Raman microscopy (RM) is a vibrational spectroscopic technique capable of obtaining sensitive measurements of molecular composition, structure, and dynamics from a very small sample volume (~1 µm). In this work, a RM system was developed for future applications in cellular radiobiology, the study of the effects of ionizing radiation on cells and tissues, with particular emphasis on the capability to investigate the internal molecular composition of single cells (10-50 µm in diameter). The performance of the RM system was evaluated by imaging 5 µm diameter polystyrene microbeads dispersed on a silicon substrate. This analysis has shown that RM of single cells is optimized for this system when using a 100x microscope objective and a 50 µm confocal collection aperture. Quantitative measurements of the spatial, confocal, and spectral resolution of the RM system have been obtained using metal nanostructures deposited on a flat silicon substrate. Furthermore, a spectral investigation of several substrate materials was successful in identifying low-fluorescence quartz as a suitable substrate for RM analysis of single cells. Protocols have been developed for culturing and preparing two human tumor cell lines, A549 (lung) and DU145 (prostate), for RM analysis, and a spectroscopic study of these two cell lines was performed. Spectra obtained from within cell nuclei yielded detectable Raman signatures from all four types of biomolecules found in a human cell: proteins, lipids, carbohydrates, and nucleic acids. Furthermore, Raman profiles and 2D maps of protein and DNA distributions within single cells have been obtained with micron-scale spatial resolution. It was also found that the intensity of Raman scattering is highly dependent on the concentration of dense nuclear material at the point of Raman collection. RM shows promise for studying the interactions of ionizing radiation with single cells, and this work has been successful in providing a foundation for the development of future radiobiological RM experiments.

Le traitement du lymphome B non hodgkinien (NHL) est généralement basé sur la combinaison d’un anticorps monoclonal anti-CD20, le rituximab, et de la chimiothérapie. Cependant, de nombreux patients deviennent réfractaires au ciblage du récepteur CD20. Dans cette thèse, l’effet d’un nouvel anticorps monoclonal anti-CD37 conjugué au Luthétium-177 (177Lu-lilotomab, Betalutin®) est étudié dans des modèles précliniques de lymphome non hodgkinien et comparé au rituximab radiomarqué au luthétium-177 (177Lu-rituximab). Nous avons développé une approche radiobiologique qui distingue les effets cytotoxiques dus à l’anticorps monoclonal et dus aux rayonnements ionisants dans des lignées cellulaires de lymphome humain. Cette méthode a permis de montrer qu’in vitro, le rituximab et le 177Lu-rituximab étaient plus cytotoxiques que le lilotomab et le 177Lu-lilotomab dans la lignée cellulaire radiorésistante Ramos (modèle du lymphome de Burkitt). Inversement, le 177Lu-lilotomab et le 177Lu-rituximab ont montré la même cytotoxicité dans la lignée cellulaire radiosensible DOHH2 (modèle de lymphome folliculaire transformé). Leur cytotoxicité était plus faible dans la lignée cellulaire Rec-1 (modèle du lymphome du manteau) que dans les cellules DOHH2. Ces résultats ont été confirmés in vivo sur des souris traitées par injection intraveineuse après xénogreffe sous-cutanée de cellules Ramos ou DOHH2. Le 177Lu-lilotomab et le 177Lu-rituximab ont montré la même efficacité thérapeutique chez les souris xénogreffées avec les cellules radiosensibles DOHH2, alors que le lilotomab non radiomarqué était moins efficace que le rituximab. Inversement, chez les souris xénogreffées avec les cellules radiorésistantes Ramos, la plus faible efficacité du 177Lu-lilotomab comparé au 177Lu-rituximab peut seulement être compensée par une augmentation de dose absorbée à la tumeur par le 177Lu-lilotomab. Mécanistiquement, la réponse cellulaire des tumeurs aux radiations dépend