This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.   Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.   Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. / <p>QC 20160509</p>

Subcellular Localization of Tobacco SABP2 under Normal and Stress Conditions Salicylic acid (SA), a phytohormone, plays an important role in plant physiology. SA mediated innate immune pathway is an important pathway for plant immunity against pathogens. Plants resisting pathogen infection synthesize higher levels of Methyl Salicylate (MeSA), which is then converted to SA by the esterase activity of Salicylic Acid Binding Protein 2 (SABP2). The high level of the converted SA leads to enhanced pathogen resistance. The study of subcellular localization of a protein is critical in explaining its potential biochemical functions. SABP2 tagged with eGFP was expressed transiently in Nicotiana benthamiana leaves. The SABP2-eGFP expressing leaves were challenged with bacterial and viral pathogens and observed under confocal microscopy. Fluorescent signals were seen throughout the cell and more concentrated towards the cell periphery. To verify the localization, mCherry fluorescent organelle markers with specific targeting sequences were used. The results indicate that the SABP2 is likely a cytoplasmic protein, and there is no change in its localization upon infection by plant pathogens.

Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.

La localisation d’ARNm est un processus cellulaire post-transcriptionnel qui assure le transport de l’ARNm vers une région subcellulaire spécifique. Dans différents organismes, plusieurs ARNm ont été décrits d’être localisés, et des études systématiques ont été réalisées uniquement chez la drosophile. Afin d’analyser la localisation des ARNm dans un modèle de mammifères, nous avons développé deux approches. La première approche est une nouvelle technique, smiFISH (single molecule inexpensive Fluorescent In Situ Hybridization), qui utilise des sondes primaires non marquées et une sonde secondaire fluorescente. Cette technique, qui permet d’analyser les ARNm au niveau endogène, est flexible, résolutive et peu coûteuse. La deuxième approche est l’analyse de centaines d’ARNm dans une collection de lignées cellulaires HeLa, par l’utilisation d’un seul jeu de sondes. Ces deux stratégies ont montré qu’environ 8% des ARNm sont localisés dans les cellules HeLa, et ont permis de découvrir de nouvelles classes de localisation d’ARNm chez les mammifères, jamais décrites auparavant. En analysant les gènes de la famille des moteurs moléculaires, nous avons trouvé que six ARNm ont des localisations particulières, parmi lesquels KIF1C présente une co-localisation entre l’ARNm et la protéine, suggérant un rôle crucial dans certains processus biologiques cellulaires. / The mRNA localization is a post-transcriptional process which allows the transport of mRNA to a specific subcellular region. In different organisms, several mRNAs have been described to be localized, but systematic studies have been only performed in Drosophila. In order to analyze the mRNA localization in a mammalian model, we developed two approaches. The first approach is a new technique, smiFISH (single molecule inexpensive Fluorescent In Situ Hybridization), which uses unlabeled primary probe and a fluorescent secondary probe. This technique, which allows the analysis of mRNA at the endogenous level, is flexible and inexpensive. The second approach is the analysis of hundreds of mRNA using a collection of HeLa cell lines, with a single set of probes. Both strategies allowed us to show that about 8% of mRNA are localized in HeLa cells. In addition, we discovered new classes of mRNA localization in mammals, never described before. By analyzing the genes of the molecular motors family, we found that six mRNAs have specific localization patterns, including KIF1C for which mRNA and protein co-localize, suggesting a crucial role in certain cell biological processes.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21161号 / 農博第2287号 / 新制||農||1060(附属図書館) / 学位論文||H30||N5135(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Tesis por compendio / [EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species. / [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas. / [CA] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora / Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477 / Compendio

