RALF é um peptídeo hormonal de aproximadamente 5kDa presente em diferentes espécies do reino vegetal regulando negativamente a expansão celular. AtRALF1 é uma isoforma específica de raiz das 37 presentes em Arabidopsis thaliana que regula negativamente o crescimento de raízes seguido de uma mobilização de Ca+2 intracelular e inibição na secreção de prótons (H+). Neste trabalho foi caraterizado a localização e tráfego intracelular do peptídeo AtRALF1. / RALF is a 5kDa peptide hormone ubiquitous in different species of the plant kingdom that regulates cell expansion. AtRALF1 is a root-specific isoform of 37 present in Arabidopsis thaliana that negatively regulates root growth by intracellular calcium mobilization and inhibition of proton secretion (H+). In this work was studied the localization and intracellular trafficking of the AtRALF1 peptide.

A pulchellina é uma glicoproteína heterodimérica com duas cadeias, pertencente à família das proteínas inativadoras de ribossomos (RIPs) do tipo 2. A cadeia A é enzimaticamente ativa e é capaz de remover uma adenina da porção 28S do rRNA; a cadeia B é uma lectina que se liga a resíduos de D-Galactose terminais, presentes na membrana. Das 4 isoformas da pulchellina (PI, PII, PIII, PIV), PII é a mais tóxica in vivo, sendo a atividade catalítica da cadeia A similar para todas as isoformas. A interação da cadeia B com os glicoreceptores de membrana e seu conseguinte processo de endocitose é crucial para que cadeia A tóxica entre na célula e torne-se disponível para atuar no seu sítio ribossomal. Assim, visando explorar e encontrar potenciais diferenças no mecanismo de ligação à célula e de endocitose das isoformas, foram realizados experimentos usando microscopia confocal com as toxinas marcadas com Alexa flúor® em células HeLa e MV3. As imagens obtidas mostraram que PII localiza-se na região perinuclear das células enquanto PIV predomina na região cortical. Esses resultados sugeriram que as isoformas apresentam distintos mecanismos de entrada e transporte nas células. Para esclarecer tal questão, a ação da pulchellina em células HeLa tratadas com diversas drogas que atuam em diferentes rotas endocíticas e de translocação, foi monitorada. Os resultados de inibição de síntese proteica mostraram que as células sofrem proteção contra a pulchellina na presença de brefeldina A, indicando que a pulchellina necessita ser transportada via Golgi para executar sua função. Inibidores de glicosilação como tunicamicina, swainsonine e inibidores de síntese proteica, como a puromicina e cicloheximidina sensibilizaram as células à PII e PIV, mas em diferentes taxas. Por outro lado, a puromicina e a cicloheximidina não afetaram a taxa de endocitose das isoformas, o que indica que a pulchellina na ausência dos inibidores compete pelo transporte ou processamento de glicoproteínas recém-sintetizadas. Experimentos de ligação e captação da pulchellina mostraram que PII apresenta 30% menos afinidade pela superfície de células HeLa que PIV, além de apresentar menor taxa endocítica. Esses dados corroboram estudos de FCS (espectroscopia de correlação e fluorescência) que identificaram que a difusão de PIV em células HeLa é maior que de PII. Nos experimentos realizados com inibidores de dinamina, ambas isoformas tiveram as suas taxas de endocitose aumentadas, indicando um efeito compensatório para via endocítica independente de dinamina. Em células incubadas com PDMP e neuraminidase, PIV mostrou uma associação às células reduzida, enquanto PII não se alterou, indicando que PIV pode necessitar de esfingolipídeos e glicocomplexos contendo ácido siálico para ligar e se internalizar nas células testadas. Para investigar essa diferença na interação foram realizados ensaios in vitro de DSC (Calorimetria Diferencial de Varredura) e SPR (ressonância plasmônica de superfície) com as isoformas isoladas. Esses ensaios mostraram que PIV e PII apresentam interações distintas com o gangliosídeo GM1, sendo que a PIV interage mais hidrofobicamente e com uma maior taxa de associação com GM1 que a PII. / Pulchellin is a heterodimeric toxin found in Abrus pulchellus seeds. It is a type 2 ribosome inactivating protein, which consists of a toxic A-chain linked to a sugar binding B-chain. The B-chain mediates its