L'ostéoporose est la maladie métabolique la plus fréquente qui touche l'os. Plusieurs substances actives sont utilisées pour le traitement pharmacologique de cette maladie. Cependant, ce sont les bisphosphonates et surtout l'alendronate de sodium, qui sont prescrits en première intention. L'alendronate de sodium est, en effet, très efficace mais présente une faible absorption quand il est administré par la voie orale. Sa solubilité dans l'eau est de 20 mg/ml. Il présente en outre une faible biodisponibilité (de 0,6 à 0,7%). Cette substance active est aussi à l'origine d'effets indésirables d'irritation au niveau de l'oesophage, l'estomac et l'intestin. Ces effets sont dus à un contact local des cristaux de la substance active avec la muqueuse. L'approche d'encapsulation des substances actives dans des particules polymériques a permis d'obtenir plusieurs bénéfices thérapeutiques comme l'amélioration de la biodisponibilité et la diminution des effets indésirables. Dans la première partie de notre étude, on a réalisé l'encapsulation de l'alendronate dans des nanoparticules à base de poly-epsilon-caprolactone en utilisant la nanoprécipitation et l'émulsion double. Les nanoparticules obtenues ont une forme sphérique et une taille comprise entre 200 et 450 nm. Le meilleur pourcentage d'encapsulation a été de 34% et il a été obtenu avec la technique d'émulsion double. Ceci confirme que cette méthode est plus adaptée à l'encapsulation des molécules hydrophiles. Le profil de libération in vitro a montré deux phases : une première phase de libération relativement rapide et une deuxième phase beaucoup plus lente. L'analyse par modélisation mathématique a montré que la libération in vitro de l'alendronate se fait par diffusion et relâchement des chaines polymériques / Osteoporosis is the most frequent metabolic disease that affects bone. Many actives have been used as pharmacological treatment of this disease. However, bisphosphonates, especially, alendronate sodium, are indicated as first line regimen. Alendronate is highly efficient but presents low absorption after oral administration. Its solubility in water is 20 mg/ml. It has also poor bioavailability (0.6-0.7%). In addition, this active could lead to many side effects, which are mainly related to the esophagus, the stomach and the intestine. Such effects are linked to a local contact of drug crystals with the mucosa. Encapsulation of active molecules allowed the obtaining of many advantages over conventional pharmaceutical forms such as, bioavailability and tolerance enhancement. In the first part of our study, we managed to encapsulate alendronate sodium in poly-epsilon-caprolactone nanoparticles via two techniques: nanoprecipitation and double emulsion. Obtained nanoparticles presented a spherical form. Their size ranged between 200 and 450 nm. The highest encapsulation efficiency value was 34% and was obtained via double emulsion technique. This confirms that double emulsion is more suitable for hydrophilic drugs encapsulation. In vitro release profile showed two phases: first phase of burst release and a second more prolonged phase. Mathematical modeling showed that alendronate in vitro release occurs by drug diffusion and polymer chain relaxation. In the second experimental part, we managed to find a more interesting alternative. In fact, we opted for the use of chitosan which is a natural hydrophilic polymer. One of the obtained advantages is the avoidance of organic solvents use. In addition, this approach allowed the enhancement of encapsulation efficiency as this value increased to 70%. The used technique is ionic gelation. It is a simple encapsulation technique that is based on the transformation of a dissolved polymer to a gel-like state

A alta variabilidade na resposta aos tratamentos superovulatórios e na produção de embriões tem sido relacionada a causas multifatoriais. Além disso, são restritas as informações que relacionam fatores correlatos ao embrião à concepção. Dessa forma, o objetivo do presente estudo foi avaliar a influência de variáveis relacionadas às doadoras (categoria animal e época do ano de produção do embrião) na eficiência de programas de superovulação (SOV), assim como na eficiência reprodutiva de receptoras em programas de transferência de embrião. Foram avaliados dados de 1.562 protocolos de SOV (609 em vacas em lactação e 953 em novilhas da raça Holandesa) e 4.076 transferências de embrião (receptoras Holandesas em lactação). A taxa de SOV (número de doadoras com dois ou mais CL) foi semelhante entre novilhas e vacas em lactação (89,7 vs. 91,9%, respectivamente; P = 0,26). A época do ano, também, não influenciou a taxa de SOV (época quente = 89,3% vs. época não quente = 92,2%; P = 0,09). Doadoras lactantes apresentaram maior número de CL (10,6 ± 0,6 vs. 7,5 ± 0,4; P < 0,0001), de estruturas recuperadas (7,6 ± 0,6 vs. 4,6 ± 0,4; P < 0,0001) e taxa de recuperação (77,6 vs. 58,7%; P < 0,0001) que novilhas. Doadoras superovuladas na época quente apresentaram menor número de CL (8,3 ± 0,5 vs. 9,7 ± 0,5; P = 0,03), de estruturas recuperadas (5,3 ± 0,5 vs. 6,7 ± 0,5; P = 0,04) e taxa