Most women die of ovarian metastasis rather than the effects of the primary tumor. However, little is known about the factors that support the survival and secondary outgrowth of exfoliated ovarian cancer cells. In addition to genetic and molecular factors, the unique environment of the peritoneal cavity exposes ovarian cells to biophysical forces, particularly fluid shear stress (FSS). These biomechanical forces, only recently identified as a hallmark of cancer, induce rapid signaling events in attached and aggregated cells, a process termed mechanotransduction. The cellular responses to these forces and their impact on tumor initiation, progression, and metastasis are not understood. In order to delineate these phenomena, dynamic and syngeneic cell models are needed that represent the development of the disease and can be used in relevant engineered testing platforms. Thus, in an interdisciplinary approach, this work bridges molecular and cancer biology, device engineering, fluid mechanics, and biophysics strategies. The results demonstrated that even a low level of continual FSS significantly and differentially affected the viability of epithelial ovarian cancer cells of various stages of progression over time, and enhanced their aggregation, adhesion, and cellular architecture, traits of more aggressive disease. Furthermore, benign cells that survived FSS displayed phenotypic and genotypic changes resembling more aggressive stages of the disease, suggesting an impact of FSS on early stages of tumor development. After identifying a biological affect, we designed an in vitro testing platform for controlled FSS investigations, and we modeled the system fluid mechanics to understand the platform's performance capability. A cylindrical platform divided into annular sections with lid-driven flow was selected to allow continuous experiments sustainable for long durations. Tuning of the lid speed or fluid height resulted in a wide range of FSS magnitudes (0- 20 N/m2) as confirmed by analytical and numerical modeling. Further, detailed numerical modeling uncovered that FSS magnitudes experienced by cell aggregates were larger than previously observed, suggesting an even larger role of FSS in ovarian cancer. Finally, we built and engineered the designed platform to investigate changes in benign and cancer cells as a function of time and FSS magnitude. Device precision was balanced with biological consistency needs, and a novel platform was built for controlled FSS investigations. This work provides a foundational understanding of the physical environment and its potential links to ovarian cancer progression and metastatic potential. / Ph. D. / Most women die of ovarian metastasis rather than the effects of the primary tumor. However, little is known about the factors that support the survival and secondary outgrowth of exfoliated ovarian cancer cells. In addition to genetic and molecular factors, the unique environment of the peritoneal cavity exposes ovarian cells to biophysical forces, particularly fluid shear stress (FSS). These biomechanical forces, only recently identified as a hallmark of cancer, induce rapid signaling events in attached and aggregated cells, a process termed mechanotransduction. The cellular responses to these forces and their impact on tumor initiation, progression, and metastasis are not understood. In order to delineate these phenomena, dynamic and syngeneic cell models are needed that represent the development of the disease and can be used in relevant engineered testing platforms. Thus, in an interdisciplinary approach, this work bridges molecular and cancer biology, device engineering, fluid mechanics, and biophysics strategies. The results demonstrated that even a low level of continual FSS significantly and differentially affected the viability of epithelial ovarian cancer cells of various stages of progression over time, and enhanced their aggregation, adhesion, and cellular architecture, traits of more aggressive disease. Furthermore, benign cells that survived FSS displayed phenotypic and genotypic changes resembling more aggressive stages of the disease, suggesting an impact of FSS on early stages of tumor development. After identifying a biological affect, we designed an in vitro testing platform for controlled FSS investigations, and we modeled the system fluid mechanics to understand the platform’s performance capability. A cylindrical platform divided into annular sections with lid-driven flow was selected to allow continuous experiments sustainable for long durations. Tuning of the lid speed or fluid height resulted in a wide range of FSS magnitudes (0 − 20 N/m² ) as confirmed by analytical and numerical modeling. Further, detailed numerical modeling uncovered that FSS magnitudes experienced by cell aggregates were larger than previously observed, suggesting an even larger role of FSS in ovarian cancer. Finally, we built and engineered the designed platform to investigate changes in benign and cancer cells as a function of time and FSS magnitude. Device precision was balanced with biological consistency needs, and a novel platform was built for controlled FSS investigations. This work provides a foundational understanding of the physical environment and its potential links to ovarian cancer progression and metastatic potential.