binding to the galactose residues on the cellular membrane in a process that is then followed by an endocytic uptake. Once the A-chain reaches the cytosol it inhibits protein synthesis leading to cell death. In order to explore pulchellin isoforms II and IV (PII and PIV) cell entry and transport mechanisms, experiments monitoring toxin labelled with Alexafuor® in MV3 and HeLa cells were performed using confocal microscopy. We have investigated the pulchellin action in pre-treated HeLa cells with several drugs, targeting different endocytic and translocation routes. Confocal images showed PII tends to be localized in cells cortical region and PIV tend to be localized in cell\'s perinuclear region, suggesting that isoforms have different cell entry and transport mechanisms. The protein synthesis inhibition results showed that brefeldin A protects cells against the toxic effect of pulchellin, which indicates the pulchellin needs to be transported to Golgi to perform its toxic effect. When HeLa cells were incubated with protein synthesis inhibitors, such as puromycin and cycloheximidine and glycosilation inhibitors such as tunicamycin, swainsonine, they were sensitized to pulchellin, but to different extent for PII and PIV. Binding and uptake experiments showed that PII exhibits 30% less affinity than PIV on HeLa cells surface, PII also has lower endocytic rate than PIV in the cells. These data corroborate with FCS (Fluorescence Correlation Spectroscopy) results, which identified that PIV diffuses faster than PII into the celIs. Dynamine inhibitors increased endocytosis rates in both isoforms, indicating that pulchellin is upregulating the dynamine-independent endocytosis, possibly pulchellin is being internalized into the cells by alternative endocytic routes. When HeLa cells were incubated with PDMP and neuraminidase, PIV showed a reduced cell association compared with PII and control, indicating that PIV may require glycocomplexes and sphingolipids containing sialic acid to enter into the cells. DSC (Differential Scanning Calorimetry) and SPR (Surface Plasmon Ressonance) experiments using biomimetic membranes were performed using GM1 ganglioside to check this interaction. The results showed PIV and PII interact with GM1. This results also evidence PIV interact more hidrophobically and with a higher association rate on GM1 than PII.

Evidências epidemiológicas indicam que a presença de microalbuminúria prediz maior freqüência de eventos cardiovasculares e mortalidade em hipertensos essenciais. A microalbuminúria pode ser decorrente do aumento da permeabilidade glomerular e/ou da diminuição da reabsorção desta macromolécula no túbulo proximal. Todavia não é sabido se os mecanismos que regulam a reabsorção de albumina em túbulo proximal renal encontram-se alterados na hipertensão essencial. Este trabalho teve como objetivo investigar as bases moleculares da microalbuminúria associada à hipertensão arterial essencial, focando na reabsorção tubular de albumina. Para tanto, avaliamos a evolução temporal da excreção urinária de albumina em ratos espontaneamente hipertensos (SHR) com 6 semanas de idade (pressão arterial sistólica, PAS, = 105 ± 4 mmHg), 14 semanas de idade (PAS = 180 ± 2 mmHg) e 21 semanas de idade (PAS = 202 ± 2 mmHg). Ratos normotensos Wistar da mesma idade serviram de controle. Observou-se que a excreção urinária diária de albumina aumentou progressivamente com o aumento da pressão arterial em SHR (10,5 ± 1,9; 92 ± 7,0 e 154 ± 27 g/dia, em SHR com PAS média igual a 105, 180 e 202 mmHg respectivamente). Este aumento progressivo não foi observado em ratos normotensos com idade correspondente, indicando que este fenômeno é decorrente do aumento da pressão arterial e não pode ser atribuído ao aumento da idade dos animais durante o período estudado. A análise das proteínas urinárias por eletroforese em gel de poliacrilamida (SDS-PAGE) mostrou que SHR excretam proteínas do tamanho da albumina ou menores (< 70kDa), padrão típico de proteinúria tubular. Adicionalmente, verificou-se que os níveis de expressão dos receptores endocíticos megalina e cubilina, bem como do canal para cloreto ClC-5 diminuem progressivamente no córtex renal de SHR com o aumento da pressão arterial. Observou-se também uma diminuição