de la réponse apoptotique des cellules et de la réduction de l’arrêt en G2/M du cycle cellulaire via les phosphorylations médiées par WEE-1 et MYT-1 de la kinase dépendante des cyclines-1 (CDK1) sur la tyrosine 15 et thréonine 14. Ces résultats indiquent que l’interaction synergistique entre les effets cytotoxiciques du 177Lu et du lilotomab dans les tumeurs montrant une réduction des niveaux de phosphorylations de CDK1 peut compenser le manque d’efficacité thérapeutique du lilotomab comparé au rituximab. / Currently, B-cell Non-Hodgkin Lymphoma (NHL) treatment relies on the anti-CD20 antibody rituximab and chemotherapy. However, some patients become refractory to this therapy. Here, the effect of the novel anti-CD37 antibody-radionuclide conjugate 177Lu-lilotomab (Betalutin®) was investigated in NHL preclinical models and compared to 177Lu-labeled rituximab (anti-CD20 antibody). We developed a radiobiological approach that discriminates between the cytotoxic effects of unlabeled antibodies and of radiation in human lymphoma cell lines. This method allowed showing that in vitro, rituximab and 177Lu-rituximab were more cytotoxic than lilotomab and 177Lu-lilotomab in the radioresistant Ramos Burkitt’s lymphoma cell line. Conversely, 177Lu-rituximab and 177Lu-lilotomab had similar efficacy in the radiosensitive follicular lymphoma DOHH2 cell line. Their cytotoxicity was lower in mantle cell lymphoma Rec-1 cells that are less radiosensitive than DOHH2 cells. These results were confirmed in vivo in mice treated by intravenous injection of these antibodies after subcutaneous xenografts of Ramos or DOHH2 cells. 177Lu-lilotomab and 177Lu-rituximab showed the same therapeutic efficacy in mice xenografted with radiosensitive DOHH2 cells, although unlabeled lilotomab was less efficient than rituximab. Conversely, in mice xenografted with radioresistant Ramos cells, the lower efficacy of 177Lu-lilotomab compared with 177Lu-rituximab could only be compensated by increasing 177Lu-lilotomab tumor absorbed dose. Mechanistically, the tumor cell response to radiation depended on the cell apoptotic response and reduction of G2/M cell cycle arrest through WEE-1 and MYT1-mediated phosphorylation of cyclin-dependent kinase-1 (CDK1) at tyrosine 15 and threonine 14. These results indicate that the synergistic interaction between 177Lu irradiation and lilotomab cytotoxic effects in tumors with reduced CDK1 phosphorylation levels can correct the lower therapeutic efficacy of lilotomab compared with rituximab.

Made available in DSpace on 2014-10-09T12:53:16Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:31Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Orientador: Frab Norberto Boscolo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-22T03:00:44Z (GMT). No. of bitstreams: 1 Almeida_SolangeMariade_D.pdf: 5623908 bytes, checksum: b26c68566d3a5cc890cf18e225a7c0c1 (MD5) Previous issue date: 1997 / Resumo: O presente trabalho teve como finalidade estudar o efeito de baixas doses de radiação de elétrons no processo de reparação tecidual em ratos. Para tanto, os animais sofreram um procedimento cirúrgico, onde foi produzida uma ferida retangular, medindo 2,3 cm por 1,4 cm, na sua região dorsal anterior. No momento da irradiação, as feridas produzidas foram protegidas, sendo irradiada somente uma região corresponde a 1,0 cm lateralmente à cada borda da ferida, com todo o restante do corpo do animal também protegido. A irradiação foi realizada para um grupo de animais, imediatamente após a abertura da ferida. O outro grupo sofreu a irradiação 3 dias após esse procedimento. O processo de reparação tecidual foi estudado aos 2, 4, 7, 11, 14, 17 e 21 dias após o procedimento cirúrgico para o primeiro grupo, enquanto para o segundo grupo de animais, a reparação tecidual foi avaliada 5, 7, 10, 14, 17, 20 e 24 dias também após a abertura da ferida. Cada grupo irradiado foi comparado com. grupos controles correspondentes, os quais não sofreram irradiação. O processo