[ES] A pesar de los nuevos avances en el tratamiento del cáncer de pulmón, su tasa de incidencia y mortalidad siguen en cabeza en todo mundo. Concretamente, el cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón, siendo su supervivencia a 5 años muy reducida. En base a dicho escenario, el objetivo principal de este trabajo es el de caracterizar de manera exhaustiva los exosomas secretados por las células del CPNM. Se sabe que estas microvesículas están involucradas en números procesos celulares, por lo que pueden contener gran cantidad de información acerca de las características moleculares del tumor. Para ello se han empleado cultivos primarios y líneas comerciales crecidas en diferentes condiciones, así como muestras de sangre periférica obtenida de los pacientes con CPNM. Un primer screening llevado a cabo en los exosomas secretados in vitro, ha permitido obtener un gran número de mRNAs y miRNAs relacionados con diferentes procesos biológicos y vías de señalización. Además, algunos genes como FDFT1 y SNAI1 han destacado por su sobreexpresión en exosomas procedentes de las células crecidas en formación de tumoresferas (modelos 3D), las cuales están enriquecidas en población de células madre tumorales. A su vez, otros marcadores presentes en el interior de estas microvesículas, se han mostrado relacionados con dos de los subtipos histológicos más frecuentes: adenocarcinoma (LUAD) y carcinoma escamoso (LUSC). Posteriormente, para validar los hallazgos obtenidos en exosomas, los marcadores más significativos fueron analizados in silico en una cohorte de muestras de tejido, compuesta por 661 pacientes con CPNM (TCGA database). Estos resultados han revelado una asociación entre la expresión del gen SNAI1 y la supervivencia de estos pacientes (OS y RFS p<0.05). Además, los genes XAGE1B, SEPP1 y TTF-1 (previamente determinados en exosomas), mantienen una relación significativa con el grupo de pacientes LUAD; mientras que CABYR, RIOK3 y CAPRIN1 se mantienen sobrexpresados en LUSC (Mann-Whitney test p<0.05). Estos marcadores también se han analizado en una cohorte de 186 pacientes con CPNM procedentes del Hospital General Universitario de Valencia, donde se corroboró la asociación de SNAI1 con la supervivencia de los pacientes en estadios tempranos (RFS en pacientes LUAD, p<0.05), así como la sobreexpresión de CABYR y RIOK3 en pacientes LUSC, y de XAGE1B y TTF-1 en LUAD. Por otra parte, el aislamiento de los exosomas presentes en la sangre periférica de pacientes en estadios avanzados, ha permitido identificar otros marcadores asociados a caracterísiticas clínico-patológicas relevantes. A su vez, el contenido de estas microvesículas ha sido empleado para la detección de mutaciones génicas ligadas al manejo clínico del CPNM. En resumen, los resultados obtenidos en este trabajo ponen de manifiesto el potencial de los exosomas como fuente de biomarcadores para el estudio de las diferentes etapas de desarrollo del CPNM. Estas microvesículas ofrecen una visión completa y en tiempo real, de las características de la enfermedad, pudiendo ser aisladas de forma repetida y mediante técnicas mínimamente invasivas. / [CA] A pesar dels avanços recents en el tractament del càncer de pulmó, les seues taxes d'incidència i mortalitat continuen sent altes a nivell mundial. Concretament, el càncer de pulmó de cèl·lules no petites (CPNM) representa gairebé el 85% de tots els càncers de pulmó, amb una taxa de supervivència a 5 anys molt limitada. Donat aquest escenari, l'objectiu principal d'aquest estudi és caracteritzar de manera exhaustiva els exosomes secretats per les cèl·lules de CPNM. Aquestes microvesícules estan involucrades en nombrosos processos tumorals i poden contenir una gran quantitat d'informació sobre les característiques moleculars de la malaltia. Per aconseguir-ho, es van utilitzar cultius primaris i línies cel·lulars (cultiu en diferents condicions), juntament amb mostres de sang perifèrica obtingudes de pacients amb CPNM. Un cribratge inicial en exosomes secrets in vitro va permetre identificar una quantitat significativa de mARNs i miARNs relacionats amb diversos processos biològics i vies de senyalització. A més, alguns gens com FDFT1 i SNAI1 van destacar per la seua sobreexpressió en exosomes derivats de cèl·lules crescuts en formació de tumorsferes (models 3D), que estan enriquides en poblacions de cèl·lules mare tumorals. A més, s'han trobat marcadors en aquestes microvesícules associats amb dos dels subtipus histològics més comuns: adenocarcinoma (LUAD) i carcinoma escamós (LUSC). Posteriorment, per validar els resultats obtinguts en exosomes, es van analitzar in silico els