significativa na expressão de uma outra macromolécula importante no processo de endocitose mediada por receptor em túbulo proximal renal, a v-H+-ATPase. Entretanto, a diminuição da expressão protéica da subunidade B2 desta ATPase foi estatisticamente significante apenas em SHR com 21 semanas comparado aos com 6 semanas de idade. Não foram encontradas alterações no padrão de expressão de componentes estruturais da barreira glomerular como a nefrina e podocina. Em suma, o nosso estudo demonstra que o aumento da excreção urinária de proteínas, especialmente de albumina, está associado com uma menor expressão de componentes essenciais do aparelho endocítico do túbulo proximal renal. É tentador especular que a disfunção da via endocítica no túbulo proximal renal possa ser o principal mecanismo subjacente ao desenvolvimento de microalbuminúria na hipertensão / Epidemiological evidences indicate that the presence of microalbuminuria predicts a higher frequency of cardiovascular events and mortality in essential hypertensive patients. Microalbuminuria may arise from increased glomerular permeability and/or reduced proximal tubular reabsorption of albumin. However, it remains to be determined whether the mechanisms that regulate the renal proximal tubular reabsorption of albumin are altered in essential hypertension. The purpose of this work was to investigate the molecular basis of microalbuminuria in essential hypertension, focusing on the renal tubular reabsorption of albumin. To this end, we evaluated the temporal evolution of urinary albumin excretion in spontaneously hypertensive rats (SHR) at 6 weeks of age (systolic arterial pressure, SAP, = 105 ± 4 mmHg), 14 weeks of age (SAP = 180 ± 2 mmHg) and 21 weeks of age (SAP = 202 ± 2 mmHg). Age-matched normotensive Wistar rats were used as controls. It was observed that the daily urinary excretion of albumin progressively increased with blood pressure in SHR from 6 to 21 weeks of age (10.5 ± 1.9, 92 ± 7.0 and 154 ± 27 g in SHR with 105, 180 and 202 mmHg of average SAP, respectively). This progressive increase in microalbuminuria has not been observed in age-matched normotensive Wistar rats, indicating that this phenomenon cannot be attributed to age progression over the studied period. SDS-PAGE analysis of urinary proteins showed that microalbuminuric SHR virtually excreted proteins of the size of albumin or smaller (< 70kDa), typical of tubular proteinuria. Additionally, it was verified that the protein expression levels of the endocytic receptors megalin and cubilin as well as of the chloride channel ClC-5 progressively decreased in the renal cortex of SHR from 6 to 21 weeks of age. Moreover, it was observed reduction of expression of another macromolecule that plays an important role in the process of receptor mediated endocytosis in the renal proximal tubule, the v-H+- ATPase, was reduced. However, reduced cortical expression of the B2 subunit of the v- H+-ATPase, was only statistically significant in 21-wk-old vs. 6-wk-old SHR. Expression levels of structural components of the glomerular barrier such as nephrin and podocin were unchanged. To sum up, our study demonstrates that the increase in urinary protein excretion, especially of albumin, is associated with lower expression of key components of the apical endocytic apparatus in the renal proximal tubule. It is tempting to speculate that dysfunction of the apical endocytic pathway in the renal proximal tubule may be the major mechanism underlying development of microalbuminuria in essential hypertension

Abstract Skeletal muscles are composed of long, multinucleated cells called myofibers, which are highly differentiated cells and therefore unique in structure. In the present study the organization of the endocytic and exocytic pathways in isolated rat skeletal myofibers was defined with confocal and electron microscopic methods. In isolated myofibers the I band areas were shown to be active in endocytosis. The sorting endosomes were distributed in a cross-striated fashion while the recycling and late endosomal compartments were located to perinuclear areas and interfibrillar spaces, where they followed the course of microtubules. Protein trafficking in the different stages of muscle cell differentation