de reparação tecidual foi avaliado pelos seguintes métodos: coloração pela hematoxilina - eosina, que possibilitou avaliar a mortologia do tecido de granulação; reação histoquímica de metacromasia pelo azul de toluidina pH 4, podendo assim ser avaliada a síntese de glicosaminoglicanas e por fim, impregnação argêntica, onde foi observada a síntese de colágeno, através da microscopia de polarização (birrefringência). Os resultados obtidos mostraram que 1,0 Gy de radiação de elétrons com um feixe de 6 MéV, usou um retardo no processo de reparação tecidual, quando aplicado imediatamente e 3 dias após a abertura da ferida, sendo que quando comparados os dois grupos irradiados, para os dias 7, 14 e 17 , o efeito na reparação tecidual foi mais acentuado no grupo que sofreu irradiação 3 dias após a abertura da ferida / Abstract: The present search had the purpose to study the low dose electron irrradiation effect in the process of tissue repair in rats. In such a way, the animais were submitted to a surgical procedure, in which a rectangular wound was performed, measuring 2.3cm X 1.4cm on the fore dorsal area. At the moment of irradiation, the wounds were protected so that only an area near 1.0cm laterally to each b9rder of the wound was i rrad iated , being protected ali the rest ofthe animal body. The irradiation was performed in one group of animais immediately after the wounding procedure. The other group was irradiated three days after wounding. The process of tissue repair was studied at 2, 4, 7, 11, 14, 17 and 21 days after the surgical procedure on the first group, while for the other group of animais, tissue repair was evaluated at 5, 7, 10, 14, 17, 20 and 24 days, also after wounding. Each irradiated group was compared to corresponding control groups, which did not were submited irradiation. The tissue repair process was evaluated by the following methods: staining by haematoxylin-eosin in order to evaluating granulation tissue morphology; histochemical reaction of metachromasia by toluidin pH 4.0, so that it was possible to evaluate the synthesis of glucosaminglucans and at last, the silver impregnation, in which it was studied the collagen synthesis bymeans of polarizing microscopy. The results obtained showed that 1.0 Gy of electron irradiation with a 6 MeV beam caused a delay in the process of tissue repair, when applied immediately after and at three days after wounding. The comparison of both irradiated groups at days 7, 14 and 17, have showed that the effect on tissue repair was stronger on the group that received irradiation 3 days after wounding / Doutorado / Radiologia / Doutor em Odontologia

Made available in DSpace on 2014-10-09T12:53:16Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:31Z (GMT). No. of bitstreams: 0 / Neste trabalho, medidas microdosimétricas foram realizadas usando um contador proporcional equivalente à tecido - TEPC. com uma cavidade esférica de diâmetro de 1.27 cm. O TEPC foi preenchido com gás propane puro, C3HS, à pressão de 5,6 kPa (42 Torr), que é equivalente a 1,3 μm em diâmetro de unidade de densidade do tecido. O instrumento de medida microdosimétrica foi irradiado com radiação de nêutrons rápidos do reator de pequisa do Nuclear Science Center da Texas A&M University, em College Station,-Texas. Os feixes de nêutrons rápidos foram emitidos com três diferentes valores de potência. 0,5, 1,0, e 2,0 kVV, durante 1 hora para alto ganho e o mesmo tempo para baixo ganho, totalizando 2 horas para cada potência, com 0,0083 Gy/min de taxa de dose. O neutron foi filtrado usando o sistema de irradiação de néutrons rápidos fortemente nitrados (FNIS). do Nuclear Science Center, para obter uma redução da contaminação da radiação de neutron por radiação gama e assim obter espectros microdosimetricos de neutrons como, distribuição de freqüência de energia lineal e distribuição de dose de energia lineal, com boa precisão, e outras grandezas como, freqüência média de energia lineal, dose média de energia lineal, dose absorvida, dose equivalente e fator de qualidade médio de neutron rápido. Os resultados obtidos foram satisfatórios, com os espectros microdosimetricos de nêutrons mostrando uma contaminação de radiação gama abaixo de 5 %, especialmente para distribuição de dose de energia lineal. Os resultados obtidos neste trabalho foram comparados com outros da literatura científica, que usaram outros procedimentos para a redução da contaminação do neutron por radiação gama. estando em concordância com eles. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Doctorat en Sciences de la santé publique / info:eu-repo/semantics/nonPublished

Les faisceaux de photons produits par les accélérateurs d'électrons linéaires médicaux sont plats, grâce à un cône égalisateur. Les technologies ont évolué et la présence d'un cône n'est plus indispensable. On parle alors de faisceaux FFF (flattening filter free). Les faisceaux FFF présentent des débits de dose plus élevés, des profils de dose hétérogènes, des spectres énergétiques différents et une diminution de la dose hors-champ. Cette thèse a eu pour but d'étudier les caractéristiques des faisceaux FFF, ainsi que l'impact de leur utilisation thérapeutique. Plusieurs thématiques ont été. Des expériences d'irradiation in vitro ont tout d'abord permis de s'assurer que les débits de dose FFF n'ont pas d'impact radiobiologique sur la réponse des cellules irradiées. Une large revue de la littérature a permis de corroborer ces résultats. Afin de maitriser les caractéristiques physiques des faisceaux FFF, des mesures ont été faites avec différents détecteurs. Les effets du spectre et du débit de dose sur la calibration en dose ont aussi été étudiés. Les faisceaux FFF ont été modélisés dans deux TPS. Les modèles ont été comparés entre les deux types de faisceaux et entre les deux TPS. La mise en place des traitements stéréotaxiques a aussi été l'occasion d'appréhender la dosimétrie des petits faisceaux. Nous avons étudié des cas VMAT de cancer de la prostate et des cas de stéréotaxies 3D de tumeurs pulmonaires. La comparaison donne un avantage aux faisceaux FFF. La maitrise de la physique et de la biologie des haut débits a permis de débuter les traitements FFF à l'IPC. Des études comparatives nous permettent aujourd'hui d'adapter leur utilisation au cas par cas. / In medical linear electron accelerators, photon beams profiles are homogenised using flattening filters. Technologies have evolved and the presence of this filter is no longer necessary. Flattening filter free (FFF) beams exhibit higher dose rates, heterogeneous dose profiles, modified energy spectra and lower out-of-field dose. This PhD aimed at studying the characteristics of unflattened beams, as well as their impact in clinical utilization. Several subjects were thoroughly investigated: radiobiology, dosimetry, quality controls, modelling and treatment planning. In vitro experiments ensured that the high dose-rate of FFF beams had not a radiobiological impact. A wide review of the literature was conducted to corroborate these results. In order to understand thoroughly the characteristics of FFF beams, measurements were conducted using several detectors. The effect of the spectra and dose rates of unflattened beams on dose calibration were also studied. FFF beams were modeled in two TPSs. The methods, results and model parameters have been compared between the available beam qualities as well as between both TPSs. Furthermore, the implementation of stereotactic treatments technique was the occasion to investigate small beam dosimetry. Prostate cancer cases treated with VMAT and pulmonary tumors treated with stereotactic 3D beams were also studied. The comparison of dose distributions and treatment metrics give advantage to FFF beams. Mastering physical and biological aspects of flattening filter free beams allowed the IPC to start FFF treatments. Comparative studies have since resulted in a deeper understanding on the pertinent use of these beams.