de recuperação (65,7 vs. 72,3 %; P = 0,007) que doadoras superovuladas na época não quente. A taxa de fertilização (47,9 vs. 82,4%; P < 0,0001), de embriões viáveis (31,5 vs. 67,4%; P < 0,0001) e embriões viáveis grau I e II (15,4 vs. 42,1%; P < 0,0001) foram inferiores em doadoras lactantes. Semelhantemente, inferiores taxas de embriões viáveis (44,6 vs. 54,0%; P = 0,002) e embriões viáveis grau I e II (21,4 vs. 32,8%; P < 0,0001) foram verificadas durante a época quente do ano. No entanto, a queda na taxa de embriões grau I e II durante a época quente foi mais acentuada em doadoras lactantes (21,7 e 10,7%), quando comparada à observada em novilhas (46,2 e 38,1%; P = 0,02). Apesar da época do ano não ter influenciado a taxa de fertilização (época quente = 64,9 vs. época não quente = 70,0%; P = 0,07), foi observado relação negativa com a temperatura máxima média durante os 15 dias do protocolo de SOV (P = 0,006). A taxa de embriões viáveis também se mostrou negativamente relacionada com a temperatura máxima durante os 15 dias do protocolo de SOV (P = 0,03). O número de embriões viáveis foi semelhante entre as categorias (novilhas = 3,8 ± 0,3 vs. vacas em lactação = 3,3 ± 0,4; P = 0,16), porém foi inferior quando produzido durante a época quente (2,8 ± 0,3 vs. 4,4 ± 0,4; P = 0,03). A taxa de concepção dos embriões oriundos de doadoras em lactação foi maior aos 31 (36,0 vs. 30,7%; P = 0,001) e aos 45 dias de gestação (28,3 vs. 23,1%; P = 0,001). No entanto, a categoria da doadora não influenciou a perda gestacional (novilhas = 18,2% vs. vacas em lactação = 16,6%; P = 0,49). Ainda, as taxas de concepção aos 31 dias (30,5 vs. 31,7%: P = 0,47) e aos 45 dias (25,3 vs. 25,1%; P = 0,94) e perda gestacional (17,4 vs. 22,1%; P = 0,07) das receptoras não diferiram conforme a época de produção dos embriões, época quente e não quente, respectivamente. Também não foi verificado diferença nas taxas de concepção aos 31 dias (31,5 vs. 30,5%: P = 0,66) e aos 45 dias (25,1 vs. 25,4%; P = 0,89) e perda gestacional (18,4 vs. 16,5%; P = 0,49) das receptoras conforme a época de transferência dos embriões, época quente e não quente, respectivamente. Porém, a taxa de concepção aos 31 (P = 0,02) e aos 45 dias (P = 0,02) tiveram relação negativa com a média da temperatura máxima entre o estro e a TE. Em conclusão, apesar das doadoras em lactação apresentarem maior resposta superovulatória, as novilhas apresentaram semelhante número de embriões viáveis, porém maior taxa de embriões viáveis. A resposta ao tratamento superovulatório e a produção de embriões foram inferiores durante a época quente do ano. Embriões originados de doadoras Holandesas lactantes apresentaram maior taxa de concepção aos 31 e aos 45 dias de gestação do que embriões originados de novilhas. Apesar da época do ano não influenciar na taxa de concepção e na perda gestacional, verificou-se relação negativa da temperatura máxima entre o estro e a TE na taxa de concepção. / The high variability in the response to superovulatory treatments and embryo production has been related to multifactorial causes. Furthermore, there are restricted data that correlates factors related to the embryo to the conception. Thus, the objective of the present study was to evaluate the influence of variables related to donors (animal type and season of embryo production) in the efficiency of superovulation (SOV) programs and reproductive performance of dairy cows submitted to embryo transfer. We evaluated data from 1,562 SOV protocols (609 in lactating Holstein cows and 953 in heifers) and 4,076 embryo transfers (lactating Holstein recipient). The SOV rate (number of donor with two or more CL) was similar among lactating cows and heifers (89.7 vs. 91.9%, respectively; P = 0.26). Also, the season did not influence the SOV rate (warm season = 89.3% vs. not warm season = 92.2%; P = 0.09). Lactating donor had higher number of CL (10.6 ± 0.6 vs. 7.5 ± 0.4; P <0.0001), of recovered structures (7.6 ± 0.6 vs. 4.6 ± 0.4; P <0.0001) and recovery rate (77.6 vs. 58.7%; P <0.0001) than heifers. Donors superovulated during warm season had fewer number of CL (8.3 ± 0.5 vs. 9.7 ± 0.5; P = 0.03), of recovered structures (5.3 ± 0.5 vs. 6, 7 ± 0.5; P = 0.04) and recovery rate (65.7 vs. 72.3%; P = 0.007). The fertilization rate (47.9 vs. 82.4%; P <0.0001), viable embryos rate (31.5 vs. 67.4%; P <0.0001) and embryos grade I and II rate (15.4 vs. 42.1%; P <0.0001) were lower in lactating donor. Similarly, lower viable embryos rate (44.6 vs. 54.0%; P = 0.002) and embryos grade I and II rate (21.4 vs. 32.8%; P <0.0001) were observed during the warm season. However, the decrease in the embryos grade I and II rate during the warm period was more pronounced in lactating donors (21.7 and 10.7%) compared to that observed in heifers (46.2 and 38.1%; P = 0.02). Although the season did not influence fertilization rate (warm season = 64.9 vs. not warm season = 70.0%, P = 0.07), it was observed a negative relationship with the average maximum temperature during the 15 days of the SOV protocol (P = 0.006). The viable embryos rate was also negatively correlated with the maximum