was also analyzed. The studies with L6 myoblasts and myotubes showed that during myogenesis varying fractions of different viral glycoproteins were sorted from the endoplasmic reticulum (ER) into a specific compartment that did not recycle with the Golgi apparatus. This compartment is suggested to be the sarcoplasmic reticulum (SR). The studies with living muscle cells showed further changes in vesicle trafficking taking place during myogenesis. With GFP-tagged tsO45G protein, transport containers were detected in 20% of the infected myofibers, while all infected L6 myoblasts or myotubes showed intense movement of corresponding structures. We also detected significant differences between the pre-and post-Golgi traffickings in myofibers. When the distribution of the ER in adult myofibers was studied, the confocal microscopic data showed that the labeling patterns of the rough endoplasmic reticulum (RER) and the SR markers were different. Blocking of different cargo proteins in the RER revealed two discrete distribution patterns, neither of them identical with the SR. The collected electron microscopic data supported the idea that in mature myofibers there are two separate RER compartments. We suggest that the RER compartment capable of export function located around the myonuclei and on the Z lines, while the non-exporting RER compartment localized to terminal cisternae and probably took care of the synthesis of the SR proteins.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

L’endocytose permet l’internalisation d’éléments présents dans le milieu extracellulaire tels que les nutriments. Ce processus prend place dans la membrane plasmique. Les courbures de la membrane jouent un rôle essentiel dans l’endocytose pour générer une invagination initiale, un puits, puis une vésicule qui se sépare ensuite de la membrane plasmique pour fusionner avec les compartiments intracellulaires. Il existe plusieurs voies d’endocytose qui peuvent être classées selon des critères tels que la taille des vésicules produites, la médiation par un récepteur ou la présence d’un manteau recouvrant les vésicules. La voie d’endocytose la mieux caractérisée est celle dépendante de la clathrine. Mon laboratoire d’accueil travaille sur l’entrée du récepteur de l’interleukine 2 (IL-2R). Ce récepteur peut entrer de façon constitutive ou induite en présence de son ligand l’IL-2. Les deux voies sont indépendantes de la clathrine. Lors de mon arrivée dans le laboratoire, ces voies étaient encore peu caractérisées, notamment les facteurs induisant les courbures membranaires restaient à identifier. Ces facteurs doivent être particulièrement impliqués car les vésicules contenant l’IL-2R sont dépourvues de manteaux. Un crible par interférence à ARN, réalisé avant mon arrivée avait permis de proposer des protéines candidates pouvant courber les membranes. La première partie de ma thèse a consisté à confirmer l’importance de certaines protéines issues de ce crible puis à étudier leurs rôles dans la voie constitutive de l’IL-2R. Parmi ces protéines confirmées, deux familles de facteurs étaient particulièrement intéressantes pour leur capacité à courber les membranes, les phospholipases D et les endophilines. Ces dernières ont permis d’identifier une nouvelle voie d’entrée nommée « Fast Endophilin Mediated Endocytosis » FEME dans laquelle l’endophiline joue un rôle essentiel et qui est empruntée par de nombreux récepteurs transmettant le signal. La voie FEME partage plusieurs facteurs communs avec la voie d’endocytose induite de l’IL-2R. Pour finir, mes travaux de thèse ont porté sur l’orchestration de l’endophiline et de la dynamine dans la voie d’endocytose constitutive de l’IL-2R. Ces deux facteurs sont impliqués en fin d’endocytose, pour scinder les vésicules de la membrane plasmique. Cependant, ces deux protéines n’ont pas la même orchestration. Nos travaux montrent une action distincte de l’endophiline et de la dynamine dans les voies d’endocytose dépendante et indépendante de la clathrine. / Endocytosis allows the uptake of elements from the extracellular fluid such as nutriments. This process takes place at the plasma membrane. The membrane curvatures play an important role in endocytosis for the production of initial invagination to form