marcadors més significatius en una cohort de teixit de CPNM de la base de dades TCGA. Aquests resultats van revelar una associació entre l'expressió del gen SNAI1 i la supervivència dels pacients (OS i RFS, p <0,05). A més, l'expressió dels gens XAGE1B, SEPP1 i TTF-1 (prèviament identificats en exosomes) va mantenir una relació significativa amb el grup LUAD, mentre que CABYR, RIOK3 i CAPRIN1 van continuar sobreexpressats en els pacients de LUSC (prova de Mann-Whitney, p <0,05). Aquests marcadors també es van analitzar en una cohort de 186 pacients amb CPNM de l'Hospital General Universitari de València, on es va confirmar l'associació de l'expressió de SNAI1 i la supervivència dels pacients en estadi precoç (RFS en pacients de LUAD, p <0,05), així com la sobreexpressió de CABYR i RIOK3 en pacients de LUSC, i de XAGE1B i TTF-1 en LUAD. D'altra banda, els exosomes presents en mostres de sang de la cohort d'estadis avançats van permetre la identificació d'altres biomarcadors associats a característiques clíniques rellevants dels pacients. A més, la càrrega exosomàtica també es va utilitzar per detectar mutacions genètiques relacionades amb el tractament clínic del CPNM. En resum, els resultats obtinguts en aquesta tesi destaquen el potencial dels exosomes com a font de biomarcadors per a l'estudi de les diferents etapes del desenvolupament del CPNM. Aquestes microvesícules ofereixen una visió completa i en temps real de les característiques moleculars de la malaltia i poden ser obtingudes de manera repetida i amb una mínima invasió. / [EN] Despite recent advancements in lung cancer treatment, its incidence and mortality rates remain high worldwide. Specifically, non-small cell lung cancer (NSCLC) accounts for nearly 85% of all lung cancers, with a 5-year survival rate of 20%. Given this scenario, the primary objective of this study is to comprehensively characterize the exosomes secreted by NSCLC cells. These microvesicles are known to be involved in numerous tumoral processes, potentially containing a wealth of information about the molecular characteristics of the disease. To achieve this, primary cultures and cell lines, along with peripheral blood samples obtained from NSCLC patients were used. An initial screening in exosomes secreted in vitro allowed the identification of a significant number of mRNAs and miRNAs, related to various biological processes and signaling pathways. Moreover, some genes such as FDFT1 and SNAI1 stood out due to their overexpression in exosomes derived from cells grown in tumorspheres formation (3D models), which are enriched in cancer stem cell population. Additionally, markers found within these microvesicles were associated with two of the most common histological subtypes: adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Subsequently, to validate the findings seen in exosomes, the most significant markers were analyzed in silico in an NSCLC tissue cohort from the TCGA database. These results revealed an association between the expression of SNAI1 and patient survival (OS and RFS, p<0.05). Furthermore, XAGE1B, SEPP1, and TTF-1 expression (previously identified in exosomes) maintained a significant relationship with the LUAD group, while CABYR, RIOK3, and CAPRIN1 remained overexpressed in LUSC patients (Mann-Whitney test, p<0.05). These markers were also analyzed in a cohort of 186 NSCLC patients from the University General Hospital of Valencia. The association of SNAI1 expression and the survival of early-stage patients (RFS in LUAD patients, p<0.05) was confirmed, as well as the overexpression of CABYR and RIOK3 in LUSC patients, and of XAGE1B and TTF-1 in LUAD. Furthermore, exosomes present in blood samples of the advanced-stage cohort, allowed the identification of other biomarkers associated with clinically relevant characteristics of the patients. Moreover, exosomal cargo was also used to detect gene mutations related to the clinical management of NSCLC. In summary, the results obtained in this thesis highlight the potential of exosomes as a source of biomarkers for the study of the different stages of NSCLC development. These microvesicles offer a comprehensive and real-time view of the disease's molecular features and can be obtained repeatedly and in a minimally invasive way. / Duréndez Sáez, ME. (2024). Liquid Biopsy in non-small cell lung cancer: exosomes as a tool for the study of biomarkers [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203438

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-06-22T11:39:54Z No. of bitstreams: 0 / Made available in DSpace on 2016-06-22T11:39:54Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:11/21708-6