temperature during the 15 days of the SOV protocol (P = 0.03). The number of viable embryos was similar between categories (heifers = 3.8 ± 0.3 vs. lactating cows = 3.3 ± 0.4; P = 0.16), but was lower when produced during the warm season (2.8 ± 0.3 vs. 4.4 ± 0.4; P = 0.03). The conception rate of embryos from lactating donors was higher at 31 (36.0 vs. 30.7%; P = 0.001) and at 45 days of pregnancy (28.3 vs. 23.1%; P = 0.001). However, the donor category did not influence pregnancy loss (heifers = 18.2% vs. lactating cows = 16.6%; P = 0.49). Still, the recipients conception rate at 31 days (30.5 vs. 31.7%; P = 0.47) and at 45 days of pregnancy (25.3 vs. 25.1%; P = 0.94) and pregnancy loss (17.4 vs. 22.1%; P = 0.07) did not differ according to the embryo production season; warm and not warm season, respectively. Also,no difference was verified in recipients conception rates at 31 days (31.5 vs. 30.5%; P = 0.66) and at 45 days (25.1 vs. 25.4%; P = 0.89) and pregnancy loss (18.4 vs. 16.5%; P = 0.49) according to the embryo transfer season; warm and not warm season, respectively. However, conception rate at 31 (P = 0,02) and 45 days (P = 0.02) had a negative relationship with the average maximum temperature between estrus and TE. In conclusion, despite the lactating donors had higher superovulatory response, heifers presented similar number of viable embryos, but higher viable embryos rate. The superovulatory response and the embryo production were lower during the warm season. Embryos from lactating Holstein donors had higher conception rate at 31 and 45 days of gestation than embryos from heifers. Although the season did not influence the conception and pregnancy loss rates, there was a negative relationship between average maximum temperature during estrus and TE and conception rate.

The use of computer programs to predict drug absorption in humans and to simulate dissolution profiles has become a valuable tool in the pharmaceutical area. The objective of this study was to use in silico methods through software GastroPlusTM and DDDPlusTM to simulate drug absorption curves and dissolution profiles, and to establish in vitro-in vivo correlations (IVIVCs). The work presented herein is divided into five chapters and includes the drugs ketoprofen, pyrimethamine, metronidazole, fluconazole, carvedilol and doxazosin. In Chapter 1, simulated plasma curves for ketoprofen matrix tablets are presented and IVIVC was established. The use of simulated intrinsic dissolution tests for pyrimethamine and metronidazole as a tool for biopharmaceutics classification is detailed in Chapter 2. In Chapter 3, simulation of plasma curves for fluconazole capsules with different dissolution profiles is demonstrated as a tool for biowaiver. IVIVC studies were also conducted for carvedilol immediate-release tablets from dissolution profiles in Chapter 4. Chapter 5 covers the application of simulated dissolution tests for development of doxazosin extended-release formulations. Simulation of plasma curves and IVIVC using the software GastroPlusTM as well as intrinsic dissolution tests and dissolution profiles using the software DDDPlusTM proved to be a tool of wide application in predicting biopharmaceutical characteristics of drugs and formulations, allowing the reduction of time and costs of experimental laboratory work. / O uso de programas de computador para prever a absorção de fármacos em humanos e simular perfis de dissolução tem se tornado uma ferramenta bastante valiosa na área farmacêutica. O objetivo deste trabalho foi utilizar métodos in silico por meio dos programas de computador GastroPlusTM e DDDPlusTM para simular curvas de absorção de fármacos, perfis de dissolução e estabelecer correlações in vitro-in vivo (CIVIVs). O material aqui apresentado é constituído por cinco capítulos incluindo os fármacos cetoprofeno, pirimetamina, metronidazol, fluconazol, carvedilol e doxazosina. No capítulo 1 são apresentadas curvas plasmáticas simuladas para comprimidos matriciais de cetoprofeno, sendo estabelecida a CIVIV. A utilização de simulações de ensaios de dissolução intrínseca para os fármacos pirimetamina e metronidazol como uma ferramenta para classificação biofarmacêutica é detalhada no capítulo 2. No capítulo 3, a simulação de curvas plasmáticas a partir de cápsulas de fluconazol contendo diferentes perfis de dissolução é demonstrada como uma ferramenta para bioisenção. Estudos de CIVIV foram também realizados para comprimidos de liberação imediata de carvedilol a partir dos perfis de dissolução no capítulo 4. Já o capítulo 5 trata da aplicação de simulações de ensaios de dissolução para o desenvolvimento de formulações de liberação prolongada de doxazosina. As simulações das curvas plasmáticas, assim como a CIVIV, obtidas com o auxílio do programa GastroPlusTM, além dos ensaios de dissolução intrínsica e os perfis de dissolução obtidos por meio do uso do programa DDDPlusTM apresentaram-se como ferramentas de grande aplicação na previsão de características biofarmacêuticas sobre os fármacos e formulações, permitindo redução de tempo e custo com trabalho experimental em laboratório.