a pit that will be then separate from the plasma membrane and will go to the intracellular compartments. Several routes of endocytosis exist and can be classified depending on vesicles size formed, receptor mediated endocytosis or coat on vesicles. The well-known characterized endocytosis pathway is the clathrin mediated one. My lab is working on interleukin 2 receptor (IL-2R) entry. This receptor can enter either constitutively or upon induction by the ligand IL-2. Both uptake pathways are independent of clathrin. When I arrived in the lab, those pathways were still under characterization, in particular the factors inducing the membrane curvature. Their role should be important since IL-2R containing vesicles are coated free. A small interfering RNA screen performed before my phD, allowed to identify new candidates. The first part of my thesis was to verify the involvement of some of them in the IL-2R constitutive pathway and then to study their role in this pathway. Among them, 2 families of proteins were particularly interesting as they can curve membranes, phospholipases D and endophilins. The endophilin allowed the discovery of a new route called “Fast Endophilin Mediated Endocytosis” (FEME) in which it plays an essential role and which is used by numerous receptors that transmit signal. The FEME pathway shares several factors that are common with the IL-2 induced endocytosis pathway. Then, I conducted a work on the orchestration of endophilin and dynamin during the constitutive IL-2R endocytosis. Both factors are recruited at the end of the mechanism, to separate the vesicles from the plasma membrane. However, both proteins do not have the same orchestration. Our works show a distinct action of endophilin and dynamin in clathrin dependent and independent endocytosis.

Les brassinostéroïdes (BR) sont des hormones stéroïdes végétales qui jouent un rôle dans la croissance et le développement des plantes. Ils sont perçus à la surface cellulaire par le récepteur kinase BRI1 (BR insensitive 1) situé au niveau de la membrane plasmique. La voie de signalisation des BRs implique la cis et trans-phosphorylation de BRI1 et de son corécepteur BAK1 (BRI1 Associated Receptor Kinase 1) et aboutit à la déphosphorylation des facteurs de transcription BZR1 (Brassinazole Resistant 1) et BES1 (bri1-EMS-Supressor 1) et l’activation des réponses génomiques aux BRs. Les mécanismes permettant la dé-activation du complexe de perception des BRs sont encore peu connus mais peuvent impliquer la déphosphorylation de BRI1, la fixation de régulateur négatif comme BKI1, et l’endocytose de BRI1. Mon travail de doctorat a consisté à étudier les mécanismes d’endocytoses et de dégradation de BRI1 et leurs régulations par les conditions environnementales.L’ubiquitination est une modification post-traductionnelle impliquant l’attachement d’un polypeptide d’ubiquitine (Ub) sur une ou plusieurs lysines (K) d’une protéine cible. L’ubiquitine elle-même peut être sujette à l’ubiquitination, créant ainsi des chaînes de poly-ubiquitine (polyUb) qui peuvent adopter des topologies différentes selon le résidu K de l’ubiquitine impliqué dans la formation de la chaîne. Parmi ces chaînes de polyUb, la chaîne impliquant la lysine-63 (K63) est connue pour être impliquée dans la dégradation des protéines par endocytose chez les levures et les mammifères. Néanmoins, peu de choses sont connues sur l’endocytose dépendante de l’ubiquitine chez les plantes. Durant la première partie de ma thèse, j’ai démontré que BRI1 est modifié in vivo par des chaînes de polyUb K63 et j’ai pu identifier des sites putatifs d’ubiquitination dans BRI1. En utilisant une forme artificiellement ubiquitinée de BRI1 ainsi qu’une forme non ubiquitinable de BRI1, j’ai montré que l’ubiquitination joue un rôle sur l’internalisation de BRI1 à la surface cellulaire et est essentielle pour son adressage à la vacuole. Par ailleurs, j’ai établi que la dynamique de la protéine BRI1 médiée par son ubiquitination joue un rôle important dans le contrôle des réponses des BR.La deuxième partie de ma thèse m’a permis de découvrir une connexion entre l’endocytose dépendante de l’ubiquitine de BRI1 et la réponse des plantes à une élévation de température. J’ai notamment montré que l’accumulation