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-06-22T11:39:54Z No. of bitstreams: 0 / Made available in DSpace on 2016-06-22T11:39:54Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A deficiência de hormônio de crescimento (DGH) é a deficiência mais comum entre os hormônios pituitários. A terapia utilizada atualmente consiste de injeções diárias de hormônio de crescimento humano recombinante (r-hGH), entretanto esta terapia apresenta alguns inconvenientes, como a necessidade de frequentes injeções de r-hGH durante um longo período de vida, dependendo da severidade da deficiência, e o alto custo do hormônio, em razão dos dispendiosos processos de purificação. Uma alternativa ao tratamento padrão seria aquele no qual fossem evitados estes tipos de inconvenientes e o processo de liberação da proteína fosse sustentável, por um longo período e promovesse níveis normais e sustentáveis do fator de crescimento semelhante à insulina I (IGF-I), o principal mediador dos efeitos do GH. Uma alternativa é a terapia gênica in vivo, baseada na administração de DNA plasmidial em diversos órgãos/tecidos, seguida de eletroporação. É considerada uma metodologia bastante promissora e que tem sido alvo de vários estudos para diversos tipos de deficiências sistêmicas. Neste trabalho foram realizadas diversas administrações de um plasmídeo contendo o gene do hormônio de crescimento humano, nos músculos quadríceps exposto ou tibial anterior sem exposição, seguidas de eletroporação, em camundongos anões e imunodeficientes (lit/scid) com 40-80 dias de idade, na tentativa de obter uma correção fenotípica do nanismo, mediante a avaliação de parâmetros de crescimento. A administração deste plasmídeo no músculo tibial anterior, em camundongos com a idade inicial de 40 dias, foi capaz de proporcionar uma normalização dos níveis de mIGF-I, quando comparados aos dos camundongos não-deficientes de GH. Além disso, foram obtidos valores de catch-up dos parâmetros de crescimento longitudinal de 36-77%. Visando uma maior eficiência na expressão de GH, foram construídos plasmídeos parentais, e a partir destes, foram produzidos minicírculos de DNA com os promotores do CMV e Ubiquitina C e com os cDNAs de hGH e mGH. Estes minicírculos de DNA foram transfectados em células HEK 293 e foram até 2 vezes mais eficientes em relação aos plasmídeos convencionais com o promotor do CMV. Estes dados são bastantes promissores e abrem caminho para ensaios mais eficientes, utilizando este tipo de protocolo de terapia gênica para a DGH, visando uma normalização de todos os parâmetros de crescimento. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:11/21708-6

Mast cells contribute to the activities of innate and adaptive branches of the immune system. They participate in pro-inflammatory responses to a wide range of pathogens, such as parasites, bacteria, and other foreign agents. These beneficial properties are in contrast to the contribution of mast cells to certain pathologies, such as asthma, allergy, autoimmune disorders, anaphylaxis, and systemic mastocytosis. Thorough knowledge of mast cell biology in health and disease is critical for the development of new therapeutic approaches. However, molecular mechanisms that control mast cell development and function are still incompletely defined. Our preliminary data indicate that the transcription factor C/EBP is a key player in mast cell biology. Here, using in vitro and in vivo models, we determine how C/EBP regulates the commitment of hematopoietic progenitors towards mast cells, and modulates mast cells function. These efforts provide novel insights to the role of C/EBP in hematopoiesis, and contribute to a better understanding of the mechanisms governing mast cell biology. Key words Mast cells, C/EBP, transcription factors, bone marrow-derived mast cell cultures, mast cell development, Cebpg conditional knockout mice

In 2D cell cultures uptake/release of various metabolic analytes such as glucose, lactate or metabolic by-products like hydrogen peroxide from/to the extracellular environment results in concentration gradients. The magnitude, direction, and time scales of these gradients carries information that is essential for internal cellular processes and/or for communication with neighboring cells. This PhD research work focusses on the design, fabrication and characterization of electrochemical microelectrode arrays (MEAs) optimized to be positioned in commonly used 2D cell culture setups. Importantly, by simultaneously measuring accurate concentration transients and associated gradients/uxes near the cell surface (surface concentration) the capability