O estudo dos efeitos da heterogeneidade de tecidos nas doses administradas em radioterapia tem sido alvo de diversos trabalhos. A maioria deles mostra medidas \"in vitro\" em fantomas homogêneos nos quais são introduzidos materiais que simulam a presença de ossos ou de cavidades de ar. No presente trabalho optou-se por um estudo \"in vivo\" para o qual utilizou-se como instrumento de medida a dosimetria termoluminescente, bastante apropriada por motivos bem conhecidos. Foram escolhidos oito pacientes com tumor na região pélvica, tratados com raios x do Acelerador Linear de 4 MeV do Instituto de Radioterapia Osvaldo Cruz. A relação entre as doses medidas na entrada e na saída do feixe quando comparada ao valor previsto para fantomas homogêneos, indica a influência da heterogeneidade na distribuição da dose. A análise das medidas efetuadas nos oito pacientes, mostrou que a dose administrada ao centro do tumor pode ser 8% menor do que aquela planejada sem levar-se em conta a referida heterogeneidade. / Several authors investigated the effect of the heterogeneity of tissue in the dose distribution in a radiation therapy. Practically all of them carried out \"in vitro\" measurements using a solid body immersed in a water phantom, in order to simulate the inhomogeneity, such as bone, air cavity, etc. In the present work, \"in vivo\" measurements were performed utilizing thermoluminescent dosimeters, whose appropriateness and convenience are well known. Eight patients at Instituto de Radioterapia Oswaldo Cruz were selected, that were under irradiation treatments in their pelvic region. The ratio between body entry radiation dose and the corresponding exit dose, when compared to the same ratio for a homogeneous phantom, gives the influence of the heterogeneity of the tissue the radiation crosses. The results found in those eight patients have shown that \"in vivo\" measurements present a ratio about 8% smaller than in homogenous phantom case.

Le cortex moteur primaire et le striatum permettent la planification et la sélection de mouvements. La dopamine régule l'activité des neurones dans ces deux structures. La perte des neurones à dopamine projetant de la substance noire compacte vers le striatum est à l'origine de troubles moteurs observés dans la maladie de Parkinson. Nous avons caractérisé le contrôle par la dopamine des neurones du cortex moteur primaire chez la souris et avons démontré que les fibres dopaminergiques innervent préférentiellement la représentation des membres antérieurs dans les couches corticales profondes. Nous avons montré que la dopamine module localement l’activité électrophysiologique des neurones cortico-striataux via les récepteurs D2. Ces résultats montrent que la dopamine peut exercer un contrôle direct sur la motricité au niveau des neurones du cortex moteur primaire. Nous avons par la suite déterminé le potentiel des thérapies cellulaires dans un modèle animal de la maladie de Parkinson. Les approches actuelles privilégient la greffe ectopique de neurones à dopamine dans la région cible, le striatum. Nous avons choisi une approche alternative consistant à pratiquer la greffe au niveau de la région lésée, la substance noire compacte. Nous avons montré chez la souris que la lésion des neurones dopaminergiques altère les propriétés électrophysiologiques des neurones du striatum et que la greffe homotopique de neurones entraîne une meilleure récupération de ces caractéristiques électrophysiologiques que la greffe ectopique dans le striatum.Ces résultats ouvrent des perspectives d'étude des effets de la greffe homotopique sur l'activité des autres structures contrôlant la motricité. / Primary motor cortex and striatum are involved in movement planification and selection. Dopamine regulates the neuronal activity of these two structures. The motor impairments observed in Parkinson's disease originates from the loss of dopamine neurons projecting from the substantia nigra pars compacta to the striatum.We characterized the dopaminergic control of the neurons of primary motor cortex in mice and we demonstrated that dopaminergic fibers preferentially innervate the forelimb representation map in the deep cortical layers. Furthermore, we demonstrated that dopamine locally modulates the electrophysiological activity of the cortico-striatal neurons through D2 receptors. These results show that dopamine can directly control motor function by influencing neuronal activity in primary motor cortex.Thereafter, we determined the potential of cell replacement therapies in an animal model of Parkinson's disease. In most studies, the transplanted dopamine neurons have been placed within the striatum. We have chosen an alternative approach by grafting neurons into the lesioned nucleus, substantia nigra. We showed in mice that the lesion of dopaminergic neurons impaired the electrophysiological properties of the striatal neurons. Whereas these properties are not fully restored with an intra-striatal transplant, all the electrophysiological characteristics are recovered with an intra-nigral graft. This result opens new perspectives to study the homotopic graft effects on the activity of the other structures controlling motor function.