de la protéine BRI1 est réduite dans les racines lorsque les plantes sont cultivées à une température plus élevée (i.e. 26°C). Cependant, la forme non ubiquitinable de BRI1 ne répond pas à cette élévation de la température suggérant une implication de l’endocytose dépendant de l’ubiquitine dans de telles conditions. Cette déstabilisation de BRI1 observée à température plus élevée se traduit au niveau moléculaire par une inhibition de la voie de signalisation des BRs et une hypersensibilité à des traitements BRs exogènes. Les plantes répondent à une montée subite de la température par une augmentation de la longueur de l’hypocotyle, des pétioles et de la racine principale ; processus largement sous le contrôle de l’auxine. J’ai accumulé au cours de ma thèse des évidences génétiques et génomiques montrant que la déstabilisation du BRI1 et l’inhibition de la signalisation des BR à plus hautes températures contrôle l’élongation des racines indépendamment de l’auxine. En conclusion, les résultats obtenus indiquent que BRI1 intègre les informations de température et de signalisation des BRs pour ajuster la croissance des racines lors de variations des conditions environnementales. / Brassinosteroids (BRs) are plant steroid hormones that play important roles on plant growth and development, and that are perceived at the plasma membrane (PM) by the receptor kinase BRI1 (BR insensitive 1). The perception of BRs by BRI1 triggers the cis and trans-phosphorylation of BRI1 and its co-receptor BAK1 (BRI1 Associated Receptor Kinase 1) initiating this way the BR signaling cascade that culminates with the de-phosphorylation of the transcription factors BZR1 (Brassinazole Resistant 1) and BES1 (bri1-EMS-Supressor 1), allowing these transcription factors to activate the transcription of thousands of BR-responsive genes. The mechanisms allowing the deactivation of the BR receptor complex are still elusive but may involve dephosphorylation, binding to negative regulators such as BKI1 and endocytosis. My PhD work focused on the endocytosis and the degradation of BRI1, and its regulation by environmental conditions.Ubiquitination is a post-translational modification that consists of the attachment of ubiquitin (Ub) polypeptides to one or several lysine (K) of a target protein. Ubiquitin itself can undergo self-ubiquitination thereby creating polyubiquitin (polyUb) chains that can harbor different topologies depending on the lysine residue from ubiquitin involved in the chain formation. Among these, polyUb chains involving lysine-63 (K63) are known to be involved in the degradation of proteins by endocytosis in yeast and mammals, while very little is known in plants. On a first part of my thesis, I demonstrated that BRI1 is post-translationally modified by K63 poly-ubiquitin chains and I identified putative ubiquitination sites in BRI1. Using both artificial ubiquitination of BRI1 and the generation of an ubiquitination-defective BRI1, I showed that ubiquitination plays a role on the internalization of BRI1 from the cell-surface, and is essential for its vacuolar targeting. Furthermore, ubiquitin-mediated BRI1 protein dynamics was also shown to play an important role in the control of BR responses by stabilizing BRI1 at the PM.The second part of the PhD uncovered a connection between BRI1 ubiquitin-mediated endocytosis and plant responses to elevated ambient temperature. I showed that BRI1 protein accumulation is lower at elevated temperature (i.e. 26°C) in roots, while the ubiquitin-defective form of BRI1 failed to respond, indicating a role of the BRI1 ubiquitin-mediated endocytosis in temperature responses. This temperature-mediated destabilization of BRI1 correlates with a downregulation of BR signaling and BR responses. Plants respond sudden rise in temperature by increasing their primary root length, a process that is under the control of auxin. Importantly, I accumulated genetic and genomic evidence that BRI1 destabilization and downregulation of BR signaling controls root elongation upon elevated ambient growth temperature, with little or no contribution of auxin. Altogether, our results establish that BRI1 integrates temperature and BR signaling to regulate root growth upon long-term changes in environmental conditions.

