Dissertations / Theses on the topic 'Plant autophagy'

Autophagy is a major catabolic pathway in which cell components get sequestered in a double membrane vesicle, transported to the vacuole, degraded by vacuolar hydrolases and recycled.  Through this process, cells ensure cell homeostasis and remobilise nutrients. The autophagic flux can be enhanced as an adaptive stress response, improving plants resistance against stress, reducing aging and ultimately increasing yield. However, autophagy regulation in plants remains poorly understood.  Novel plant-specific modulators can be used in a chemical genetic approach for identification of proteins involved in the autophagy pathway. Furthermore, autophagy enhancers can find their application in agriculture for improved plant fitness. Known autophagy modulators have severe off-target effects, affecting plant growth and development. A recent screening identified two potential autophagy modulators. We developed a novel method for photoaffinity labelling and pulldown assay in Arabidopsis thaliana to identify potential interactors of the modulators. The identification of autophagy-related proteins will help to further elucidate the autophagic pathway in plants. The effect of the new autophagy enhancers on plant growth and development was analysed by automated growth assays. In comparison with a currently available autophagy enhancer, treated plants showed higher viability, indicating possible further applications for the new autophagy modulators in planta.

L’agent pathogène Phytophthora parasitica est un oomycète qui a des effets dévastateurs sur l’agriculture et les écosystèmes naturels. En tant qu'organisme hémi-biotrophe, il infecte les racines des plantes en établissant d'abord un contact intime avec les cellules hôtes (biotrophie) avant de les tuer (nécrotrophie) et de terminer son cycle d'infection. Pour contrôler ces processus, les oomycètes sécrètent des protéines effectrices, qui sont internalisées dans les cellules végétales par un motif de translocation (appelé RxLR-EER) pour manipuler la physiologie et les réponses immunitaires de l'hôte. Les études des échanges moléculaires entre Phytophthora parasitica et la plante qui ont été menées par le laboratoire d'accueil ont permis d'identifier un effecteur RxLR, dénommé Avh195. La séquence en acides aminés de l'effecteur est caractérisée par la présence de cinq motifs AIM (« ATG8 Interacting Motive ») qui indiquent une interaction potentielle avec la protéine centrale de l’autophagie, ATG8. Avh195 co-localise avec la fraction membranaire de l'ATG8, et un système double-hybride en levure permettant la détermination d’interactions entre protéines membranaires, a confirmé une interaction non sélective entre Avh195 et plusieurs isoformes d'ATG8. La caractérisation de la perturbation de l'autophagie dépendante de Avh195 a été réalisée dans l'algue unicellulaire Chlamydomonas reinhardtii après génération de lignées transgéniques surexprimant l'effecteur. Les analyses par cytométrie de flux ont révélé que Avh195 ne modifie pas la physiologie et la « fitness » de l'algue dans des conditions de croissance normales et pendant l'autophagie induite par la rapamycine. La microscopie électronique à transmission a révélé que l'effecteur provoque dans les cellules de l’algue un retard dans le flux autophagique, se traduisant par une réduction de la coalescence et de la clairance des vacuoles et une forte accumulation d'amidon dans les chloroplastes. Cependant, ce phénotype est transitoire et seulement légèrement lié aux modifications de la régulation transcriptionnelle de la machinerie autophagique. L'analyse de la fonction effectrice chez les plantes a montré que Avh195 retarde le développement de la mort cellulaire hypersensible, déclenchée par un éliciteur d’oomycète. Cette activité dépend de trois AIM sur cinq, ce qui renforce encore l’importance de l’interaction Avh195-ATG8 pour la fonction de l’effecteur. La surexpression stable d'Avh195 chez A. thaliana a permis de déterminer que l'effecteur n'altère pas les réponses immunitaires des plantes, mais favorise globalement le développement de l'agent pathogène, accélérant le passage de la biotrophie à la nécrotrophie au cours de l'infection. À notre connaissance, le travail présenté dans cette thèse représente la première preuve qu'un effecteur d’oomycète possède une activité transitoire, ciblant de manière non sélective la protéine ATG8 dans différents organismes photosynthétiques pour ralentir le flux autophagique, favorisant ainsi le mode de vie hémi-biotrophe d'un agent pathogène<br>The plant pathogen Phytophthora parasitica is an oomycete with devastating impact on both agriculture and natural ecosystems. As a hemi-biotrophic organism it infects the roots of plants first establishing an intimate contact with host cells (biotrophy) before killing them (necrotrophy) and completing its infection cycle. To control these processes, oomycetes secrete effector proteins, which are internalized in plant cells by a translocation motif (called RxLR-EER) to manipulate the physiology and the immune responses of the host. Studies of the molecular exchanges between Phytophthora parasitica and the plant that were conducted by the hosting laboratory led to the identification of an RxLR effector, designed to as Avh195. The amino acid sequence of the effector is characterized by the presence of five AIMs (ATG8 interacting motifs), that indicate a potential interaction with the autophagic core protein, ATG8. Avh195 colocalizes with the membrane-bound fraction of ATG8, and a yeast two-hybrid system, which allows to determine interactions between membrane proteins, confirmed a non-selective interaction between Avh195 and several ATG8 isoforms. The characterization of Avh195-dependent autophagy perturbation was carried out in the unicellular alga Chlamydomonas reinhardtii after generation of transgenic lines overexpressing the effector. Analyses by flow cytometry revealed that Avh195 does not modify the physiology and fitness of the alga, both under normal growth conditions and during rapamycin-induced autophagy. Transmission electron microscopy of cells revealed that the effector provokes a delay in the autophagic flux, manifested as a reduced coalescence and clearance of autophagic vacuoles and a strong accumulation of starch in chloroplasts. However, this phenotype was transient and only slightly related to modifications in the transcriptional regulation of the autophagic machinery. The analysis of effector function in planta showed that Avh195 delays the development of hypersensitive cell death, which is triggered by an oomycete elicitor. This cell death-delaying activity is dependent on three out of five AIMs, further consolidating the importance of the Avh195-ATG8 interaction for the function of the effector. The stable overexpression of Avh195 in A. thaliana allowed to determine that the effector does not impair plant defense responses, but overall promotes the development of the pathogen, accelerating the switch from biotrophy to necrotrophy during infection. To our knowledge, the work presented in this thesis represents the first evidence for an oomycete effector to possess a transitory activity, which targets in a non-selective manner the protein ATG8 in different organisms from the green lineage to slow down autophagic flux, thus promoting the hemibiotrophic life style of a pathogen