A alta variabilidade na resposta aos tratamentos superovulatórios e na produção de embriões tem sido relacionada a causas multifatoriais. Além disso, são restritas as informações que relacionam fatores correlatos ao embrião à concepção. Dessa forma, o objetivo do presente estudo foi avaliar a influência de variáveis relacionadas às doadoras (categoria animal e época do ano de produção do embrião) na eficiência de programas de superovulação (SOV), assim como na eficiência reprodutiva de receptoras em programas de transferência de embrião. Foram avaliados dados de 1.562 protocolos de SOV (609 em vacas em lactação e 953 em novilhas da raça Holandesa) e 4.076 transferências de embrião (receptoras Holandesas em lactação). A taxa de SOV (número de doadoras com dois ou mais CL) foi semelhante entre novilhas e vacas em lactação (89,7 vs. 91,9%, respectivamente; P = 0,26). A época do ano, também, não influenciou a taxa de SOV (época quente = 89,3% vs. época não quente = 92,2%; P = 0,09). Doadoras lactantes apresentaram maior número de CL (10,6 ± 0,6 vs. 7,5 ± 0,4; P < 0,0001), de estruturas recuperadas (7,6 ± 0,6 vs. 4,6 ± 0,4; P < 0,0001) e taxa de recuperação (77,6 vs. 58,7%; P < 0,0001) que novilhas. Doadoras superovuladas na época quente apresentaram menor número de CL (8,3 ± 0,5 vs. 9,7 ± 0,5; P = 0,03), de estruturas recuperadas (5,3 ± 0,5 vs. 6,7 ± 0,5; P = 0,04) e taxa de recuperação (65,7 vs. 72,3 %; P = 0,007) que doadoras superovuladas na época não quente. A taxa de fertilização (47,9 vs. 82,4%; P < 0,0001), de embriões viáveis (31,5 vs. 67,4%; P < 0,0001) e embriões viáveis grau I e II (15,4 vs. 42,1%; P < 0,0001) foram inferiores em doadoras lactantes. Semelhantemente, inferiores taxas de embriões viáveis (44,6 vs. 54,0%; P = 0,002) e embriões viáveis grau I e II (21,4 vs. 32,8%; P < 0,0001) foram verificadas durante a época quente do ano. No entanto, a queda na taxa de embriões grau I e II durante a época quente foi mais acentuada em doadoras lactantes (21,7 e 10,7%), quando comparada à observada em novilhas (46,2 e 38,1%; P = 0,02). Apesar da época do ano não ter influenciado a taxa de fertilização (época quente = 64,9 vs. época não quente = 70,0%; P = 0,07), foi observado relação negativa com a temperatura máxima média durante os 15 dias do protocolo de SOV (P = 0,006). A taxa de embriões viáveis também se mostrou negativamente relacionada com a temperatura máxima durante os 15 dias do protocolo de SOV (P = 0,03). O número de embriões viáveis foi semelhante entre as categorias (novilhas = 3,8 ± 0,3 vs. vacas em lactação = 3,3 ± 0,4; P = 0,16), porém foi inferior quando produzido durante a época quente (2,8 ± 0,3 vs. 4,4 ± 0,4; P = 0,03). A taxa de concepção dos embriões oriundos de doadoras em lactação foi maior aos 31 (36,0 vs. 30,7%; P = 0,001) e aos 45 dias de gestação (28,3 vs. 23,1%; P = 0,001). No entanto, a categoria da doadora não influenciou a perda gestacional (novilhas = 18,2% vs. vacas em lactação = 16,6%; P = 0,49). Ainda, as taxas de concepção aos 31 dias (30,5 vs. 31,7%: P = 0,47) e aos 45 dias (25,3 vs. 25,1%; P = 0,94) e perda gestacional (17,4 vs. 22,1%; P = 0,07) das receptoras não diferiram conforme a época de produção dos embriões, época quente e não quente, respectivamente. Também não foi verificado diferença nas taxas de concepção aos 31 dias (31,5 vs. 30,5%: P = 0,66) e aos 45 dias (25,1 vs. 25,4%; P = 0,89) e perda gestacional (18,4 vs. 16,5%; P = 0,49) das receptoras conforme a época de transferência dos embriões, época quente e não quente, respectivamente. Porém, a taxa de concepção aos 31 (P = 0,02) e aos 45 dias (P = 0,02) tiveram relação negativa com a média da temperatura máxima entre o estro e a TE. Em conclusão, apesar das doadoras em lactação apresentarem maior resposta superovulatória, as novilhas apresentaram semelhante número de embriões viáveis, porém maior taxa de embriões viáveis. A resposta ao tratamento superovulatório e a produção de embriões foram inferiores durante a época quente do ano. Embriões originados de doadoras Holandesas lactantes apresentaram maior taxa de concepção aos 31 e aos 45 dias de gestação do que embriões originados de novilhas. Apesar