águas. Tais respostas foram descritas através da caracterização das estruturas ovarianas como: (NTF) Número total de folículos; (DFOLIPR) Diâmetro folicular médio no início do protocolo; (DFOLOV) Diâmetro do folículo ovulatório; (DCL) Diâmetro do corpo lúteo; (SCL) Escore de ecogenicidade do corpo lúteo; Taxa de gestação e Taxa de ovulação no D9 obtidas em duas estações do ano. Foram utilizadas 40 novilhas girolando com idades entre 18-24 meses, divididas em dois grupos de 20 animais, sendo (G1) de estação seca e (G2) grupo de estação de águas. Durante as avaliações foram encontradas diferenças quanto ao número total de folículos 8,07±0,25 e 8,83±0,26 nas duas estações avaliadas. O diâmetro folicular médio no início do protocolo não mostrou diferenças, porém, houve diferença entre as estações do ano (P< 0,01), quanto ao diâmetro folicular ovulatório sendo o diâmetro médio para a estação seca 11,88 ± 0,4mm enquanto para a estação das águas foi 10,13 ± 0,36mm. Houve nesse estudo maior freqüência de folículos que ovularam em torno de 11 a 12mm na estação seca e 10 a 11mm na estação das águas. O diâmetro médio do corpo lúteo no presente estudo não diferiu entre as estações (P>0,05), com 10,46±0,41mm para a estação seca e 10,49±0,34mm, com maior freqüência de CLs de 11 a 12mm em ambas as estações. Os escores de corpo lúteo também não mostraram diferenças (P>0,05) sendo 2,6 ± 0,16 para seca e 2,31 ± 0,11 para águas. A taxa de gestação também mostrou diferença entre as estações com valores de 45% e 11% nas estações seca e águas respectivamente. Taxa de ovulação no D9 não diferiu (P>0,05) entre as estações apesar de ser numericamente diferente entre os tratamentos. Os valores de THI diferiram entre as estações seca e águas (P<0,01). Foi possível concluir que as duas estações avaliadas, seca e águas, mostraram diferenças quanto morfometria folicular e taxas de gestação.

Depuis plusieurs années, la vectorisation de molécules est considérée comme la stratégie la plus prometteuse pour améliorer la pénétration cutanée des principes actifs et pour cibler et contrôler leur libération, augmentant ainsi l'efficacité thérapeutique des traitements tout en limitant leurs effets secondaires. Les nanoparticules polymériques ont fait l'objet de nombreuses études car elles possèdent une très grande stabilité et une capacité supérieure aux autres vecteurs à libérer les principes actifs de façon prolongée. Récemment, le laboratoire de Pharmacie Galénique de Besançon a montré, in vivo, que les nanoparticules polymériques de diamètre inférieure ou égale à 100 nm pénétraient spécifiquement dans la peau inflammée de la souris, alors qu'aucune pénétration n'était observée dans la peau saine. Le premier objectif de cette thèse était de confirmer ces hypothèses dans la peau inflammée d'un autre animal, le porc. Les résultats de notre étude in vivo confirment l'absence de pénétration des nanoparticules polymériques dans la peau saine du porc et montrent une pénétration taille dépendante dans la peau inflammée. Le second objectif poursuivi était de vérifier ces résultats chez l'Homme. Pour cela, une preuve de concept a été mise en place dans le service de Dermatologie du CHRU de Besançon. Les premiers résultats de cette étude clinique semblent confirmer l'absence de pénétration des nanoparticules polymérique de I 00 nm dans la peau des volontaires sains et dans la peau non lésée des patients souffrant de dermatite atopique. A l'inverse, une forte pénétration des nanoparticules est observée au niveau des plaques d'eczéma des patients. Si les résultats de l'étude clinique se confirment, nous prévoyons d'encapsuler un anti­inflammatoire ou un immunosuppresseur pour vérifier l'intérêt thérapeutique de ces vecteurs en médecine humaine dans le traitement de la dermatite atopique. Parallèlement, une évaluation pourrait être réalisée en médecine vétérinaire pour le traitement de cette pathologie fréquente chez le chien, et dont le traitement actuel repose sur l'administration per os de corticoïdes à l'origine de nombreux effets secondaires. / For several years now, nanocarriers have been considered as the most promising strategy to improve skin penetration of active ingredients. Moreover, these carriers are more efficient at targeting and controlling drug release into the skin, which leads to increased treatment efficiency