of the device to quantify kinetic rates and distinguish mechanisms involved in various cellular processes is demonstrated. An in-situ transient calibration technique suitable for amperometric MEAs is developed and the technique is validated by quantitatively measuring dynamic concentration profiles with varying spatial (100-800 µm) and time (s to hrs.) scales set up from an electrically controlled diffusion reaction system. With the proposed MEA design and technique three physiological applications are demonstrated. Firstly, the position able 1D MEA was employed real time to quantitatively measure the hydrogen peroxide scavenging rates from astrocyte vs glioblastoma cell cultures. With the ability to extract to dynamic surface concentration and fluxes, the cell lines were shown to have hydrogen peroxide uptake rates dependent on local surface concentrations. Moreover, the cancerous glioblastoma cells demonstrated an upregulated linear peroxide scavenging mechanism as compared to astrocytes. For the next phase, spatial scales of 1D MEA device along the size and functionalization scheme of the electrodes in the MEA was further modified to selectively sense glucose and lactate to enable extracellular metabolic profiling of cancer vs normal cell lines. Secondly, measurement of glucose concentration profiles demonstrated an increased glucose uptake rate in glioblastoma as compared to astrocytes. Additionally, sigmoidal (allosteric) vs Michaelis - Menten glucose uptake kinetics was observed in glioblastoma vs astrocytes. Moreover, the presence of a glucose sensing mechanism was observed in glioblastoma cells due to the dependence of the glucose uptake rate on initial exposed concentration rather than surface concentration. Finally, simultaneous multi-analyte (glucose and lactate) gradient measurements were performed on genetically modified mouse pancreatic cancer cell lines. While glucose uptake rate was shown to increase with increasing extracellular glucose concentration for one of the cell lines, the lactate release rate was observed to be independent of the initial extracellular glucose dose.

A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar, com o uso de inibidores do ciclo celular, o período do ciclo em que isto ocorre; b) verificar se o aumento da sensibilidade a pH baixo está relacionado com a expansão celular ou com alterações no potencial osmótico da célula; c) examinar o efeito da aplicação de H2O2 ou ascorbato sobre a resposta de células sensíveis a pH baixo; d) testar a hipótese de que a sensibilidade a pH baixo pode ser revertida por meio de um choque hipo-osmótico prévio; e) avaliar o possível papel da modulação oxidativa da parede celular na reversão de sensibilidade das células a pH baixo expostas ao choque hipo-osmótico. A retomada do ciclo celular é necessária para que ocorra a alteração de sensibilidade a pH baixo, pois a remoção de auxina (2,4-D) ou a adição de bloqueadores de canais de K+ impediu ou atrasou, respectivamente, a alteração na sensibilidade a pH baixo. O uso de inibidores do ciclo celular demonstrou que as células de BY-2 se tornam mais sensíveis a pH baixo durante o final da fase G1 mas antes do ponto de checagem da transição G1/S do ciclo celular. A aplicação de H2O2, diminuiu a suscetibilidade das células a pH baixo, ao contrário da aplicação de ascorbato. Foi demonstrado que a aplicação prévia de tratamento hipo-osmótico por 60 min reverteu a sensibilidade de células a pH baixo. A aplicação de inibidores de NAPDH oxidase da membrana plasmática e de peroxidases revelou a participação destas enzimas na reversão de sensibilidade das células a pH baixo, indicando a possibilidade de geração de ROS e de modulação oxidativa da parede. Embora já tenha sido descrito que ocorre uma explosão oxidativa com choque hipo-osmótico, ainda não havia sido demonstrado a conseqüência disto. Este trabalho fornece indícios de que uma explosão oxidativa poderia modificar a parede tornando-a mais resistente e a célula menos suscetível a pH baixo / Soil acidity is a major factor limiting plant growth worldwide. Although aluminum toxicity, which occurs only at low pH, has been extensively studied, little attention has been given to stress caused by low pH. There are marked differences in the sensitivity of cells to low pH which are contingent on the growth and developmental stage of the cells. These differences should be explored to further the understanding of the factors governing sensitivity and tolerance to low pH. In at least some cases, the susceptibility of cells to low pH is related to derangements in the wall of growing cells, which can cause ruptures or