Orientador: Mario Antonio Bernal Rodriguez / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-30T22:42:12Z (GMT). No. of bitstreams: 1 TelloCajiao_JohnJames_M.pdf: 2614936 bytes, checksum: e5bdfc91b42434b003cad0b5fa850afb (MD5) Previous issue date: 2016 / Resumo: O objetivo deste trabalho é estudar a influência da conformação do DNA na probabilidade de dano direto produzido por partículas ionizantes. Além disso, os fundamentos mecanicísticos do modelo Linear-Quadrático são investigadas através de um modelo biofísico desenvolvido neste trabalho, baseado na TADR (Teoria da Ação Dual da Radiação). Para este fim, três modelos geométricos do material genético foram construídos. Os modelos têm resolução atomística e levam em conta ? 10^9 pares de base (bps) nas configurações A,B e Z do DNA. A partir de um único bp, os diferentes níveis organizacionais no interior do núcleo da célula foram criados por meio de transformações lineares. Em seguida, o código Monte Carlo (MC) GEANT4-DNA foi usado para simular o transporte de prótons de 0.5, 1, 5, 7 e 10 MeV assim como de partículas alfa de 2, 5, 7 e 10M eV . O número de partículas em cada caso é de tal modo que as doses absorvidas estão entre 0.5 ? 16Gy. Os três modelos foram consistentes com as dimensões das estruturas reais. Em particular, os modelos foram compatíveis com a exigência de que o diâmetro da cromatina seja de 30 nm, bem como com os volumes bp reportados em outros trabalhos. Os rendimentos tanto das quebras totais quanto das quebras duplas (TSBY e DSBY) foram obtidos para cada qualidade de radiação. Além disso, a probabilidade de impacto (SHP) definida como a razão entre o volume do DNA e o volume núcleo, foi calculada teoricamente e a partir das simulações. O modelo biofísico em conjunto com as simulações MC forneceu o número de lesões letais (N_LL) em função da dose, para prótons de 0,5 e 10 MeV, e para partículas alfa de 2 e 10 MeV . Os N_LL puderam ser divididos em aqueles criados por uma única trajetória e aqueles originados pela interacção de duas trajetórias. Concluiu-se que o TSBY é praticamente determinada pela SHP e depende fracamente da qualidade de radiação incidente. No entanto, o DSBY mostrou forte dependência tanto da conformação do DNA quanto da qualidade de radiação. Isto é devido à relação entre a capacidade de agrupamento das deposições de energia para uma radiação dada e o empacotamento do DNA. Por outro lado, a análise dos mecanismos de produção de dano, baseada na TADR e testada com o modelo biofísico desenvolvido, mostraram que os efeitos de uma única trajetória (de primeira ordem) dependem linearmente com a dose. Além disso, os efeitos inter-trajetórias seguem um comportamento quadrático com a dose, com um termo linear que influencia o mecanismo de primeira ordem. Isto significa que o comportamento linear-quadrático do N_LL com a dose, tem fundamentos mecanicistas, pelo menos, na primeira fase do dano / Abstract: The aim of this work is to study the influence of the DNA conformation on the probability of direct damage induction by ionizing particles. Also, the mechanistic grounds of the Linear-Quadratic radiobiological model are investigated through the eyes of a home-made biophysical model based on the DRAT (Dual Radiation Action Theory). To this end, three geometrical models of the genetic material were constructed. The models have atomistic resolution and account for ? 10^9 base pairs (bps) in the A-, B- and Z-DNA configurations. Starting from a single bp, the different organizational levels inside the cell nucleus were created by means of linear transformations. Next, the Monte Carlo (MC) code GEANT4-DNA was used to simulate the transport of protons of 0.5, 1, 5, 7 and 10 MeV , and alpha particles of 2, 5, 7 and 10 MeV. The number of particles in each case is such that the absorbed doses range between 0.5 Gy and 16 Gy. The three models proved to be consistent with the dimensions of the real structures. In particular, the models were compatible with the 30 nm chromatin fiber diameter requirement as well as with the bp volumes reported in other works. The Total and Double Strand Break Yields (TSBY and DSBY) were obtained for every radiation quality. Also, the Site-Hit Probability (SHP) defined as the total target to the nucleus volume ratio, was computed theoretically and from the simulations. The biophysical model in conjunction with the MC simulations furnished the number of lethal lesions (N_LL) as a function of dose, for protons of 0.5 and 10 MeV , and for alpha particles of 2 and 10 MeV . The N_LL could be split into those created by a single track and those originated by interaction of two tracks. It is concluded that the TSBY is practically determined by the SHP and depends weakly on the incident radiation quality. Nevertheless, the DSBY showed strong dependence on both the DNA conformation and the radiation quality. This is due to the interplay between the energy deposition clustering capacity of a given radiation and the DNA spatial packing. On the other hand, the analysis of the mechanisms of damage production based on the DRAT and tested with the biophysical model developed, showed that single-track (first order) effects depend linearly on the dose. Moreover, inter-track effects follows a quadratic behavior with the dose, having a linear term that influences the first order mechanism. This means that the Linear-Quadratic behavior of the N_LL with the dose, has mechanistic groundings at least at the first stage of the damage / Mestrado / Física / Mestre em Física / 1370449/2014 / CAPES