Les plantes, étant des organismes sessiles, sont fréquemment confrontées à une grande variété de stress environnementaux. Ces conditions peuvent conduire à l'accumulation d'agrégats de protéines ou au disfonctionnement de multiples organites intracellulaires. Pour faire face à ces conditions, les plantes ont mis au point des mécanismes d'adaptation sophistiqués qui permettent le recyclage des composants intracellulaires. Ces mécanismes sont essentiels pour les remodelages métaboliques nécessaires à un recyclage efficace des nutriments ainsi qu'à l'élimination des composants nocifs pour la cellule comme des organites endommagés. L'un de ces mécanismes est l'autophagie, une voie de dégradation intracellulaire qui utilise des vésicules à double membrane qui encapsulent des portions du cytoplasme et le délivrent à la vacuole où elles sont dégradées. L'autophagie repose sur la formation de ces vésicules spécialisées, appelées autophagosomes (AP). Les AP sont des vésicules uniques dans le système endomembranaire, d'abord parce qu'elles sont constituées d'une double couche lipidique, et ensuite parce qu'elles ne bourgeonnent pas à partir d'un compartiment déjà existant. La biogenèse des AP est un processus en plusieurs étapes impliquant une machinerie centrale (protéines ATG) qui intervient dans la formation de novo d'une membrane initiale ; puis, par l'addition de lipides, cette membrane s’agrandit et devient une structure en forme de coupe avec des bords fortement incurvés lui permettant d’engloutir la cargaison autophagique. Une fois la cargaison engloutie, ses bords fusionnent afin de fermer la structure, qui circule ensuite vers la vacuole, où sa membrane externe fusionne avec la membrane de la vacuole ce qui libère la membrane interne et la cargaison à l'intérieur de la vacuole pour sa dégradation. Ainsi, la biogenèse des AP repose sur de nombreux événements de remodelage membranaire, d'abord pour initier la membrane initiale, puis pour maintenir sa forme très incurvée tout en assurant son expansion, et enfin pour sceller les structures matures et promouvoir sa fusion ultérieure à la vacuole. Dans les membranes biologiques, les lipides, grâce à leurs propriétés physico-chimiques, définissent des caractéristiques importantes telles que la fluidité, la courbure ainsi que les champs électrostatiques des membranes. Par conséquent, le rôle crucial des lipides dans l'autophagie a émergé ces dernières années. Chez les plantes, on ne connait encore que très peu de choses sur la composition lipidique des membranes autophagiques et les fonctions des lipides dans la formation des AP restent largement méconnu. Mon travail de thèse a consisté à identifier des acteurs lipidiques et protéiques impliquées dans l'autophagie chez les plantes dans le but de caractériser leur fonction dans le processus. En effectuant un criblage d'inhibiteurs enzymatiques nous avons analysé l'impact de l'inhibition de la synthèse de différentes espèces de lipides sur l'autophagie. En utilisant cette approche, nous avons identifié différents candidats lipidiques importants pour l'autophagie des plantes. Notamment, nous avons identifié le phosphatydilinositol-4-phosphate (PI4P) comme étant critique pour la formation des APs. En l'absence dePI4P, la formation des AP est stoppée à un stade très précoce, ce qui entraîne un blocage total dans le processus. De plus, nous avons obtenu des informations précieuses pour mieux comprendre la formation des APs chez les plantes. En particulier, nos résultats suggèrent que la membrane plasmique (PM) semble jouer un rôle important dans la formation de ces structures. Dans leur ensemble, nos résultats ont confirmé notre hypothèse initiale: les lipides ne sont pas seulement des éléments inertes qui constituent les membranes autophagiques ;ils semblent plutôt jouer des rôles distincts et avoir des fonctions spécifiques dans le processus<br>Plants, being sessile organisms, are frequently confronted to a plethora of environmental stresses and harsh conditions. Enduring these conditions can lead to the accumulation of protein aggregates or organelles that become dysfunctional. To withstand these conditions, plants have evolved sophisticated adaptation mechanisms for the recycling of intracellular components. These mechanisms are essential for the metabolic transitions required for efficient nutrient use, as well as proper disposal of protein aggregates or damaged organelles. One of these mechanisms is autophagy, an intracellular degradation pathway that employs specialized double membrane vesicles that encapsulate cytosolic material and delivers it to the vacuole for degradation. Autophagy relies on the formation of these specialized vesicles, called autophagosomes (APs). APs are unique vesicles in the endomembrane system, first because they are made of a double lipid bilayer, and second because they do not but from a pre-existing compartment. AP biogenesis is a multistep process implicating a core machinery (ATG proteins) that mediate the de novo formation of an initial membrane; then, by the addition of lipids, this membrane expands into a cup-shaped structure with highly curved edges to engulf autophagic cargo. Upon completion, the rims of the structure seal and form a mature AP that traffics to the vacuole, where its outer membrane fuses with the tonoplast releasingthe inner membrane and cargo inside the vacuole. Thus, AP biogenesis relies on numerous membrane remodeling events, first to initiate the initial membrane, then to maintain the highly curved shape of the structure while ensuring its expansion, and finally to seal the mature structures and its subsequent fusion to the vacuole. Lipids, thanks to their physicochemical properties define important membrane features such as its, fluidity, curvature and electrostatics. Hence, evidence showing the crucial role of lipids in autophagy has emerged in the recent years. In plants however, little is known about the lipid composition of autophagic membranes and thus, about the functional contribution of lipids in plant autophagy. My PhD thesis consisted on identifying crucial lipids for plant autophagy with an aim to characterize their function in the process. By performing a lipid-related enzymes inhibitor screen in which we assayed the impact of inhibiting the synthesis of specific lipids on autophagy, we identified different lipid candidates important for plant autophagy. Notably, we identified the phosphatydil-inositol-4-phosphate (PI4P) as being critical for the formation of APs. In the absence of PI4P, AP formation is stalled at a very early stage resulting in a block in the process. Furthermore, we have obtained valuable insights to better understand the AP formation. In plants, particularly, our results suggest that the plasma membrane (PM) plays important roles in the formation of these structures. Taken together, our results confirmed that lipids are more than just building blocks constituting the autophagic membranes; rather, they seem to play distinct and specific roles in the pathway. Finally, this thesis highlights how lipids are key actors for the autophagic process and thus for plants adaptations to adverse and stressful environmental conditions

L’agent pathogène Phytophthora parasitica est un oomycète qui a des effets dévastateurs sur l’agriculture et les écosystèmes naturels. En tant qu'organisme hémi-biotrophe, il infecte les racines des plantes en établissant d'abord un contact intime avec les cellules hôtes (biotrophie) avant de les tuer (nécrotrophie) et de terminer son cycle d'infection. Pour contrôler ces processus, les oomycètes sécrètent des protéines effectrices, qui sont internalisées dans les cellules végétales par un motif de translocation (appelé RxLR-EER) pour manipuler la physiologie et les réponses immunitaires de l'hôte. Les études des échanges moléculaires entre Phytophthora parasitica et la plante qui ont été menées par le laboratoire d'accueil ont permis d'identifier un effecteur RxLR, dénommé Avh195. La séquence en acides aminés de l'effecteur est caractérisée par la présence de cinq motifs AIM (« ATG8 Interacting Motive ») qui indiquent une interaction potentielle avec la protéine centrale de l’autophagie, ATG8. Avh195 co-localise avec la fraction membranaire de l'ATG8, et un système double-hybride en levure permettant la détermination d’interactions entre protéines membranaires, a confirmé une interaction non sélective entre Avh195 et plusieurs isoformes d'ATG8. La caractérisation de la perturbation de l'autophagie dépendante de Avh195 a été réalisée dans l'algue unicellulaire Chlamydomonas reinhardtii après génération de lignées transgéniques surexprimant l'effecteur. Les analyses par cytométrie de flux ont révélé que Avh195 ne modifie pas la physiologie et la « fitness » de l'algue dans des conditions de croissance normales et pendant l'autophagie induite par la rapamycine. La microscopie électronique à transmission a révélé que l'effecteur provoque dans les cellules de l’algue un retard dans le flux autophagique, se traduisant par une réduction de la coalescence et de la clairance des vacuoles et une forte accumulation d'amidon dans les chloroplastes. Cependant, ce phénotype est transitoire et seulement légèrement lié aux modifications de la régulation transcriptionnelle de la machinerie autophagique. L'analyse de la fonction effectrice chez les plantes a montré que Avh195 retarde le développement de la mort cellulaire hypersensible, déclenchée par un éliciteur d’oomycète. Cette activité dépend de trois AIM sur cinq, ce qui renforce encore l’importance de l’interaction Avh195-ATG8 pour la fonction de l’effecteur. La surexpression stable d'Avh195 chez A. thaliana a permis de déterminer que l'effecteur n'altère pas les réponses immunitaires des plantes, mais favorise globalement le développement de l'agent pathogène, accélérant le passage de la biotrophie à la nécrotrophie au cours de l'infection. À notre connaissance, le travail présenté dans cette thèse représente la première preuve qu'un effecteur d’oomycète possède une activité transitoire, ciblant de manière non sélective la protéine ATG8 dans différents organismes photosynthétiques pour ralentir le flux autophagique, favorisant ainsi le mode de vie hémi-biotrophe d'un agent pathogène<br>The plant pathogen Phytophthora parasitica is an oomycete with devastating impact on both agriculture and natural ecosystems. As a hemi-biotrophic organism it infects the roots of plants first establishing an intimate contact with host cells (biotrophy) before killing them (necrotrophy) and completing its infection cycle. To control these processes, oomycetes secrete effector proteins, which are internalized in plant cells by a translocation motif (called RxLR-EER) to manipulate the physiology and the immune responses of the host. Studies of the molecular exchanges between Phytophthora parasitica and the plant that were conducted by the hosting laboratory led to the identification of an RxLR effector, designed to as Avh195. The amino acid sequence of the effector is characterized by the presence of five AIMs (ATG8 interacting motifs), that indicate a potential interaction with the autophagic core protein, ATG8. Avh195 colocalizes with the membrane-bound fraction of ATG8, and a yeast two-hybrid system, which allows to determine interactions between membrane proteins, confirmed a non-selective interaction between Avh195 and several ATG8 isoforms. The characterization of Avh195-dependent autophagy perturbation was carried out in the unicellular alga Chlamydomonas reinhardtii after generation of transgenic lines overexpressing the effector. Analyses by flow cytometry revealed that Avh195 does not modify the physiology and fitness of the alga, both under normal growth conditions and during rapamycin-induced autophagy. Transmission electron microscopy of cells revealed that the effector provokes a delay in the autophagic flux, manifested as a reduced coalescence and clearance of autophagic vacuoles and a strong accumulation of starch in chloroplasts. However, this phenotype was transient and only slightly related to modifications in the transcriptional regulation of the autophagic machinery. The analysis of effector function in planta showed that Avh195 delays the development of hypersensitive cell death, which is triggered by an oomycete elicitor. This cell death-delaying activity is dependent on three out of five AIMs, further consolidating the importance of the Avh195-ATG8 interaction for the function of the effector. The stable overexpression of Avh195 in A. thaliana allowed to determine that the effector does not impair plant defense responses, but overall promotes the development of the pathogen, accelerating the switch from biotrophy to necrotrophy during infection. To our knowledge, the work presented in this thesis represents the first evidence for an oomycete effector to possess a transitory activity, which targets in a non-selective manner the protein ATG8 in different organisms from the green lineage to slow down autophagic flux, thus promoting the hemibiotrophic life style of a pathogen