bursting of the cells, as has been clearly demonstrated in expanding root hairs. On the other hand, the oxidative metabolism and generation of reactive oxygen species (ROS) can modulate cell wall extensibility by breaking or making bonds within and between cell wall polymers. In tobacco (Nicotiana tabacum) cv. BY-2 cells, there is a sharp increase in sensitivity to low pH at the end of the lag phase of the cell culture, which occurs between 12 and 24 h of subculture. The objectives of this study were: a) determine if the changes in sensitivity to low pH occurred during the restart of the cell cycle and, by employing cell cycle inhibitors, at which points of the cycle does this occur; b) examine if the changes in sensitivity to low pH are related to cell expansion or changes in osmotic potential of the cell; c) examine how the application of H2O2 or ascorbate affects the response of cells to low pH; d) test the hypothesis that sensitivity of cells to low pH can be reverted by the previous application of a hypo-osmotic shock; e) evaluate the possible role of oxidative modulation of the cell wall in hypo-osmotic-induced reversal of the sensitivity of cells to low pH. The restart of the cell cycle was shown to be necessary for the change in sensitivity to low pH occur, since the absence of auxin (2,4-D) or the addition of K+ channel blockers prevented or delayed this change, respectively. The use of cell cycle inhibitors demonstrated that BY-2 cells become sensitive to low pH at the end of G1 but before the G1/S transition restriction point of the cell cycle. Exogenous H2O2, but not ascorbate, reduced the effect of low pH on sensitive cells. Sensitive cells submitted to 60 min hypo-osmotic treatment became insensitive to low pH. This reversal of sensitivity depended on the activity of plasma membrane NADPH oxidase and peroxidase, as evidenced by the use of DPI and SHAM, inhibitors of these enzymes, respectively. This suggests that ROS is generated and that oxidative modifications of the cell wall occur. Although hypo-osmotic treatments have been shown to generate an oxidative burst, its purpose or implication has not yet been shown. This study provides evidence that an oxidative burst might modify and strengthen the cell wall, making cells less susceptible to low pH

No description available.

A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar, com o uso de inibidores do ciclo celular, o período do ciclo em que isto ocorre; b) verificar se o aumento da sensibilidade a pH baixo está relacionado com a expansão celular ou com alterações no potencial osmótico da célula; c) examinar o efeito da aplicação de H2O2 ou ascorbato sobre a resposta de células sensíveis a pH baixo; d) testar a hipótese de que a sensibilidade a pH baixo pode ser revertida por meio de um choque hipo-osmótico prévio; e) avaliar o possível papel da modulação oxidativa da parede celular na reversão de sensibilidade das células a pH baixo expostas ao choque hipo-osmótico. A retomada do ciclo celular é necessária para que ocorra a alteração de sensibilidade a pH baixo, pois a remoção de auxina (2,4-D) ou a adição de bloqueadores de canais de K+ impediu ou atrasou, respectivamente, a alteração na sensibilidade a pH baixo. O uso de inibidores do ciclo celular demonstrou que as células de BY-2 se tornam mais sensíveis a pH baixo durante o final da fase G1 mas antes do ponto de checagem da transição G1/S do ciclo celular. A aplicação de H2O2, diminuiu a suscetibilidade das células a pH baixo, ao contrário da aplicação de ascorbato. Foi demonstrado que a aplicação prévia de tratamento hipo-osmótico por 60 min reverteu a sensibilidade de células a pH baixo. A aplicação de inibidores de NAPDH oxidase da membrana plasmática e de peroxidases revelou a participação destas enzimas na reversão de sensibilidade das células a pH baixo, indicando a possibilidade de geração de ROS e de modulação oxidativa da parede. Embora já tenha sido descrito que ocorre uma explosão oxidativa com choque hipo-osmótico, ainda não havia sido demonstrado a conseqüência disto. Este trabalho fornece indícios de que uma explosão oxidativa poderia modificar a parede tornando-a mais resistente e a célula menos suscetível a pH baixo / Soil acidity is a major factor limiting plant growth worldwide. Although aluminum toxicity, which occurs only at low pH, has been extensively studied, little attention has been given to stress caused by low pH. There are marked differences in the sensitivity of cells to low pH which are contingent on the growth and developmental stage of the cells. These differences should be explored to further the understanding of the factors