O urânio (U) é um elemento tóxico radioativo encontrado na natureza, normalmente presente na água e nos alimentos e acumula-se preferencialmente em ossos. Nestes, a medula óssea constitui o alvo com o maior risco radiobiológico. Foram utilizados 60 ratos wistar recém desmamados, com vinte e dois dias de vida. Destes, trinta e cinco foram tratados com ração suplementada de 50ppm (parte por milhão) de Nitrato de Uranila e vinte e cinco foram mantidos como controle. Os animais tratados foram separados em seis grupos com cinco animais cada e os grupos controle com três animais. Foi feita a eutanásia dos 5 animais de cada grupo alimentado com urânio e 3 animais de cada grupo de controle com intervalo de tempo de 3 e 4 dias para avaliar alterações histopatológicas, hematológicas, na densidade mineral óssea e medir o teor de urânio acumulado em ossos, em função do tempo, utilizando a técnica de registro de traços de fissão SSNTD (Solid State Nuclear Track Detector). Nas avaliações histopatológicas foi observada congestão, fibrose e necrose hepática, degeneração vacuolar e desarranjo cordonal dos hepatócitos. Essas alterações iniciaram-se em animais alimentados durante três dias com ração contendo U e se intensificaram nos animais tratados durante onze dias, sugerindo que tenha ocorrido combinação de efeitos toxicológicos e radiobiológicos. Foi observada degeneração vacuolar, cilindros hialinos, fibrose e necrose nos rins dos animais alimentados com ração suplementada de U, a partir de quatorze dias de alimentação, decorrentes da nefrotoxicidade do Nitrato de Uranila. Foi observado que não ocorre alteração da densidade mineral óssea no curto prazo; porém, os animais tratados durante 21 e 28 dias, ou seja, expostos ao U por período mais longo, tiveram a densidade mineral óssea diminuída. Ocorreu substancial acúmulo de urânio nos ossos, onde foi observado 1,139 ± 0,057 ppm em ossos e 0,705 +- 0,092 ppm em dentes. Os animais dos grupos controle apresentaram teor de urânio praticamente constante no decorrer do estudo. Não foi observada alteração do teor de urânio em ração comercial. / Uranium (U) is a radioactive toxic element found in the environment, naturally present in water and food, with preference for accumulation in bone. In the latter, marrow is the target with the highest radiobiological risk. It was carried out a study with sixty Wistar rats, twenty two days old, starting at the post weaning period. From this total, thirty five animals fed with chow containing Uranyl Nitrate at a concentration of 50 ppm (parts per million) were selected as the treated group, while the remaining twenty five were the control group. Treated animals were divided into six groups with five animals each plus six control groups with three animals each. Five animals of the treated group and three of the control group were sacrificed at intervals of four days to observe histopathologic, hematologic, and bone mineral density (BMD) alterations, as well as to measure the uranium content in bone as function of time, using the Solid State Nuclear Track Detector technique. It was observed congestion, vacuolar degeneration, hepatocytes misalignment, fibrosis and necrosis in liver. These alterations were initiated in treated animals fed for three days with diets containing U and intensified in the animals treated for eleven days, suggesting the occurrence of an intertwining between radiobiological and toxicological effects. It was also observed vacuolar degeneration, hyaline cylinders, fibrosis and necrosis in the kidneys of the treated animals, all initiated after fourteen days of treatment, and these effects were attributed to the nephrotoxic character of the Uranyl Nitrate. It was found out that the BMD was not altered in the short range term of treatment, that is, treatments of twenty-one and twenty-eight days, but appreciably reduced in the long range term. There was substantial accumulation of uranium in bones and teeth, where it was measured concentrations of 1.139 ± 0.057 ppm and 0.705 ± 0.092 ppm, respectively. The uranium concentration in the bones of animals of the control group were low and approximately constant.