da época do ano não influenciar na taxa de concepção e na perda gestacional, verificou-se relação negativa da temperatura máxima entre o estro e a TE na taxa de concepção. / The high variability in the response to superovulatory treatments and embryo production has been related to multifactorial causes. Furthermore, there are restricted data that correlates factors related to the embryo to the conception. Thus, the objective of the present study was to evaluate the influence of variables related to donors (animal type and season of embryo production) in the efficiency of superovulation (SOV) programs and reproductive performance of dairy cows submitted to embryo transfer. We evaluated data from 1,562 SOV protocols (609 in lactating Holstein cows and 953 in heifers) and 4,076 embryo transfers (lactating Holstein recipient). The SOV rate (number of donor with two or more CL) was similar among lactating cows and heifers (89.7 vs. 91.9%, respectively; P = 0.26). Also, the season did not influence the SOV rate (warm season = 89.3% vs. not warm season = 92.2%; P = 0.09). Lactating donor had higher number of CL (10.6 ± 0.6 vs. 7.5 ± 0.4; P <0.0001), of recovered structures (7.6 ± 0.6 vs. 4.6 ± 0.4; P <0.0001) and recovery rate (77.6 vs. 58.7%; P <0.0001) than heifers. Donors superovulated during warm season had fewer number of CL (8.3 ± 0.5 vs. 9.7 ± 0.5; P = 0.03), of recovered structures (5.3 ± 0.5 vs. 6, 7 ± 0.5; P = 0.04) and recovery rate (65.7 vs. 72.3%; P = 0.007). The fertilization rate (47.9 vs. 82.4%; P <0.0001), viable embryos rate (31.5 vs. 67.4%; P <0.0001) and embryos grade I and II rate (15.4 vs. 42.1%; P <0.0001) were lower in lactating donor. Similarly, lower viable embryos rate (44.6 vs. 54.0%; P = 0.002) and embryos grade I and II rate (21.4 vs. 32.8%; P <0.0001) were observed during the warm season. However, the decrease in the embryos grade I and II rate during the warm period was more pronounced in lactating donors (21.7 and 10.7%) compared to that observed in heifers (46.2 and 38.1%; P = 0.02). Although the season did not influence fertilization rate (warm season = 64.9 vs. not warm season = 70.0%, P = 0.07), it was observed a negative relationship with the average maximum temperature during the 15 days of the SOV protocol (P = 0.006). The viable embryos rate was also negatively correlated with the maximum temperature during the 15 days of the SOV protocol (P = 0.03). The number of viable embryos was similar between categories (heifers = 3.8 ± 0.3 vs. lactating cows = 3.3 ± 0.4; P = 0.16), but was lower when produced during the warm season (2.8 ± 0.3 vs. 4.4 ± 0.4; P = 0.03). The conception rate of embryos from lactating donors was higher at 31 (36.0 vs. 30.7%; P = 0.001) and at 45 days of pregnancy (28.3 vs. 23.1%; P = 0.001). However, the donor category did not influence pregnancy loss (heifers = 18.2% vs. lactating cows = 16.6%; P = 0.49). Still, the recipients conception rate at 31 days (30.5 vs. 31.7%; P = 0.47) and at 45 days of pregnancy (25.3 vs. 25.1%; P = 0.94) and pregnancy loss (17.4 vs. 22.1%; P = 0.07) did not differ according to the embryo production season; warm and not warm season, respectively. Also,no difference was verified in recipients conception rates at 31 days (31.5 vs. 30.5%; P = 0.66) and at 45 days (25.1 vs. 25.4%; P = 0.89) and pregnancy loss (18.4 vs. 16.5%; P = 0.49) according to the embryo transfer season; warm and not warm season, respectively. However, conception rate at 31 (P = 0,02) and 45 days (P = 0.02) had a negative relationship with the average maximum temperature between estrus and TE. In conclusion, despite the lactating donors had higher superovulatory response, heifers presented similar number of viable embryos, but higher viable embryos rate. The superovulatory response and the embryo production were lower during the warm season. Embryos from lactating Holstein donors had higher conception rate at 31 and 45 days of gestation than embryos from heifers. Although the season did not influence the conception and pregnancy loss rates, there was a negative relationship between average maximum temperature during estrus and TE and conception rate.