governing sensitivity and tolerance to low pH. In at least some cases, the susceptibility of cells to low pH is related to derangements in the wall of growing cells, which can cause ruptures or bursting of the cells, as has been clearly demonstrated in expanding root hairs. On the other hand, the oxidative metabolism and generation of reactive oxygen species (ROS) can modulate cell wall extensibility by breaking or making bonds within and between cell wall polymers. In tobacco (Nicotiana tabacum) cv. BY-2 cells, there is a sharp increase in sensitivity to low pH at the end of the lag phase of the cell culture, which occurs between 12 and 24 h of subculture. The objectives of this study were: a) determine if the changes in sensitivity to low pH occurred during the restart of the cell cycle and, by employing cell cycle inhibitors, at which points of the cycle does this occur; b) examine if the changes in sensitivity to low pH are related to cell expansion or changes in osmotic potential of the cell; c) examine how the application of H2O2 or ascorbate affects the response of cells to low pH; d) test the hypothesis that sensitivity of cells to low pH can be reverted by the previous application of a hypo-osmotic shock; e) evaluate the possible role of oxidative modulation of the cell wall in hypo-osmotic-induced reversal of the sensitivity of cells to low pH. The restart of the cell cycle was shown to be necessary for the change in sensitivity to low pH occur, since the absence of auxin (2,4-D) or the addition of K+ channel blockers prevented or delayed this change, respectively. The use of cell cycle inhibitors demonstrated that BY-2 cells become sensitive to low pH at the end of G1 but before the G1/S transition restriction point of the cell cycle. Exogenous H2O2, but not ascorbate, reduced the effect of low pH on sensitive cells. Sensitive cells submitted to 60 min hypo-osmotic treatment became insensitive to low pH. This reversal of sensitivity depended on the activity of plasma membrane NADPH oxidase and peroxidase, as evidenced by the use of DPI and SHAM, inhibitors of these enzymes, respectively. This suggests that ROS is generated and that oxidative modifications of the cell wall occur. Although hypo-osmotic treatments have been shown to generate an oxidative burst, its purpose or implication has not yet been shown. This study provides evidence that an oxidative burst might modify and strengthen the cell wall, making cells less susceptible to low pH

L’objectif de cette thèse était de mettre au point une analyse critique de vecteurs polymères utilisés pour la thérapie photodynamique (PDT) et de faire le lien avec l’efficacité thérapeutique observée. Pour cela, une analyse complète des vecteurs a été réalisée par des techniques classiques comme la diffusion dynamique de la lumière ou la microscopie électronique, mais aussi grâce au fractionnement flux-force, technique peu utilisée jusqu’à présent dans le domaine des auto-assemblages polymères. Dans un deuxième temps, les auto-assemblages ont été utilisés comme vecteurs d’un photosensibilisateur, le Phéophorbide a, et l’efficacité thérapeutique évaluée en travaillant sur culture cellulaire 2D et 3D de lignées HCT116 (cancer du colon) ou FaDu (cancer tête et cou). Différents vecteurs polymères simples ont tout d’abord été examinés, à savoir des micelles ou des polymersomes à base de copolymères diblocs amphiphiles comme le poly(oxyde d’éthylène-b--caprolactone), le poly(oxyde d’éthylène-b-lactide) ou le poly(oxyde d’éthylène-b-styrène). Ceci a permis d’obtenir des vecteurs présentant des tailles et des morphologies variables. Les résultats en PDT ont montré des comportements différents et une meilleure efficacité en 3D pour les systèmes à base de PEO-PDLLA. La technique de fractionnement flux-force asymétrique (AsFlFFF) a particulièrement été utilisée pour ces vecteurs afin de démontrer la pureté des auto-assemblages. Les connaissances acquises dans cette première partie ont permis de caractériser des vecteurs faits à base de mélanges d’auto-assemblages micelles/vésicules. Ceux-ci ont révélé des phénomènes d’antagonisme ou de synergie dans l’efficacité en PDT, démontrant l’existence de processus complexes au niveau de la réponse cellulaire.Des auto-assemblages figés par réticulation ont aussi été développés, caractérisés et examinés en PDT. Ils se sont avérés extrêmement intéressants pour la PDT sur les cultures cellulaires en 3D, démontrant une efficacité accrue comparée aux systèmes simples. La comparaison de ces résultats avec ceux obtenus en culture 2D pour les mêmes objets a de plus permis de mettre en évidence la différence entre