Salinity stress is one of the main challenges for crop growth and production. The estimated loss of crop yield due to salinity stress is up to 20% worldwide each year. Plants have evolved an array of mechanisms to defend themselves against salinity stress. A key aspect of plant responses to salinity stress is the engagement of a nitrosative burst that results in nitric oxide (NO) accumulation. A major mechanism for the transfer of NO bioactivity is S-nitrosylation which is a modification of the reactive thiol group of a rare but highly active cysteine residue within a protein through the addition of a NO moiety to generate an S-nitrosothiol (SNO). S-nitrosylation can result in altered structure, function and cellular localisation of a protein. Our findings suggest that S-nitrosylation is a key regulator of plant responses to salinity stress. Glutathione (GSH), a tripeptide cellular antioxidant, is S-nitrosylated to form S-nitrosoglutathione (GSNO), which functions as a stable store of NO bioactivity. Cellular GSNO levels are directly controlled by S-nitrosoglutathione reductase (GSNOR), thereby, regulating global SNO levels indirectly. The absence of this gene results in high levels of SNOs. In Arabidopsis, previous research has shown that loss-of-function mutation in GSNOR1 results in pathogen susceptibility (Feechan et al., 2005). In our study, we investigated salt tolerance in gsnor1-3 plants. We have found that this line is salt sensitive at various stages of their life cycle. Interestingly, classical salt stress signalling pathways are fully functional in gsnor1-3 plants. We have also explored non-classical pathways involved in salt tolerance. Autophagy is a cellular catabolic process which is involved in the recycling and degradation of unwanted cellular materials under stressed and non-stressed conditions. We have demonstrated that gsnor1-3 plants have impaired autophagy during salt stress. An accumulation of the autophagy marker NBR1 supports the lack of autophagosome formation. We hypothesised that S-nitrosylation might regulate upstream nodes of autophagosome formation. Our study demonstrated that at least one key player involved in autophagosome biogenesis is regulated by S-nitrosylation. ATG7, an E1-like activating enzyme, which regulates ATG8-PE and ATG12-ATG5 ubiquitin like conjugation systems, is S-nitrosylated in vitro and in vivo. S-nitrosylation of ATG7 impairs its function in vitro. We showed that S-nitrosylation of ATG7 is mediated by GSNO. Interestingly, ATG7 is also transnitrosylated by thioredoxin (TRX), another important redox regulatory enzyme. We suggest that similar mechanisms might exist in planta. Finally, work in this study revealed that S-nitrosylation of Cys558 and Cys637 cause the inhibition of ATG7 function. In aggregate, this study revealed a novel mechanism for the redox-based regulation of autophagy during salt stress.

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第21829号<br>農博第2342号<br>新制||農||1068(附属図書館)<br>学位論文||H31||N5201(農学部図書室)<br>京都大学大学院農学研究科地域環境科学専攻<br>(主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博<br>学位規則第4条第1項該当

Recent studies have shown that T.loliiformis, a resurrection grass, may use the tight regulation of PCD pathways like autophagy to facilitate desiccation tolerance. The aim of this project was to further investigate the mechanisms that T.loliiformis use to suppress PCD and survive prolonged periods of water deficit. This study provides additional insight on how T.loliiformis attains desiccation tolerance that significantly contributes to our knowledge on the pathways and mechanisms used by resurrection plants. The findings from this study will hopefully be employed to harness drought tolerant properties of resurrection grasses to improve the resilience of economically important crops.

Pancreatic cancer is known to be one of the most life-threatening cancers characterized by aggressive local invasion and distant metastasis. The high basal level of autophagy in pancreatic cancer may be responsible for the low chemotherapeutic drug response rate and poor disease prognosis. However, the clinical application of autophagy inhibitors was unsatisfactory due to their toxicity and minimal single-agent anticancer efficacy. Hence, oncologists begin to consider the tumor microenvironment when exploring new drug targets. In the present study, the anti-tumorigenic mechanisms of two major phytochemicals derived from Chinese medicinal herbs had been investigated against pancreatic cancer development. Calycosin is a bioactive isoflavonoid of the medicinal plant Astragalus membranaceus. Our results have shown that calycosin inhibited the growth of various pancreatic cancer cells both in vitro and in vivo by inducing cell cycle arrest and apoptosis. Alternatively, calycosin also facilitated MIA PaCa-2 pancreatic cancer cell migration in vitro and increased the expression of epithelial-mesenchymal transition (EMT) biomarkers in vivo. Further mechanistic study suggests that induction of the Raf/MEK/ERK pathway and facilitated polarization of M2 tumor-associated macrophage in the tumor microenvironment both contribute to the pro-metastatic potential of calycosin in pancreatic cancer. These events appear to be associated with calycosin-evoked activation of TGF-β signaling, which may explain the paradoxical drug actions due to the dual roles of TGF-β as both tumor suppressor and tumor promoter in pancreatic cancer development under different conditions. Isoliquiritigenin (ISL) is a chalcone obtained from the medicinal plant Glycyrrhiza glabra, which can be a precursor for chemical conversion to form calycosin. Results have shown that ISL decreased the growth and EMT of pancreatic cancer cells in vitro, probably due to modulation of autophagy. ISL-induced inhibition of autophagy subsequently promoted reactive oxygen species (ROS) production, leading to induction of apoptosis in pancreatic cancer cells. Such phenomenon also contributed to the synergistic growth-inhibitory effect in combined treatment with the orthodox chemotherapeutic drug 5-fluorouracil. In addition, ISL-induced tumor growth inhibition in vivo was further demonstrated in a tumor xenograft mice model of pancreatic cancer. ISL promoted apoptosis and inhibited autophagy in the tumor tissues. Study on immune cells indicates that ISL could reduce the number of myeloid-derived suppressor cells (MDSCs) both in tumor tissue and in peripheral blood, while CD4+ and CD8+ T cells were increased correspondingly. In vitro test has revealed that ISL inhibited the polarization of M2 macrophage along with its inhibition of autophagy in M2 macrophage. These immunomodulating effects of ISL had reversed the pro-invasive role of M2 macrophage in pancreatic cancer.In conclusion, calycosin acts as a "double-edged sword" on the growth and metastasis of pancreatic cancer, which may be related to the dual roles of TGF-β and its influence on the tumor microenvironment. Alternatively, ISL consistently inhibited the growth and metastatic drive of pancreatic cancer through regulation of autophagy and reprogramming of the immune system. The differential modes of action of these compounds have provided new insights in the development of effective pancreatic cancer treatment adjuvants.