and reduced side effects. Polymeric nanoparticles have been the object of an increasing number of studies due to their good physicochemical stability and prolonged release of active ingredients which is superior to any other carriers. Recently, the Laboratory of Pharmaceutical Engineering at Besançon proved in vivo, that polymeric nanoparticles with a diameter smaller or equivalent to I 00 nm specifically penetrated in inflamed skin of mice whilst no penetration was observed in healthy skin. The first aim ofthis thesis was to confirm this hypothesis on another animal's inflamed skin, in instance the pig. Our results confirm the poor penetration of polymeric nanoparticles in healthy skin ofpigs and show various degree ofpenetration depending on the size of the nanoparticles into the inflamed skin area. The second objective ofthis work was to evaluate the skin penetration of our polymeric nanoparticles in humans. A proof of concept has been developed in the Department of Dermatology at Besancon University Hospital. The first results ofthis clinical trial tend to confim1 the greater penetration ofour carrier, specifically in inflamed skin. In fact, no penetration of polymeric nanoparticles with a size close to 100 nm was observed in healthy skin ofvolunteers or in the non-inflamed skin of patients suffering from atopic dennatitis. Conversely, a high penetration ofthese carriers was observed in the skin lesions of patients with atopic dermatitis. If the results ofthis clinical trial are confirmed, we plan to load an anti-inflammatory or an immunosuppressive drug into the nanoparticles to evaluate the therapeutic value ofthese nanocarriers in human medicine in the treatment ofatopic dermatitis. Meanwhile, a similar study may be undertaken in veterinary medicine for the treatment of atopic dermatitis in dogs which is a common disease whose current treatment is based on the oral administration of corticosteroids and cause many undesirable side effects.

The goal of this long-term study was to better understand the reproductive biology of the female bottlenose dolphin (Tursiops truncatus) and provide a hypothesis for how dolphins may communicate reproductive readiness to one another. Utilizing conditioned dolphins in aquaria, this dissertation examined several previously unknown aspects of dolphin reproduction, including ovarian follicular dynamics during the luteinizing hormone surge, urinary prolactin levels, estrus behavior, vaginal fluid arboriform arrangement, in-situ vaginal and cervical anatomy during estrus, reversed-phase high-performance liquid chromatography (RP-HPLC) of urine samples to identify proteins and peptides that may be used in chemical communication, and a review and anatomical analysis of dolphin vibrassal crypts. The diffusely seasonal dolphin estrous cycle is not controlled by photoperiod and has a 10-day follicular and 20-day luteal phase. A brief ovulatory LH surge is followed by ovulation within 48 hours. An ethogram of 20 reproductive behaviors was developed, and all occurrences of reproductive behavior were analyzed during conceptive estrous cycles. A novel form of standing heat estrus, termed immobility, was observed, and estrus dolphins displayed genital nuzzling, active and passive mounting with other females, and an increase of standing heat intensity as LH levels rose. Prolactin plays a role in pregnancy maintenance, mammary development, allo-mothering behavior, lactation, and lactational anestrus. Dolphins are similar to sows where weaning causes a return to estrus, and in the boar effect, where days to ovulation are shortened in the presence of a mature male. Dolphin vaginal fluid showed crystallization arrangements with large open mesh patterns, conducive to sperm transport, during the estrogenic follicular phase, and closed mesh during the luteal phase. RP-HPLC analysis revealed that urine contained large amounts of peptides and proteins with peaks that change throughout the estrous cycle and with changes in social grouping. Remnant vibrissae from dolphin follicular crypts were sectioned, and it was hypothesized that trigeminal nerve endings could act similarly to those found in the nasal mucosa of terrestrial species and respond to chemical stimuli. This study provides new data to better understand the reproductive biology of a holaquatic mammal.