ces deux modèles biologiques. Enfin, des auto-assemblages à base de complexes poly-ioniques ont aussi été formés et caractérisés. Le fractionnement flux-force s’est là encore avéré efficace, mais a nécessité l’utilisation d’une injection spéciale par Frit-inlet. Leur efficacité en PDT s’est avérée faible. / The objective of this study was to critically analyze different polymer self-assemblies used for photodynamic therapy (PDT) and to link this analysis to their therapeutic efficiency. To do that, a thorough characterization of the vectors has been performed by classical techniques such as Dynamic Light Scattering or electron Microscopy, but also using flow fractionation, which has been seldomly used so far for polymeric self-assemblies. In a second step, these have been used as vectors of a photosensitizer, namely Phéophorbide a, and the therapeutic efficiency assessed on both 2D and 3D cell cultures of HCT 116 (colon cancer) and FaDu (head and neck cancer) cells. Different simple polymer vectors have first been evaluated, namely micelles and polymersomes based on diblock amphiphilic copolymers such as poly(ethylene-oxide-b--caprolactone), poly(ethylene-oxide-b-lactide) or poly(ethylene-oxide-b-styrene). This enabled obtaining vectors exhibiting various sizes and morphologies. Results in PDT showed different behaviours and a better efficiency in 3D for PEO-PDLLA. The Asymmetric Flow Field Flow Fractionation was particularly used for these systems to demonstrate their purity. The acquired expertise on this part enabled us to also characterize vectors made of known mixtures of micelles and polymersomes. These revealed antagonism and synergy effects in PDT, demonstrating the presence of complex processes for the cell response. Other self-assemblies consisting of crosslinked systems have also been developed and characterized. These were observed to be particularly efficient for PDT on 3D cell cultures. The comparison of these results with those for the 2D cell culture enabled to highlight the difference between those two biological systems. Finally, self-assemblies based on Polyion Complexes were also formed and characterized. Field Flow Fractionation was once again used as a powerful technique for this, although this implied the use of a special injection device called Frit Inlet. Their PDT efficiency however proved to be low.

Catharanthus roseus est une plante tropicale qui produit spécifiquement des alcaloïdes indoliques monoterpéniques (AIM) d’intérêt thérapeutique. Chez C. roseus, la branche terpénique incluant la voie du méthylérythritol phosphate (MEP) est considérée comme limitante et présente une régulation transcriptionnelle coordonnée en réponse aux hormones inductrices de l’accumulation alcaloïdique. Lors de ce travail, suite à des analyses bioinformatiques et à la caractérisation de promoteurs de gènes de la voie MEP, nous avons identifié de nouvelles familles de facteurs de transcription impliquées dans la régulation de la biosynthèse des AIM. Des membres de la famille des ZCT, des WRKY et des RR type B interagissent avec le promoteur du gène hds de la voie MEP et régulent son activité. Ces travaux ont permis d’approfondir les connaissances sur les réseaux transcriptionnels régulateurs de la biosynthèse des AIM. L’utilisation de ces nouveaux facteurs de transcription activateurs peut désormais être envisagée dans le cadre d’expériences d’ingénierie métabolique afin d’augmenter l’accumulation d’alcaloïdes d’intérêt pharmaceutique chez C. roseus. / Catharanthus roseus is a tropical plant producing specifically monoterpene indole alkaloids (MIA) of high interest due to their therapeutical values. In C. roseus cells, the terpenoid branch including the methyl erythritol phosphate pathway (MEP) provides the MIA terpenoid moiety and is regarded as limited for MIA biosynthesis. This branch presents a coordinated transcriptional regulation in response to hormonal signals leading to MIA production. In this context, bioinformatic analysises and functional characterization of MEP pathway gene promoters allowed the identification of new transcription factor families involved in the MIA pathway regulation. Members of ZCT proteins, WRKY and type B RR families specifically interact with the hds promoter from the MEP pathway and regulate its activity. This work permits to gain into insight the transcriptional network controlling the MIA biosynthesis. It is possible now to consider using transcription factor that act as activators and target genes from the terpenoid branch to increase the accumulation of alkaloids of pharmaceutical interest in C. roseus by metabolic engineering approaches.