During severe hypoxia (<0.01% oxygen) the protein folding machinery becomes dysfunctional, resulting in the accumulation of unfolded proteins with consequent endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) and autophagy, a process involved in the physiological turnover of cytoplasmic components. The link between the UPR and autophagy is not clearly defined. The aim of this thesis is to investigate the role of the induction of UPR under severe hypoxia in tumour survival and resistance to therapy. The results of this research suggest that the activating transcription factor 4 (ATF4), a component of the PKR-like ER kinase (PERK) pathway, fundamental in the UPR, is required for the ER-stress induced upregulation of autophagy. Mechanisms other than hypoxia for UPR induction were investigated, using the proteasome inhibitor bortezomib (BZ). BZ treatment increased ATF4 protein levels in MCF7 cells, even transfected with short-interference RNA (siRNA) against the classical UPR activator PERK, suggesting that the proteasomal stabilization is likely the main mechanism for ATF4 protein accumulation. The induction of autophagy by BZ is dependent upon the upregulation of the microtubule-associated protein 1 light chain 3B (LC3B), an autophagy marker, by ATF4 and acts as a survival mechanism. Hypoxia, UPR and autophagy markers (such as Pimonidazole, carbonic anhydrases IX (CAIX), C/EBP homologous protein (CHOP) and LC3B) were evaluated by immunohistochemical approach in spheroids, xenografts models and breast cancer samples. CHOP immunohistochemical staining was performed in breast cancer sections from a series of patients. CHOP was expressed in cells surrounding necrotic areas. No correlation were found with clinical outcome and further studies are needed.

Research in this thesis focussed on the improvement of agricultural crops in increasing water use efficiency that impacts global crop productivity. The study identified key genetic regulatory mechanisms that the resurrection plant Tripogon loliiformis utilises to tolerate desiccation. Due to the conserved nature of the pathways involved, this information can be transferred for the enhancement of drought tolerance and water use efficiency in agricultural crops. Specifically this study used high throughput sequencing, microscopy and plant transformation to further the understanding of post-transcriptional regulatory mechanisms. It was shown that T. loliiformis uses microRNAs to regulate pro-survival autophagy pathways to tolerate desiccation.