L'objectif principal de cette thèse est de développer et optimiser les algorithmes caractérisant les propriétés physiques et biologiques des mini-faisceaux de protons pour la réalisation des traitements avec modulation d'intensité. Un modèle basé sur la superposition et décomposition des mini-faisceaux en faisceaux élémentaires a été utilisé. Un nouveau modèle de description des mini-faisceaux primaires a été développé à partir de la sommation de trois fonctions gaussiennes. Les algorithmes ont été intégrés dans un logiciel de planification de traitement, puis validés expérimentalement et par comparaison avec des simulations Monte Carlo. Des approximations ont été réalisées et validées afin de réduire les temps de calcul en vue d'une utilisation clinique. Dans un deuxième temps, un travail en collaboration avec les équipes de radiobiologie de l'institut Curie a été réalisé afin d'introduire des résultats radiobiologiques dans l'optimisation biologique des plans de traitement. En effet, les faisceaux balayés sont délivrés avec des débits de dose très élevés (de 10 à 100 Gy/s) et de façon discontinue, et l'efficacité biologique des protons est encore relativement méconnue vue la diversité d'utilisation de ces faisceaux : les différents modèles disponibles et notamment leur dépendance avec le transfert d'énergie linéique ont été étudiés. De bons accords (écarts inférieurs à 3 % et 2 mm) ont été obtenus entre calculs et mesures de dose. Un protocole d'expérimentation pour caractériser les effets des hauts débits pulsés a été mis en place et les premiers résultats obtenus sur une lignée cellulaire suggèrent des variations d'efficacité biologique inférieures à 10 %, avec toutefois de larges incertitudes. / The main objective of this thesis is to develop and optimize algorithms for intensity modulated proton therapy, taking into account the physical and biological pencil beam properties. A model based on the summation and fluence weighted division of the pencil beams has been used. A new parameterization of the lateral dose distribution has been developed using a combination of three Gaussian functions. The algorithms have been implemented into a treatment planning system, then experimentally validated and compared with Monte Carlo simulations. Some approximations have been made and validated in order to achieve reasonable calculation times for clinical purposes. In a second phase, a collaboration with Institut Curie radiobiological teams has been started in order to implement radiobiological parameters and results into the optimization loop of the treatment planning process. Indeed, scanned pencil beams are pulsed and delivered at high dose rates (from 10 to 100 Gy/s), and the relative biological efficiency of protons is still relatively unknown given the wide diversity of use of these beams: the different models available and their dependence with linear energy transfers have been studied. A good agreement between dose calculations and measurements (deviations lower than 3 % and 2 mm) has been obtained. An experimental protocol has been set in order to qualify pulsed high dose rate effects and preliminary results obtained on one cell line suggested variations of the biological efficiency up to 10 %, though with large uncertainties.