Le mélanome métastatique est le cancer de la peau le plus agressif. Bien que son taux d’incidence soit inférieur à 1%, plus de 75% des décès associés à un cancer de la peau lui sont attribués. Au cours des dernières années, de nouvelles stratégies thérapeutiques ont permis d’améliorer la survie des patients. Cependant, des mécanismes de résistance à ces traitements se développent dans la majorité des cas, conduisant à une phase de rechute, et une survie à 5 ans inférieure à 20%. Des modèles d’étude expérimentaux sont nécessaires afin de comprendre les mécanismes impliqués dans l’apparition de ces résistances et développer de nouvelles stratégies thérapeutiques. Différents modèles in vitro sont actuellement utilisés pour le développement de drogues anti-tumorales, tels que celui du sphéroïde. Bien qu’il permette de reproduire l’organisation tridimensionnelle d’une tumeur, l’absence de microenvironnement tumoral empêche l’étude des interactions entre les cellules tumorales et celui-ci alors que ces facteurs jouent un rôle primordial dans la croissance tumorale et le développement de métastases. Dans ce contexte, mes travaux ont porté sur le développement et la caractérisation d’un modèle ex vivo de mélanome humain complet permettant l’étude de l’évolution d’une tumeur dans le tissu sain et l’évaluation de composés pharmacologiques. Les travaux réalisés ont tout d’abord conduit au développement d’un modèle de cancer cutané basé sur la combinaison d’un modèle de sphéroïde de lignée cellulaire de mélanome humain et du modèle de peau humaine ex vivo NativeSkin®, développé par la société Genoskin. Une procédure a été développée et validée pour permettre l’implantation reproductible d’un sphéroïde dans le derme des explants de peau. Parallèlement, j’ai développé une approche d’imagerie in situ par microscopie à feuille de lumière après transparisation des modèles. J’ai également développé une stratégie d’analyse d’images permettant la caractérisation quantitative de l'évolution du sphéroïde implanté en 3 dimensions et de suivre la dispersion des cellules du tumorales au sein de l’explant de peau. La caractérisation histologique du modèle implanté a révélé de façon très inattendue une perte progressive de l’intégrité du sphéroïde après implantation, associée à une diminution rapide de la prolifération des cellules le constituant et l’apoptose massive des cellules situées à sa périphérie. Ce phénomène a été observé de façon similaire lors de l’implantation de sphéroïdes produits à partir de différents types cellulaires. Afin de comprendre ces résultats, j’ai étudié l’implication potentielle de différents paramètres dans l’induction de la mortalité cellulaire observée tels que les conditions d’implantation, les facteurs synthétisés par le modèle et la contrainte mécanique exercée par le derme. Les résultats obtenus suggèrent que les facteurs sécrétés par les modèles après implantation du sphéroïde ont un effet antiprolifératif sur les sphéroïdes de mélanome et qu’ils induisent la mortalité des cellules situées à sa périphérie. Par ailleurs, l’application d’une contrainte mécanique extérieure sur les sphéroïdes de mélanome entraîne la perte de la cohésion de leur structure. Enfin, l’implantation de sphéroïdes dans le derme de biopsies de peau préalablement desséchées, induisant une perte de la viabilité cellulaire, a conduit à des résultats opposés à ceux observés avec de la peau normale : la structure des sphéroïdes reste cohésive et la prolifération des cellules est maintenue en périphérie du sphéroïde sans qu’aucune apoptose massive ne soit observée. L'ensemble de ces travaux semble suggérer que la mortalité du sphéroïde pourrait être, en partie, la conséquence d’une contrainte mécanique exercée par la peau sur le sphéroïde et/ou de facteurs produits par la peau durant sa culture. Ces données ouvrent des perspectives intéressantes dans le domaine de l’ingénierie tissulaire pour l’évaluation pharmacologique de composés thérapeutiques. / Malignant melanoma is the most aggressive form of skin cancer. Although it only occurs in less than 1%, it is responsible for more than 75% of skin cancer-related deaths. Furthermore, melanoma incidence has constantly increased during the last decades. New therapies such as targeted therapy and immunotherapy have emerged over the past years, significantly improving the overall survival rates of patients with advanced melanoma stages. However, resistance to those treatments develops in most cases, leading to relapse with a 5-years survival of those patients under 20%. Experimental models are needed in order to better understand the molecular events underlying these resistance mechanisms, and to develop new therapeutic strategies. MultiCellular Tumor spheroid is an increasingly recognized 3D in vitro model for pharmacological evaluation. Although this model accurately reproduces the 3D architecture, cell-cell interaction and cell heterogeneity found in microtumor in vivo, spheroids lack tumor-microenvironment interactions, which play a key role in tumor growth and metastasis development. In this context, the aim of my project was to develop and characterize a fully ex vivo human melanoma model for the study of tumor growth within the skin and the evaluation of antitumor drugs. Our approach relies on the combination of human melanoma cell lines grown in Multicellular Tumor Spheroids and the NativeSkin® model, an ex vivo human skin model produced by the biotechnology company Genoskin. Hence, I developed and validated a method to reproducibly implant one spheroid into the dermal compartment of skin explants cultured ex vivo. In parallel I have