Os estudos em reprodução e desenvolvimento das plantas, especialmente voltados ao pistilo, são de grande interesse agronômico, econômico e científico. Em nosso laboratório, recentemente, foi identificado e caracterizado SCI1 (Stigma/style Cell-cycle Inhibitor 1), um inibidor do ciclo celular que atua de forma tecido específica no pistilo de Nicotiana tabacum L. e Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). Foi identificada a proteína NtCDKG;2 (N. tabacum Cyclin-dependent Kinase 2) como parceira de interação de NtSCI1 (N . tabacum SCI1), em um ensaio de pull-down (STRINI, 2014). A literatura aponta que os inibidores de ciclo celular regulam o ciclo através da inibição de CDK, o que sugere que NtSCI1 possa regular o ciclo celular através da inibição de NtCDKG;2. O presente estudo mostra análises detalhadas da localização de GFP-NtCDKG;2 em células epiteliais de N. benthamiana. Verificou-se que a proteína NtCDKG;2 está presente no nucleoplasma e também co-localiza em speckles nucleares. Em cultura de células BY2 expressando GFP-NtCDKG;2 de forma estável, foi observado que, durante a metáfase e anáfase, a proteína NtCDKG;2 está junto ao fuso acromático. Adicionalmente, ensaios de BiFC (Bi-molecular Fluorescence Complementation) realizados neste trabalho mostram que a interação entre as proteínas NtCDKG;2 e NtSCI1 ocorre em uma região localizada na periferia nucleolar, durante a interfase. Também foram identificadas possíveis isoformas de NtCDKG;2. A possibilidade da ocorrência de isoformas sugere que, de maneira análoga à sua homóloga em humanos, as isoformas resultantes de NtCDKG;2 possam atuar em diferentes processos. Em busca de parceiros de interação de NtCDKG;2, para identificar em que vias esta proteína atua, foi realizado um screening de uma biblioteca de cDNAs de estigmas e estiletes de N. tabacum, no sistema de duplo-híbrido em leveduras (Y2H). Através desse ensaio, foram identificados diversos parceiros envolvidos com transcrição e processamento de RNA. Dentre as proteínas identificadas, cuja interação foi confirmada neste trabalho, destaca-se a proteína NtCDKF;1, uma proteína que fosforila o CTD da RNA Polimerase II e, dessa forma, auxilia a transcrição e o splicing cotranscricional (HAJHEIDARI et al., 2012). O presente trabalho mostra também a interação entre NtCDKG;2 e a proteína NtCBP1, uma proteína que possui um papel importante na regulação inicial da transcrição de proteínas mediadoras do crescimento do tubo polínico (LI et al., 2015). xx Adicionalmente, o screening de Y2H possibilitou a identificação da interação entre NtCDKG;2 e NtRanBP1, uma proteína chave na formação do fuso acromático que, em humanos, interage com uma isoforma homóloga a NtCDKG;2, a CDK11p46 (MIKOLAJCZYK et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011). Análises in silico realizadas com a sequência de aminoácidos de NtCDKG;2 apontaram motivos de interação com proteína do tipo F-Box, ciclina, CDK, fosfatase, 14-3-3, BRCA1 e indicaram o local provável de interação do complexo CDK-Ciclina com o respectivo inibidor. Foi testada e comprovada a interação entre NtCDKG;2 e a 14-3-3D, por Y2H, uma parceira de NtSCI1. Outra lacuna que precisava ser preenchida é referente à regulação da expressão de NtSCI1. Com este intuito, foram realizadas análises in silico para identificar elementos cis-regulatórios na sequência genômica de NtSCI1. Essas análises indicaram a presença de importantes elementos cis-regulatórios relacionados à identidade meristemática (como WUSCHEL e AINTEGUMENTA), identidade do carpelo (AGAMOUS, BELL) e progressão do ciclo celular (E2F e CDC5). Algumas considerações podem ser feitas associando os resultados obtidos a estudos feitos paralelamente em nosso laboratório: 1) Compilando a localização de NtCDKG;2 em splicing speckles e sua interação com os diferentes parceiros de interação relacionados à transcrição e splicing, sugere-se que NtCDKG;2 também atue nos processos transcricionais e de splicing. 2) Considerando a localização subcelular de NtCDKG;2 durante as diferentes fases do ciclo celular, às análises in silico dessa proteína que identificaram sua possível interação com BRCA1, além da interação confirmada com a proteína NtRanBP1, é possível sugerir que NtCDKG;2 atue, direta ou indiretamente, na organização do fuso acromático de plantas. 3) Propõem-se que NtSCI1 regule a proliferação celular no pistilo através da interação com NtCDKG;2 que se dá no nucléolo das células. Dessa forma, NtSCI1 prenderia NtCDKG;2 no nucléolo e inibiria sua atuação, como na organização do fuso acromático, o que acarretaria inibição da divisão celular. 