This thesis reports the research I conducted on aspects of the pharmacology and biological activities of two natural stilbenes, Resveratrol (Rv) and Pterostilbene (Pt), major polyphenolic components of grapevines and blueberries respectively. Over the years, these two molecules have drawn attention from the scientific community thanks to their beneficial bioactivities, relevant for many areas of health care. Numerous papers describe their protective roles against the metabolic syndrome, cancer development and neurodegeneration. These striking effects nowadays are not exclusively attributed to their redox properties but also to their capacity to modulate, directly or indirectly, the activities of key proteins of interconnected cellular signalling networks. Importantly, no noxious side effects have been reported. However, in spite of their positive features, Rv and Pt are not free of shortcomings. They are subjected to an intensive phase II metabolism, largely limiting their bioavailability. Moreover, while much research has been carried out to elucidate the molecular mechanisms of action of Rv, those of Pt still largely remain to be established. During my PhD program, I addressed both these themes. I describe below: 1) the attempts to increase the bioavailability of Rv and Pt 2) my investigations on intracellular processes affected by Pt as well as the demonstration of its therapeutic potential in two different in vivo models 1) A variety of approaches has been developed to overcome the poor bioavailability of many drugs. My research group turned to the development of prodrugs for oral administration. In the case of polyphenols, a useful prodrug implies the use of protecting groups to mask the characteristic hydroxyl groups and to avoid metabolic transformations by phase II conjugative enzymes. These substituents, technically defined as pro-moieties, are attached to the backbone of the molecule through a chemical bond which can be broken in the relevant physiological environment. The choice of the pro-moiety is important for the process of absorption from the intestinal tract. The type of linkage is crucial to achieve “cargo” regeneration with appropriate kinetics. We created prodrugs for oral administration. Thus, they ought to exhibit relative stability in the gastro-intestinal tract and lability in other biological compartments (body fluids and organs). When I joined the group of Dr. Mario Zoratti, N,N-disubstituted carbamoyl derivatives of Rv bearing a PEG 350 or a sugar pro-moiety had been created. Although they increased the poor water solubility of the natural compound, these prodrugs were too stable under physiological conditions. Thus, we moved to the N-monosubstituted carbamoyl bond, expected to be less stable, for new prodrugs. Importantly, this type of chemical bond is essentially stable in acid, while it can be more easily lysed at neutral or basic pH values. A first set of compounds incorporated amino acids as pro-moieties. We hypothesized that in addition to increasing water solubility, amino acids might be recognized and transported by specific carrier systems, expressed in the enterocytes, and thus favor the intestinal absorption of the molecules. In another set of Nmonosubstituted carbamoyl derivatives polyhydroxylated groups (dihydroxypropyl or 6-deoxygalactose) were attached to all three, two or one phenolic oxygen(s) of Rv. In this case, the hope was that sugar transporters might intervene to facilitate absorption through the intestinal epithelium. All synthetized compounds displayed hydrolysis kinetics suitable for their use as prodrugs. However, absorption after oral administration to rats turned out to be rather unsatisfactory, with no evidence of an involvement of membrane carriers. In no case did galactosylated Rv derivatives reach the bloodstream of rats. The best results were obtained by administering monosubstituted aminoacid derivatives. We speculate that the extended, three-pronged structure of the fully protecting prodrugs might interfere with recognition and transport. All synthesis procedures and the characterization and pharmacokinetic evaluation of these families of compounds have been already published. The papers are included in this thesis as chapters 1 and 2. Once completed the assessment of Rv prodrugs, I started a new research project involving Pterostilbene, a more compact and possibly more effective compound than Resveratrol. This represented the main theme of my PhD program investigations. As Pt was still poorly defined from a pharmacological point of view, we first evaluated its distribution in blood and organs after oral administration (see chapter 3). Thus, we developed an appropriate method for the quantification of the molecule and its metabolites in organs after administration to rats. The resulting protocol preserves stability and allows the quantitative extraction and clarification from biological matrices of Pt itself and its metabolites, and their quantitative analysis by HPLC/UV. We used this method to determine the levels of these compounds in the blood and several other organs as a function of time after administration as a single intra-gastric dose of 88µmoles/kg of body weight. Pterostilbene levels in several organs turned out to be much higher than those measured in blood, reaching nmoles/gr (μM)-range concentrations. Moreover, Pt-4’-Sulfate was identified as the major metabolic species. Its levels were higher than those of Pt in all tissues examined except the brain. In light of this, we next aimed to prevent metabolic modifications by phase II conjugative enzymes. I exploited the experience derived from the work with the prodrugs of Rv and in collaboration with the research group of Prof. Paradisi of the Department of Chemical Sciences of the University of Padua, we developed and characterized a collection of Pt prodrugs bearing amino acids as pro-moieties, linked to the Pt 4’-OH via an Nmonosubstituted carbamate ester group. The rates of hydrolysis of all compounds fell in a range suitable for their use. Derivatives with hydrophobic aminoacid side chains were notable for the high levels of Pterostilbene they regenerated in blood after intragastric administration. We selected the most promising one and we measured its levels in rat organs, following the same methodology developed for Pterostilbene. This time, the pro-drug itself was the major specie measured in all organs considered (except the brain) but, most importantly, the levels of (regenerated) Pterostilbene were drastically increased and those of its sulfate decreased in comparison with the administration of the polyphenol as such (see chapter 4). 2) As mentioned above, the intracellular processes underlying Pt benefits are still largely undefined. Several papers suggest that the induction of autophagy may contribute importantly. I decided to investigate this aspect. Autophagy is a catabolic pathway whose dysregulation is associated with various pathological conditions. As discovered by Prof. Ballabio and co-workers it is mainly controlled by transcription factor EB (TFEB), normally inhibited by mTORC1. Thus, I evaluated the effect of Pt on the autophagy master regulator. We demonstrated that this polyphenol stimulates TFEB activity by promoting its translocation to the nucleus as well as its expression. Accordingly, Pt induced an increase of LC3B protein, an autophagy marker, and an up-regulation of TFEB lysosomal target genes. These investigations have been extended to two Pt major metabolites, Pt-4’-Sulfate and DiHydroPt, as they are the main species formed upon ingestion of the natural compound. Interestingly, while the former was ineffective, the reduced form showed an activity similar to that of the parent compound but required higher concentrations. Further studies have been carried out to clarify the upstream signalling cascade. FRET-sensor based measurements have shown that Pt is able to modestly increase the concentration of cAMP, followed by the activation of CREB. This cyclic nucleotide has been previously reported to indirectly activate AMPK, a well-recognized mTORC1 antagonist. Thus, the enhancement of this cellular axis may be responsible for the activation of TFEB. Accordingly, we observed a reduction of the activity of the mammalian target of the rapamycin. However, pharmacological interventions expected to drastically increase cAMP and AMPK activity were less effective than Pt, suggesting that this phenolic compound promotes TFEB nuclear migration by modulating more than one signalling pathway. In addition to mTORC1 inhibition, the activation of TFEB is mediated by Calcineurin. Recently, it has been demonstrated that both endogenous and exogenous ROS may promote the activity of this phosphatase. We observed in in vitro studies with cultured cells that indeed Pt increased the production of these species by mitochondria and that this phenomenon is related to TFEB migration. In light of this, it is possible to speculate that this may represent an alternative way through which this phenolic compound perturbs TFEB intracellular localization (see chapter 5). The establishment of the capability of Pt to induce autophagy in cultured cells led me to test Pt as a potential therapeutic treatment for Collagen VI (ColVI) muscular dystrophies. These disorders are mainly characterized by the presence of dysfunctional mitochondria. The concomitant deficiency of the autophagic process results in the exacerbation of the pathological conditions since the apoptotic death of myofibers occurs. As known from the literature, the use of an antisense morpholino is nowadays the best strategy to obtain zebrafish animal models with a strong phenotype of ColVI-related muscle dystrophy. Therefore, we injected into fertilized eggs a designed oligonucleotide, specifically directed against the ColVI exon 9 splicing region. This results in a frame-shift deletion of the N-terminal region of the ColVIa1 triple helical domain and therefore in a strong impairment of the organization of the muscular fibers. The data I have obtained using this model indicate that Pt treatment induces a recovery of muscular structure by more than 30%, as well as a clear recovery of motor activity.This part of the project has been carried out in collaboration with Prof. Paolo Bernardi and Dr. Marco Schiavone of the University of Padua. Although these results do not provide evidence that the amelioration of the dystrophic phenotype is due to the induction of autophagy, it is credible to suppose that the same cascade of events enhanced in vitro by Pt may be relevant also in vivo. Supporting this hypothesis, Pt provoked a significant increase of a mCherry protein, whose expression is under control of cAMP responsive elements (CRE) in a zebrafish transgenic reporter (this model has been generated by Dr. Patrizia Porazzi and Prof. Natascia Tiso of the University of Padua). Further investigations need to be performed (see chapter 6). Finally, during my graduate training I have also participated in another project still ongoing in my research laboratory. The pharmacokinetic study we performed in rats showed that Pt was particularly abundant in the brain. This observation is consistent with several papers showing a role of this phenolic compound in ameliorating performance of old rodents in behavioral tests. However, also in this context, the mechanisms accounting for these effects have been poorly characterized. My in vitro data, confirmed later in zebrafish, suggest that Pt may activate CREB following an increase of cAMP. This transcription factor has been demonstrated to play a crucial role in memory consolidation as it promotes neurogenesis in the dentate gyrus, a subarea of the hippocampus, in adult individuals. Memory, among other functions, undergoes deterioration in old age. In light of this, we set up an experimental work (see chapter 7), in collaboration with Prof. Nicoletta Berardi and the research group of Dr. Alessandro Sale, aimed to evaluate the molecular changes in the dentate gyrus and the hippocampus related to the recovery of cognitive impairments in old rats by Pt, if any. The results confirmed that after chronic administration of Pt aged animal presented a remarkable improvement in memory-related behavioral tasks. Moreover, although statistics need to be improved by adding more animals, Western blot and gene expression analyses suggest that the treatment up-regulates the activity of transcription factor CREB. An increase of mitochondrial mass has been also measured. These effects were more evident in the dentate gyrus if compared to the remaining hippocampus and strongly support the occurrence of neuronal remodeling processes. However, no differences in PSD95 levels have been observed. Further markers of synaptic plasticity will need to be taken into account in future investigations. Concluding, the studies I carried out have outlined one of the mechanisms accounting for the striking properties of the Pt, characterized its organ pharmacokinetics in the rat and allowed to boost its bioavailability. Thus, they may be helpful for the development of Pt-based therapeutic/preventive treatments.