developed in situ imaging strategies based on light-sheet microscopy (SPIM, “Selective Plane Illumination Microscopy”) after optical clearing of the implanted skin biopsies. I also developed analytic methods to allow for the quantitative characterization of the spheroids evolution in 3 dimensions as well as tumor cells dispersal within the dermis of skin explants. Histological characterization of the implanted models over time revealed a progressive loss of the spheroids integrity after implantation associated with a rapid decrease in cell proliferation and massive apoptosis of the cells located in the peripheral layers. These results were shared by implanted spheroids made from different cell types. Further experiments were conducted in order to better understand these results and evaluate the impact of different parameters on the implanted microtumors viability such as the implantation procedure conditions, factors synthesized by the model after spheroid implantation and external mechanical stress. Results suggest that factors produced by the implanted models have an antiproliferative effect on melanoma spheroids and induce mortality in the peripheral layers of the spheroids. Moreover, results show that mechanical stress applied on melanoma spheroids induces loss of their cohesion. Finally, implantation of spheroids within the dermis of previously dessicated biopsies for 7 days, causing loss of skin cells viability, led to opposite results than in normal skin: spheroids maintain both a cohesive structure and proliferation in the peripheral cells without any massive apoptosis. Overall, this work led to the validation of a methodology to reproducibly implant spheroids into an ex vivo skin explant and the setup of an optical clearing technique necessary for in situ imaging of the implanted spheroid. Histological characterization unexpectedly revealed spheroids cells death following their implantation. Results suggest that this mortality could be partly related to mechanical stress exerted on the spheroids by the skin and/or by factors produced by the skin during culture. These data open new perspectives in the research field of tissue engineering for antitumoral pharmacology.

<p>Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression <i>in vivo</i>, i.e. to accomplish <i>in vivo</i> hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. </p><p>This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced <i>in vivo</i> imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.</p><p>The present study aimed to investigate ⁷⁶Br- and ⁶⁸Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-<i>O</i>-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of <i>in vivo</i> hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.</p>

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.

Lo scopo del presente lavoro è stato quello di esaminare il potenziale di idrolisi e degradabilità dell’amido dei cereali e co-prodotti e di valutare gli aspetti collegati. L’approccio utilizzato comprende sia test in vitro che in vivo. In particolare: - Una serie d’indagini in vitro ha fornito dati sul potenziale di degradabilità enzimatica dell’amido dei cereali e co-prodotti considerando sia l’influenza della struttura dell’amido, del tipo di cereale, del rapporto amilosio:amilopectina, del processo termico, dell’insilamento, dello stadio di maturazione e delle pratiche agronomiche. - Una valutazione funzionale basata sul calcolo del potenziale di degradabilità enzimatica dell’amido tramite la stima dell’indice glicemico in vitro, combinata con un approccio matematico di modellizzazione dei dati ottenuti, è stata proposta per fornire un metodo efficace di screening dei materiali amidacei utilizzati comunamente nella formulazione per diete destinate ai suini. - Un esperimento in vitro è stato condotto per investigare la risposta glicemica post-prandiale in suini alimentati con diete che differivano nel potenziale di idrolisi enzimatica. Infine, considerando la crescente attenzione dei nutrizionisti sulla matrice proteica dei cereali (con particolare importanza data alle prolammine), i metodi comunemente utilizzati per le tecniche di estrazione delle prolammine sono stati confrontati e ri-investigati. / In the present work, it has been attempted to examine the variability in starch hydrolysis and digestion from cereal grains and co-products and to study aspects related, in order to give a better understand of the science behind the differences in grains/feeds starch digestion potential. The general approach was to use a combination of in vitro and in vivo techniques. In particular: - A series of in vitro investigations has provided data on the degradation potential of starch from cereal grains and co-products considering the influence of starch structure, cereal type, amylose-amylopectin ratio, heat processing, ensiling, stage of maturity and of agricultural practices. - A laboratory-based functional evaluation of starch value of cereal grains based on a predicted glycemic index approach, combined with a mathematical first-order exponential model, has been proposed in order to allow an efficient screening of starchy-materials entering in the pig diet formulation. - An in vivo experiment has been undertaken to investigate the postprandial plasma glucose response in pigs fed diets containing starch with a wide range in in vitro digestion patterns. Lastly, taking into account the increasing attention of swine nutritionists on prolamin proteins, the most commonly used prolamin extraction procedures were compared and reinvestigated, in order to get useful information for routine lab analysis.