4) Devido aos motivos cis-regulatórios encontrados na sequência genômica de NtSCI1 e o efeito que a proteína possui desde as fases iniciais do desenvolvimento do pistilo, sugere-se que a expressão desse gene seja regulada por elementos diretamente envolvidos no controle do término do meristema floral e nas vias de desenvolvimento de órgãos florais. / Studies on plant reproduction and development, specifically those related to the pistil, are of great agronomic, economic and scientific interest. In our laboratory, we recently identified and characterized SCI1 (Stigma/style Cell-cycle Inhibitor 1), an inhibitor of the cell cycle which acts tissuespecifically in the pistil of Nicotiana tabacum L. and Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). The NtCDKG;2 (N. tabacum Cyclin-dependent Kinase G; 2) protein was identified as an interaction partner of NtSCI1 (N. tabacum SCI1) in a pulldown assay (STRINI, 2014). The literature suggests that cell cycle inhibitors control the cycle through the inhibition of CDKs, indicating that NtSCI1 might control cell cycle by inhibiting NtCDKG;2. This study shows detailed analysis of GFP-NtCDKG;2 localization in leaf cells of N. benthamiana. The analysis shows that NtCDKG;2 is present in the nucleoplasm and also co-localizes with nuclear speckles. In BY2 cell culture stably expressing GFP-NtCDKG;2, it was observed that NtCDKG;2 is at the achromatic spindle during metaphase and anaphase. Additionally, BiFC (Bimolecular Fluorescence Complementation) assays performed in this study have shown that the interaction of NtCDKG;2 and NtSCI1 occurs in the nucleolar periphery during interphase. Putative isoforms of NtCDKG;2 were also identified. The possible occurrence of these isoforms suggests that, in a similar way to its human homologue, NtCDKG;2 putative isoforms could act in different processes. To identify in which processes this protein could act, a search for NtCDKG;2 interaction partners was performed through the screening of a N. tabacum stigma and style cDNA library in the yeast two-hybrid (Y2H) system. Several partners identified through this assay have roles in RNA transcription and processing. Among the identified partners with interaction confirmed during this work, stands out the NtCDKF;1 protein, a CDK that phosphorylates the RNA polymerase II CTD, and thus, supports transcription and co-transcriptional splicing (HAJHEIDARI et al., 2012). This study also shows the interaction of NtCDKG;2 with NtCBP1, a protein which has an important role in the transcriptional regulation of genes encoding proteins mediating pollen tube growth (LI et al., 2015). Furthermore, the Y2H screening allowed the identification of the interaction of NtCDKG;2 with NtRanBP1, a key protein in the formation of the achromatic spindle which, in humans, interacts with the CDK11p46 isoform (MIKOLAJCZYK xxii et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011), a homologue of NtCDKG;2. In silico analysis of the amino acid sequence of NtCDKG;2 revealed motifs of predicted interaction with F-box proteins, cyclins, CDKs, phosphatases, 14-3-3s, BRCA1, and also pointed the region where the CDK-cyclin complex might interact with its respective inhibitor. The interaction of NtCDKG;2 with 14-3-3D, a known partner of NtSCI1, was tested and confirmed by Y2H. Another gap that needed to be filled is related to the regulation of NtSCI1 expression. To address this issue, in silico analysis to identify cis-regulatory elements was performed in NtSCI1 genomic region. These analyses revealed the presence of important cis-regulatory elements related to meristem identity (such as WUSCHEL and AINTEGUMENTA), carpel identity (AGAMOUS, BELL), and cell cycle progression (E2F and CDC5). Taken together results from this study and parallel studies performed in our laboratory, a few remarks can be made: 1) Taken the localization of NtCDKG;2 in splicing speckles, and its interaction with different proteins involved in transcription and splicing, it is suggested that NtCDKG;2 also has roles on these processes; 2) Considering the subcellular localization of NtCDKG;2 during the different cell cycle phases, the in silico analysis of this protein that predicts its interaction with BRCA1, and the confirmed interaction with NtRanBP1 protein, it is possible to suggest that NtCDKG;2 has a direct or indirect role in the organization of the achromatic spindle in plants; 3) It is proposed that NtSCI1 regulates cell proliferation in the pistil through its interaction with NtCDKG;2, which occurs in the nucleolus. Thus, NtSCI1 could hold NtCDKG;2 in the nucleolus, inhibiting its actions, such as in the organization of the achromatic spindle, resulting in cell division arrest. 4) Due to the cis-regulatory elements found in the genomic sequence of NtSCI1, and the effect of this protein since the initial stages of pistil development, it is suggested that its expression is regulated by elements directly involved in the control of the floral meristem termination and pathways of floral organ development.