<br>Questa tesi riguarda la ricerca che ho condotto su aspetti della farmacologia e delle attività biologiche di due stilbeni, il Resveratrolo (Rv) e lo Pterostilbene (Pt), componenti polifenoliche principali rispettivamente della vite e dei mirtilli. Negli ultimi anni queste due molecole hanno attirato l’attenzione della comunità scientifica grazie alle loro attività biologiche di rilievo per molte settori della medicina. Numerosi studi descrivono le loro azioni protettive nei confronti di sindromi metaboliche, contro il cancro e la neurodegenerazione. Al giorno d’oggi, questi effetti non vengono attribuiti tanto alle loro proprietà antiossidanti quanto alla loro capacità di modulare, direttamente o indirettamente, l’attività di proteine-chiave in vie di segnalazione intracellulare tra loro interconnesse. Un punto importante è che non sono stati riportati effetti collaterali negativi del loro utilizzo. Nonostante questi aspetti positivi, Rv e Pt presentano dei punti deboli. Entrambi sono soggetti ad un intenso metabolismo di fase II che ne limita significativamente la biodisponibilità. Inoltre, mentre molto lavoro è stato condotto con il fine di elucidare i meccanismi di azione del Rv, quelli dello Pt rimangono ancora in gran parte da chiarire. Durante il mio corso di dottorato, mi sono occupata di entrambe queste problematiche. Descrivo di seguito: 1) I tentativi effettuati per aumentare la biodisponibilità del Rv e dello Pt 2) Gli studi dei processi intracellulari innescati dallo Pt e la valutazione del suo potenziale terapeutico in due modelli in vivo 1) Per far fronte al problema della scarsa biodisponibilità sono stati adottati diversi metodi. Il mio gruppo di ricerca è ricorso alla produzione di pro-farmaci. Nel caso dei polifenoli, un buon pro-farmaco implica l’utilizzo di gruppi protettori per mascherare i gruppi ossidrilici tipici di queste molecole e per evitare le modifiche metaboliche da parte degli enzimi coniugativi di fase II. Tali sostituenti, chiamati “pro-moieties”, sono legati chimicamente alla struttura stilbenica in modo reversibile nelle condizioni fisiologiche d’impiego. Essi assumono particolare importanza per l’assorbimento dei composti, mentre la tipologia del legame è cruciale affinché le molecole attive vengano rigenerate con cinetiche adeguate. Noi abbiamo voluto creare dei pro-farmaci da somministrare oralmente. Pertanto essi dovevano essere relativamente stabili nel tratto gastro-intestinale e più labili negli altri compartimenti biologici (fluidi corporei e organi). Quando ho iniziato la mia esperienza nel gruppo di ricerca del Dott. Mario Zoratti, erano stati sintetizzati dei derivati del Rv come esteri carbammici N,N-bisostituiti recanti come sostituenti (pro-moieties) PEG 350 o gruppi glicosidici. Questi profarmaci aumentavano la scarsa solubilità in acqua del composto fenolico, ma si sono rivelati troppo stabili in ambiente fisiologico per essere utilizzati come pro-drugs. Abbiamo quindi deciso di creare dei derivati carbamoilici N-monosostituiti che si prevedeva sarebbero stati meno stabili. Un aspetto importante è che questa struttura chimica è essenzialmente stabile in acido, mentre può essere idrolizzata a pH vicino alla neutralità o basico. In un primo set di composti si sono utilizzati come promoieties degli aminoacidi. Oltre ad aumentare la solubilità in acqua, ci si aspettava che gli aminoacidi potessero essere riconosciuti da specifici sistemi di trasporto espressi a livello degli enterociti favorendo l’assorbimento intestinale della molecola. In un altro gruppo di derivati carbamoilici N-monosostituiti, tutti e tre, due o uno solo degli ossigeni fenolici del Resveratrolo sono stati decorati con gruppi poliossidrilati (diidrossipropil o 6-deossigalattosil). In questo caso si sperava che i trasportatori di zuccheri potessero intervenire per facilitarne l’assorbimento attraverso l’epitelio intestinale. Come ci si attendeva, tutti i composti sintetizzati venivano idrolizzati con cinetiche compatibili con la loro utilizzazione come pro-drugs. L’assorbimento dopo somministrazione orale invece è risultato essere insoddisfacente, senza alcuna evidenza di un coinvolgimento di traslocatori di membrana. Il Rv galattosilato non ha raggiunto la circolazione sistemica in nessun caso. I risultati migliori sono stati ottenuti con i derivati amminoacidici mono-sostituiti. Ipotizziamo quindi che la struttura estesa e tripartita dei prodrugs con protezione completa interferisca con il riconoscimento ed il trasporto da parte dei sistemi di membrana. Tutte le procedure sintetiche, la caratterizzazione e la valutazione farmacocinetica di queste famiglie di composti sono già state pubblicate. Gli articoli relativi sono inclusi in questa tesi come capitoli 1 e 2. Dopo aver completato il lavoro coi pro-farmaci del Rv, ho iniziato un nuovo progetto riguardante lo Pt. Questo ha rappresentato il principale tema delle ricerche del mio programma di PhD. Poiché lo Pt era stato poco studiato da un punto di vista farmacologico, ne abbiamo dapprima valutato la distribuzione nel sangue e negli organi (capitolo 3). Abbiamo quindi messo a punto un metodo per la quantificazione di questa molecola e suoi metaboliti negli organi di ratto. Il protocollo che abbiamo sviluppato ha permesso di preservare la stabilità del composto e di ottenere un’estrazione quantitativa dello Pt e dei suoi metaboliti dalle matrici biologiche. Le analisi HPLC/UV hanno rivelato che, in seguito ad una somministrazione singola di una dose pari a 88µmoli/Kg di peso corporeo, i livelli di Pt in diversi organi erano maggiori rispetto a quelli riscontrati nel sangue, raggiungendo valori nell’ambito di varie nmoli/gr (µM). Inoltre, lo Pt-4’-solfato è stato identificato come la principale specie metabolica. I suoi livelli erano più alti rispetto a quelli dello Pt in tutti gli organi presi in considerazione tranne che nel cervello. Alla luce di questi risultati, abbiamo lavorato ulteriormente per cercare di limitare le modificazioni metaboliche da parte degli enzimi di fase II. Ho quindi sfruttato l’esperienza acquisita dal lavoro con i pro-farmaci del Rv e in collaborazione con il gruppo di ricerca della Prof. Paradisi del Dipartimento di Scienze Chimiche dell’Università di Padova, abbiamo sintetizzato e caratterizzato una serie di derivati dello Pt recanti amminoacidi come pro-moieties, collegate all’ossidrile in posizione 4’ tramite un legame carbammico mono-sostituito. Le cinetiche di idrolisi di questi composti sono risultate adatte per il loro utilizzo. I derivati con catene laterali idrofobiche si sono distinti per gli elevati livelli di Pterostilbene rigenerato nel sangue dopo somministrazione intra-gastrica ai ratti. Abbiamo pertanto selezionato il più promettente tra questi composti e ne abbiamo controllato la distribuzione negli organi, seguendo lo stesso protocollo utilizzato per lo Pt. In questo caso, il pro-farmaco per se è stato identificato come la specie predominante in tutti gli organi eccetto che nel cervello, ma soprattutto, i livelli di Pt rilasciati sono risultati notevolmente maggiori, e quelli di solfato minori, se paragonati a quelli ottenuti somministrando la molecola originaria (capitolo 4). 2) Come accennato in precedenza, i meccanismi intracellulari alla base degli effetti benefici dello Pt non sono stati ancora completamente delucidati. Alcune ricerche suggeriscono che le sue proprietà potrebbero essere attribuite all’induzione dell’autofagia. Ho quindi deciso di indagare questo aspetto (capitolo 5). L’autofagia è una via di degradazione la cui errata regolazione è associata a varie condizioni patologiche. Il Prof. Ballabio ed i suoi collaboratori hanno dimostrato che questo processo è regolato principalmente dal Fattore di Trascrizione EB (TFEB), inibito da mTORC1. Ho quindi voluto verificare gli effetti dello Pt su questo fattore di trascrizione. Ho dimostrato che questo polifenolo è in grado di stimolare l’attività di TFEB inducendo la sua migrazione nel nucleo e promuovendo la sua espressione. In accordo con quanto osservato, lo Pt ha provocato un aumento della lipidazione della proteina LC3 e dei livelli di espressione di alcuni geni lisosomiali target di TFEB. È stata valutata anche l’efficacia dei due metaboliti principali dello Pt, Pt-4’-solfato e DiidroPt, poiché essi rappresentano le specie principali ritrovate in circolo dopo somministrazione dello Pt. Mentre il primo composto è risultato essere inerte, il secondo si è mostrato attivo come lo Pt, ma a concentrazioni più alte. Ulteriori studi sono stati effettuati per esplorare le vie di segnalazione a monte di questi fenomeni. Misurazioni basate sull’utilizzo di sonde FRET hanno messo in evidenza che lo Pt incrementa la concentrazione di cAMP e attiva CREB. Come riportato in precedenti lavori, questo nucleotide ciclico può indurre indirettamente l’attivazione dell’AMPK, noto antagonista di mTORC1. La stimolazione di questo asse di segnalazione cellulare potrebbe quindi essere alla base dell’attivazione del TFEB. In accordo con questa ipotesi, abbiamo osservato una riduzione dell’attività chinasica di mTORC1. Trattamenti farmacologici volti ad aumentare le concentrazioni di cAMP o attivare l’AMPK si sono tuttavia rivelati meno efficaci dello Pt indicando che questo polifenolo induce la migrazione di TFEB al nucleo modulando diverse vie di segnalazione cellulare. La traslocazione nucleare di TFEB, oltre ad essere sotto il controllo di mTORC1, può anche essere regolata dalla Calcineurina. Di recente, è stato dimostrato che questa fosfatasi può essere attivata da ROS esogeni ed endogeni. Nel contesto di queste ricerche, ho evidenziato che lo Pt aumenta la produzione mitocondriale di queste specie e che questo fenomeno è correlato alla migrazione del TFEB. Alla luce di ciò, è possibile ipotizzare che questa rappresenti una via alternativa attraverso la quale lo Pterostilbene influenza la localizzazione subcellulare del fattore di trascrizione (capitolo 5). Lo stabilirsi dell’azione pro-autofagica dello Pt in cellule in coltura mi ha spinto a saggiare il suo potenziale terapeutico per il trattamento delle distrofie muscolari da carenza di Collagene VI. Queste malattie sono caratterizzate principalmente dall’accumulo di mitocondri non funzionali. Concomitanti difetti del processo autofagico aggravano ulteriormente le condizioni patologiche e conducono alla morte di tipo apoptotico delle miofibrille. Come indicato dalla letteratura scientifica, l’utilizzo di un morfolino antisenso è attualmente la strategia migliore per ottenere un fenotipo marcato di distrofie muscolari da carenza di Collagene VI in zebrafish. Abbiamo quindi iniettato specifici oligonucleotidi antisenso diretti contro l’esone 9 dell’mRNA codificante per il Collagene VI nelle uova fecondate di tali pesci. In questo modo abbiamo indotto una delezione “in frame” della regione N-terminale del dominio a tripla elica della molecola di Collagene VI e una profonda alterazione della struttura delle fibre muscolari. I dati che ho ottenuto indicano che il trattamento con lo Pt induce un recupero pari a più del 30% nella struttura delle fibre muscolari ed un notevole aumento dell’attività motoria. Questa parte del progetto è stata svolta in collaborazione con il Prof. Bernardi e il Dott. Marco Schiavone del Dipartimento di Scienze Biomediche dell’Università di Padova. Nonostante i nostri risultati non dimostrino direttamente che il miglioramento delle condizioni patologiche sia dovuto all’induzione dell’autofagia, è plausibile che la stessa serie di eventi che si verificano in vitro possano aver luogo anche in vivo. A supporto di questa ipotesi, abbiamo verificato che lo Pt è in grado di aumentare i livelli di una proteina mCherry la cui espressione è posta sotto il controllo di elementi influenzati dal cAMP (CRE) in una linea transgenica reporter di zebrafish (questo modello è stato generato dalla Dott.ssa Patrizia Porazzi e dalla Prof.ssa Natascia Tiso del Dipartimento di Biologia dell’Università di Padova). Tali fenomeni necessitano di essere ulteriormente approfonditi (capitolo 6). Infine, durante il mio percorso di dottorato, ho preso parte ad un altro progetto ancora in corso nel mio laboratorio di ricerca. Le analisi farmacocinetiche condotte in ratti hanno riportato che lo Pt è particolarmente abbondante nel cervello. Questa evidenza è in linea con diversi studi che dimostrano la capacità del polifenolo di migliorare le prestazioni di animali anziani in test comportamentali. I meccanismi molecolari alla base di questi effetti sono stati poco caratterizzati anche in questo contesto. Il lavoro che ho condotto in vitro, e confermato in zebrafish, dimostra che lo Pt è in grado di indurre un aumento di cAMP attivando CREB. È stato dimostrato che questo fattore di trascrizione svolge un ruolo cruciale per il consolidamento della memoria perché in grado di promuovere la neurogenesi in soggetti adulti a livello del giro dentato, una parte dell’ippocampo. La memoria, insieme ad altre funzioni cognitive, peggiora notevolmente con l’invecchiamento. Alla luce di ciò, in collaborazione con la Prof.ssa Nicoletta Berardi ed il gruppo di ricerca del Dott. Alessandro Sale (Istituto di Neuroscienze - Pisa), abbiamo messo a punto un lavoro sperimentale volto a valutare cambiamenti molecolari nel giro dentato e nell’ippocampo in seguito ad un eventuale miglioramento cognitivo in ratti anziani da parte dello Pt (capitolo 7). I risultati che abbiamo ottenuto hanno confermato che, dopo somministrazione cronica di questo composto, gli animali dimostrano un miglioramento della memoria. Inoltre, nonostante la statistica necessiti di essere ampliata aggiungendo più individui, sia analisi Western blot che RT-qPCR suggeriscono che tale trattamento ha indotto una up-regolazione di CREB. Allo stesso tempo, è stato misurato un aumento della massa mitocondriale. Questi effetti sono più evidenti a livello del giro dentato piuttosto che nella parte restante dell’ippocampo e suggeriscono fortemente il verificarsi di processi di rimodellamento neuronale. Nonostante ciò, non sono state riportate differenze nei livelli di PSD95. Per le prossime analisi saranno presi in considerazione marker diversi di plasticità sinaptica. In conclusione, le ricerche che ho condotto hanno permesso di delineare uno dei meccanismi responsabili delle proprietà dello Pt e di incrementare la sua biodisponibilità. Pertanto, essi potrebbero esse utili per lo sviluppo di futuri trattamenti terapeutici o preventivi.