OBJECTIVE: One of the major issues in current radiotherapy (RT) is the normal tissue toxicity. A smart combination of agents within the tumor would allow lowering the RT dose required while minimizing the damage to healthy tissue surrounding the tumor. We chose gold nanoparticles (GNPs) and docetaxel (DTX) as our choice of two radiosensitizing agents. They have a different mechanism of action which could lead to synergistic effect. Our first goal was to assess the variation in GNP uptake, distribution, and retention in the presence of DTX. Our second goal was to assess the therapeutic results of the triple combination, RT/GNPs/DTX. METHODS: We used HeLa and MDA-MB-231 cells for our study. Cells were incubated with GNPs (0.2nM) in the absence and presence of DTX (50nM) for 24 hrs for determination of uptake, distribution, and retention of NPs. For RT experiment, treated cells were given a 2 Gy dose of 6 MV photons using a linear accelerator. RESULTS: Concurrent treatment of DTX and GNPs resulted in over 85% retention of GNPs in tumor cells. DTX treatment also forced GNPs to be closer to the most important target, the nucleus, resulting in a significant decrease in cell survival with the triple combination of RT, GNPs, and DTX vs. RT plus DTX alone. Our experimental therapeutics results are supported by Monte Carlo simulations. CONCLUSION: The ability to not only trap GNPs at clinically feasible doses but also to retain them within the cells could lead to meaningful fractionated treatments in future combined cancer therapy. Furthermore, the suggested triple combination of RT/GNPs/DTX may allow lowering the RT dose to spare surrounding healthy tissue. ADVANCES IN KNOWLEDGE: This is the first study to show intracellular GNP transport disruption by DTX, and its advantage in radiosensitization. / Graduate / 2020-10-31

Human platelets respond to various macromolecules in the plasma. Uptake of specific ligands, and antibodies to various epitopes on the platelet plasma membrane, has been observed. The platelet canalicular system has been shown to be involved with this uptake. Recently, investigators have speculated on the role of endocytosis in platelets to account for the presence of plasma proteins such as fibrinogen and immunoglobulin within platelet organelles. Antibodies binding to cell-surface antigens on platelets can lead to a redistribution of these antigens. When antibodies, specific for platelet cell-surface receptors, bind to platelets they may either undergo endocytosis into intracellular vacuoles, or may merely become sequestered within the canalicular system of platelets. The present study investigated whether endocytosis occurs in platelets. Such a process would lead to the endocytic uptake of a fluid-phase marker and would involve internalization and recycling of cell surface membrane. A fluid-phase marker (FITC-dextran) was used to measure any constitutive endocytic activity. In addition, a suitable membrane marker was used to determine whether membrane internalization occurred. This involved a technique whereby radioactive galactose was covalently attached to cell-surface glycoconjugates. A monoclonal antibody to the platelet receptor, GPIIbIIIa, was used in conjunction with the membrane marker in order to determine if membrane internalization was involved during the subsequent redistribution of the receptor-antibody complex. Immunocytochemical techniques using electron-dense probes were employed to localise the sites to which this receptor-antibody complex became redistributed. In comparison with reported rates of endocytic uptake of fluid-phase marker in other cell types, no significant endocytic activity could be detected with platelets, after taking their relatively small volume into account. Similarly, membrane internalization was not detected with resting platelets. Following challenge of the platelets with anti-GPIIbIIIa antibody, no membrane internalization could be measured during redistribution of the receptor-antibody complex. The compartment to which the receptor-antibody complex was redistributed could be identified morphologically as the canalicular system. The present data provide evidence for a process of sequestration of receptor-antibody in the canalicular system of resting platelets. It remains possible that other mechanisms exist within the platelet system for uptake of extracellular material as this study dealt exclusively with the platelet response to a specific antibody. These results may have implications with respect to the interaction of platelets with anti-platelet antibodies in the normal state, as well as with clinical disorders involving elevated levels of platelet-associated IgG. As far as can be deduced from the available literature, these data represent the first use of a covalent membrane marker in conjunction with uptake of macromolecules to study endocytic events in human platelets.