Os estudos em reprodução e desenvolvimento das plantas, especialmente voltados ao pistilo, são de grande interesse agronômico, econômico e científico. Em nosso laboratório, recentemente, foi identificado e caracterizado SCI1 (Stigma/style Cell-cycle Inhibitor 1), um inibidor do ciclo celular que atua de forma tecido específica no pistilo de Nicotiana tabacum L. e Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). Foi identificada a proteína NtCDKG;2 (N. tabacum Cyclin-dependent Kinase 2) como parceira de interação de NtSCI1 (N . tabacum SCI1), em um ensaio de pull-down (STRINI, 2014). A literatura aponta que os inibidores de ciclo celular regulam o ciclo através da inibição de CDK, o que sugere que NtSCI1 possa regular o ciclo celular através da inibição de NtCDKG;2. O presente estudo mostra análises detalhadas da localização de GFP-NtCDKG;2 em células epiteliais de N. benthamiana. Verificou-se que a proteína NtCDKG;2 está presente no nucleoplasma e também co-localiza em speckles nucleares. Em cultura de células BY2 expressando GFP-NtCDKG;2 de forma estável, foi observado que, durante a metáfase e anáfase, a proteína NtCDKG;2 está junto ao fuso acromático. Adicionalmente, ensaios de BiFC (Bi-molecular Fluorescence Complementation) realizados neste trabalho mostram que a interação entre as proteínas NtCDKG;2 e NtSCI1 ocorre em uma região localizada na periferia nucleolar, durante a interfase. Também foram identificadas possíveis isoformas de NtCDKG;2. A possibilidade da ocorrência de isoformas sugere que, de maneira análoga à sua homóloga em humanos, as isoformas resultantes de NtCDKG;2 possam atuar em diferentes processos. Em busca de parceiros de interação de NtCDKG;2, para identificar em que vias esta proteína atua, foi realizado um screening de uma biblioteca de cDNAs de estigmas e estiletes de N. tabacum, no sistema de duplo-híbrido em leveduras (Y2H). Através desse ensaio, foram identificados diversos parceiros envolvidos com transcrição e processamento de RNA. Dentre as proteínas identificadas, cuja interação foi confirmada neste trabalho, destaca-se a proteína NtCDKF;1, uma proteína que fosforila o CTD da RNA Polimerase II e, dessa forma, auxilia a transcrição e o splicing cotranscricional (HAJHEIDARI et al., 2012). O presente trabalho mostra também a interação entre NtCDKG;2 e a proteína NtCBP1, uma proteína que possui um papel importante na regulação inicial da transcrição de proteínas mediadoras do crescimento do tubo polínico (LI et al., 2015). xx Adicionalmente, o screening de Y2H possibilitou a identificação da interação entre NtCDKG;2 e NtRanBP1, uma proteína chave na formação do fuso acromático que, em humanos, interage com uma isoforma homóloga a NtCDKG;2, a CDK11p46 (MIKOLAJCZYK et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011). Análises in silico realizadas com a sequência de aminoácidos de NtCDKG;2 apontaram motivos de interação com proteína do tipo F-Box, ciclina, CDK, fosfatase, 14-3-3, BRCA1 e indicaram o local provável de interação do complexo CDK-Ciclina com o respectivo inibidor. Foi testada e comprovada a interação entre NtCDKG;2 e a 14-3-3D, por Y2H, uma parceira de NtSCI1. Outra lacuna que precisava ser preenchida é referente à regulação da expressão de NtSCI1. Com este intuito, foram realizadas análises in silico para identificar elementos cis-regulatórios na sequência genômica de NtSCI1. Essas análises indicaram a presença de importantes elementos cis-regulatórios relacionados à identidade meristemática (como WUSCHEL e AINTEGUMENTA), identidade do carpelo (AGAMOUS, BELL) e progressão do ciclo celular (E2F e CDC5). Algumas considerações podem ser feitas associando os resultados obtidos a estudos feitos paralelamente em nosso laboratório: 1) Compilando a localização de NtCDKG;2 em splicing speckles e sua interação com os diferentes parceiros de interação relacionados à transcrição e splicing, sugere-se que NtCDKG;2 também atue nos processos transcricionais e de splicing. 2) Considerando a localização subcelular de NtCDKG;2 durante as diferentes fases do ciclo celular, às análises in silico dessa proteína que identificaram sua possível interação com BRCA1, além da interação confirmada com a proteína NtRanBP1, é possível sugerir que NtCDKG;2 atue, direta ou indiretamente, na organização do fuso acromático de plantas. 