The evolutionary success of land plants was fostered by the acquisition of the xylem vascular tissue which conducts water and minerals upwards from the roots. The xylem tissue of flowering plants is composed of three main types of cells: the sap-conducting tracheary elements (TE), the fibres which provide mechanical support and the parenchyma cells which provide metabolic support to the tissue. Both the TEs and the fibres deposit thick polysaccharidic secondary cell walls (SCWs), reinforced by a rigid phenolic polymer called lignin. The cell walls of TEs form efficient water conducting hollow tubes after the TEs have undergone programmed cell death (PCD) and complete protoplast degradation as a part of their differentiation. The work presented in this thesis studied the regulation of TE PCD by characterizing the function of the candidate PCD regulator METACASPASE 9 (MC9) in Arabidopsis thaliana xylogenic cell suspensions. These cell suspensions can be externally induced to differentiate into a mix of TEs and parenchymatic non-TE cells, thus representing an ideal system to study the cellular processes of TE PCD. In this system, TEs with reduced expression of MC9 were shown to have increased levels of autophagy and to trigger the ectopic death of the non-TE cells. The viability of the non-TE cells could be restored by down-regulating autophagy specifically in the TEs with reduced MC9 expression. Therefore, this work showed that MC9 must tightly regulate the level of autophagy during TE PCD in order to prevent the TEs from becoming harmful to the non-TEs. Hence, this work demonstrated the existence of a cellular cooperation between the TEs and the surrounding parenchymatic cells during TE PCD. The potential cooperation between the TEs and the neighbouring parenchyma during the biosynthesis of lignin was also investigated. The cupin domain containing protein PIRIN2 was found to regulate TE lignification in a non-cell autonomous manner in Arabidopsis thaliana. More precisely, PIRIN2 was shown to function as an antagonist of positive transcriptional regulators of lignin biosynthetic genes in xylem parenchyma cells. Part of the transcriptional regulation by PIRIN2 involves chromatin modifications, which represent a new type of regulation of lignin biosynthesis. Because xylem constitutes the wood in tree species, this newly discovered regulation of non-cell autonomous lignification represents a potential target to modify lignin biosynthesis in order to overcome the recalcitrance of the woody biomass for the production of biofuels.