3) Propõem-se que NtSCI1 regule a proliferação celular no pistilo através da interação com NtCDKG;2 que se dá no nucléolo das células. Dessa forma, NtSCI1 prenderia NtCDKG;2 no nucléolo e inibiria sua atuação, como na organização do fuso acromático, o que acarretaria inibição da divisão celular. 4) Devido aos motivos cis-regulatórios encontrados na sequência genômica de NtSCI1 e o efeito que a proteína possui desde as fases iniciais do desenvolvimento do pistilo, sugere-se que a expressão desse gene seja regulada por elementos diretamente envolvidos no controle do término do meristema floral e nas vias de desenvolvimento de órgãos florais. / Studies on plant reproduction and development, specifically those related to the pistil, are of great agronomic, economic and scientific interest. In our laboratory, we recently identified and characterized SCI1 (Stigma/style Cell-cycle Inhibitor 1), an inhibitor of the cell cycle which acts tissuespecifically in the pistil of Nicotiana tabacum L. and Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). The NtCDKG;2 (N. tabacum Cyclin-dependent Kinase G; 2) protein was identified as an interaction partner of NtSCI1 (N. tabacum SCI1) in a pulldown assay (STRINI, 2014). The literature suggests that cell cycle inhibitors control the cycle through the inhibition of CDKs, indicating that NtSCI1 might control cell cycle by inhibiting NtCDKG;2. This study shows detailed analysis of GFP-NtCDKG;2 localization in leaf cells of N. benthamiana. The analysis shows that NtCDKG;2 is present in the nucleoplasm and also co-localizes with nuclear speckles. In BY2 cell culture stably expressing GFP-NtCDKG;2, it was observed that NtCDKG;2 is at the achromatic spindle during metaphase and anaphase. Additionally, BiFC (Bimolecular Fluorescence Complementation) assays performed in this study have shown that the interaction of NtCDKG;2 and NtSCI1 occurs in the nucleolar periphery during interphase. Putative isoforms of NtCDKG;2 were also identified. The possible occurrence of these isoforms suggests that, in a similar way to its human homologue, NtCDKG;2 putative isoforms could act in different processes. To identify in which processes this protein could act, a search for NtCDKG;2 interaction partners was performed through the screening of a N. tabacum stigma and style cDNA library in the yeast two-hybrid (Y2H) system. Several partners identified through this assay have roles in RNA transcription and processing. Among the identified partners with interaction confirmed during this work, stands out the NtCDKF;1 protein, a CDK that phosphorylates the RNA polymerase II CTD, and thus, supports transcription and co-transcriptional splicing (HAJHEIDARI et al., 2012). This study also shows the interaction of NtCDKG;2 with NtCBP1, a protein which has an important role in the transcriptional regulation of genes encoding proteins mediating pollen tube growth (LI et al., 2015). Furthermore, the Y2H screening allowed the identification of the interaction of NtCDKG;2 with NtRanBP1, a key protein in the formation of the achromatic spindle which, in humans, interacts with the CDK11p46 isoform (MIKOLAJCZYK xxii et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011), a homologue of NtCDKG;2. In silico analysis of the amino acid sequence of NtCDKG;2 revealed motifs of predicted interaction with F-box proteins, cyclins, CDKs, phosphatases, 14-3-3s, BRCA1, and also pointed the region where the CDK-cyclin complex might interact with its respective inhibitor. The interaction of NtCDKG;2 with 14-3-3D, a known partner of NtSCI1, was tested and confirmed by Y2H. Another gap that needed to be filled is related to the regulation of NtSCI1 expression. To address this issue, in silico analysis to identify cis-regulatory elements was performed in NtSCI1 genomic region. These analyses revealed the presence of important cis-regulatory elements related to meristem identity (such as WUSCHEL and AINTEGUMENTA), carpel identity (AGAMOUS, BELL), and cell cycle progression (E2F and CDC5). Taken together results from this study and parallel studies performed in our laboratory, a few remarks can be made: 1) Taken the localization of NtCDKG;2 in splicing speckles, and its interaction with different proteins involved in transcription and splicing, it is suggested that NtCDKG;2 also has roles on these processes; 2) Considering the subcellular localization of NtCDKG;2 during the different cell cycle phases, the in silico analysis of this protein that predicts its interaction with BRCA1, and the confirmed interaction with NtRanBP1 protein, it is possible to suggest that NtCDKG;2 has a direct or indirect role in the organization of the achromatic spindle in plants; 3) It is proposed that NtSCI1 regulates cell proliferation in the pistil through its interaction with NtCDKG;2, which occurs in the nucleolus. Thus, NtSCI1 could hold NtCDKG;2 in the nucleolus, inhibiting its actions, such as in the organization of the achromatic spindle, resulting in cell division arrest. 4) Due to the cis-regulatory elements found in the genomic sequence of NtSCI1, and the effect of this protein since the initial stages of pistil development, it is suggested that its expression is regulated by elements directly involved in the control of the floral meristem termination and pathways of floral organ development.

La photothérapie dynamique (PDT) consiste en la destruction d’une tumeur par l’association de l’administration d’un photosensibilisateur et de l’exposition à la lumière visible. Ce travail comporte : i) une étude de l’ionisation et de la lipophilie d’une série de photosensibilisateurs, des méso-tétraphénylporphyrines (TPP) glycoconjuguées, ii) une évaluation de l’impact de ces deux propriétés sur la distribution plasmatique et la localisation subcellulaire du photosensibilisateur.La protonation des azotes tétrapyrroliques a été étudiée par spectroscopie électronique, combinéeà une analyse chimiométrique, tandis que la lipophilie a été évaluée par chromatographie liquide haute performance. L’impact de différents effets de substitution (position, nombre ou nature du substituant) sur ces deux propriétés physico-chimiques a été mis en évidence.Dans le plasma, les TPP glycoconjuguées se lient principalement aux lipoprotéines de haute densité. La lipophilie de ces dérivés permet d'expliquer leur affinité pour les lipoprotéines, mais pas pour l'albumine. L’étude de localisation subcellulaire, combinant approche expérimentale et modélisation, a conduit à proposer une hypothèse expliquant la localisation de la TPP(pODEGOαManOH)3 au niveau du réticulum endoplasmique, hypothèse accordant un rôle central à la lipophilie de la TPP . A l'issue de ce travail, avant d'appliquer nos hypothèses à la synthèse de nouvelles molécules, il apparaît nécessaire de mieux explorer l'impact de la distribution plasmatique et de la localisation subcellulaire sur l'efficacité PDT. / Photodynamic Therapy (PDT) is based on the destruction of a tumor tissue through a combinationof administration of a photosensitizer and exposure to visible light. This work includes : i) a study of ionization and hydrophobicity of a series of candidate sensitizers, glycoconjugated mesotetraphenylporphyrins (TPP), ii) an evaluation of the impact of those two physico-chemicalproperties on sensitizer's plasma distribution and subcellular localization. Protonation of tetrapyrrolic nitrogens has been studied by electronic spectroscopy combined with chemometric analysis whereas hydrophobicity has been evaluated by high-performance liquid chromatography. The effect of substitution modalities (position, number and nature of pendantgroups) on both physico-chemical properties has been evidenced.In human plasma, glycoconjugated TPPs mainly bind to high density lipoproteins. Hydrophobicity accounts for differences in affinities towards lipoproteins, but not towards albumin. Subcellular localization studies, combining computational and experimental approaches, led to formulate some assumptions explaining localization of TPP(pODEGOαManOH)3 in endoplasmic reticulum,assumptions centered on a major role of sensitizer's hydrophobicity. At the end of this work, before trying to use our conclusions for the design of new sensitizers, it remains necessary to better explore the effect of plasma distribution and subcellular localization on sensitizer's photo-efficiency.