There is an urgent need to develop crops that can withstand future climates. Results from this thesis demonstrated that a native Australian resurrection grass exhibits structural, physiological and metabolic strategies to tolerate drying. These strategies may be utilized for the generation of stress tolerant crops.

2-0xoglutarate/Fe(II)-dependent dioxygenases (ZOG Oxygenases) are a relatively poorly characterised enzyme family that hydroxylate biological macromolecules to regulate a variety of essential cellular processes in mammals, including; chromatin remodeling, extra-cellular matrix formation and oxygen sensing. The work in this th esis focuses on a ZOG Oxygenase termed Myc-Induced Nuclear Antigen (MINAS3). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity afits substrate(s) and their role in cancer is unknown. The aims of the resea rch that has resulted in this thesis were to; (i) develop a cell model of MINAS3 enzyme activity, (ii) apply this model to study the role of MINAS3 activity in cell transformation and cancer, and (iii) discover novel cellular processes regulated by MINA53 activity. As such, I have created an isogenic cell model consisting of K-Ras-transformed MINAS3 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINAS3. Using this model I have shown that MINAS3 activity maintains normal levels of the large ribosomal subunit (60S), and suppresses anchorage-independent growth, autophagy and gene expression. These observations suggest the existence and involvement of one or more substrates. Indeed, proteomic and biochemical analyses in collaboration with the Schofield laboratory (Chemistry, Oxford) confirmed the identity of a MINA53 substrate, the 60S ribosomal protein Rp127a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyJ hydroxylase; this represents the first discovery of a ribosomal oxygenase. The model developed here did not support a positive role for MINA53 in the transformation of MEFs. Rather it suggested that MINA53 can suppress transformation in some contexts, This prompted a wider investigation that demonstrated underexpression of MINA53 in several tumour types, and the presence of inactivating mutations in breast. ovarian and colon cancer. This thesis provides data supporting further research to understand the role of Rpl27a hydroxylation in the regulation of 60S biogenesis, autophagy and cancer. 2

L'autophagie est importante pour le recyclage et la mobilisation des éléments nutritifs dans les plantes. Plusieurs lignées sur-exprimant les gènes AtATG8a-i d’Arabidopsis ont été sélectionnées afin de déterminer l'effet de l'augmentation de l'activité autophagique sur l'efficacité de la remobilisation de l'azote. Les lignées sur-expresseur ont présenté une meilleure remobilisation de l'azote des feuilles de la rosette jusqu'aux graines qque le contrôle, mais uniquement dans des conditions de culture en nitrate pléthorique. Les lignées sur-exprimant ATG8a et ATG8g ont été les plus performantes. Une vaste collection de mutants atg, comprenant les mutants atg8a-i, a ensuite été utilisée pour mesurer la remobilisation de l’azote des rosettes, aux tiges et aux semences, afin de déterminer quels gènes ATG sont essentiels pour la remobilisation. Une attention particulière a été accordée à la famille de gènes ATG8a-i afin de déterminer si un membre de la famille ATG8 pourrait être plus spécifiquement dédié au recyclage de l'azote lors de la sénescence des feuilles pour la remobilisation. Bien que les divers mutants atg8 n’aient pas présenté de différence majeure par rapport au sauvage, l'un d'entre eux a montré un léger phénotype de sénescence précoce, suggérant que cette isoforme pourrait être plus spécialisée dans la remobilisation de l'azote. Afin d'améliorer l'activité de l'autophagie chez l'orge, nous avons surexprimé HvATG5 chez l'orge et testé la sensibilité des sur-expresseurs à plusieurs conditions de stress. Nous avons constaté que les sur-expresseurs de HvATG5 étaient plus tolérants à une faible alimentation en nitrate, à une faible teneur en soufre, et surtout moins sensibles aux effets de l'obscurité prolongée. Afin d'estimer les rôles respectifs des protéases induites par la sénescence et de l'autophagie, plusieurs mutants de protéase (sag12, rd21A, cathB3) ont été croisés avec des mutants d'autophagie (atg5, atg7. Leur contribution à la remobilisation de l'azote a été mesurée par marquage 15N<br>Autophagy is important for nutrient recycling and mobilization in plants. Several Arabidopsis AtATG8a-i overexpressing lines were selected in order to determine the effect of increasing autophagy on nitrogen remobilization efficiency. The overexpressing lines remobilized more nitrogen from the rosette leaves to the seeds but only when cultivated under high nitrate conditions. The lines overexpressing ATG8a and ATG8g were the most performant. A large collection of atg mutants including the atg8a-i mutants was then used to monitor N-remobilization from the rosettes to the stems and seeds in order to determine which ATG genes are essential for N remobilization. A special focus was dedicated to the ATG8a-i gene family in order to determine whether a member of the ATG8 family could be more specifically dedicated to N-recycling during leaf senescence for remobilization. Although the various atg8 mutants were not different from wild-type, one of them presented slight early senescence phenotype, suggesting this isoform could be more specialized in N remobilization. In order to enhance autophagy activity in barley, we overexpressed HvATG5 in barley and tested sensitivity of over-expressors to several stress conditions. We found that barley HvATG5 over-expressors were more tolerant to low nitrate supply, to low sulfur, and especially less sensitive to dark-stress effects. In order to estimate the respective roles of autophagy and senescence induced proteases, several protease mutants (sag12, rd21A, cathB3) were crossed with autophagy mutants (atg5, atg7) in order to monitor their respective contributions to nitrogen remobilization

Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α- syn) is the major component of Lewy bodies (LBs) in PD, and impairment of the autophagy-lysosomal pathway has been linked to its accumulation. In our previous study, we identified corynoxine B (Cory B), an oxindole alkaloid isolated from Uncaria rhynchophylla (Miq.) Jacks (Gouteng in Chinese), as a Beclin-1-dependent autophagy enhancer. In this work, we continued to screen autophagy enhancers from Gouteng alkaloids, and found corynoxine (Cory), an isomer of Cory B, also induces autophagy in different neuronal cell lines and primary neurons. Meanwhile, Cory promotes the formation of autophagosomes in the fat bodies of Drosophila. By inducing autophagy, Cory promotes the clearance of wild-type and A53T α-syn in inducible PC12 cells. Interestingly, different from its enantiomer Cory B, Cory induces autophagy through the Akt/mTOR pathway as evidenced by the reduced levels of phospho-TSC2, phospho-Akt, phospho-mTOR and phospho-p70 S6 Kinase. To identify the different pathway between Cory and Cory B, we performed phosphoproteomic study on N2a cells. With the help of iGPS (In vivo Group-based Prediction System), protein kinases which were significantly regulated by Cory or Cory B were predicted. Based on these kinases, we drew the detailed kinasesubstrates network regulated by Cory or Cory B. The structures of Cory and Cory B differ only in the stereochemistry at the spiro carbon; however, Cory has more effect on the CAMK, Trb and TSSK families, while CDK and CDKL families are more sensitive to Cory B. Furthermore, we established a rotenone rat model of PD via injecting rotenone into the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), and evaluated the neuroprotection of Cory and Cory B on this rat model. Motor dysfunction, decreased TH level, impairment of autophagy, aggregation of α-syn and activation of microglia were all found on this PD model, which were consistent with previous reports. After the treatment of Cory or Cory B, we found that both Cory and Cory B improve motor dysfunction, increase the TH level, and inhibit microglial activation. Both Cory and Cory B decrease the puncta number of aggregated α-syn, likely due to the induction of autophagy. All these results indicate the neuroprotection of Cory and Cory B against PD. Collectively, our findings (1) provide the original finding of Coy to be an autophagy enhancer with experimental evidences that Cory inhibited the pathway of Akt/mTOR; (2) provide cellular and animal experimental evidences for developing Cory or Cory B as anti-PD agent, by inducing autophagy in neurons; and (3) provide candidate pathways to identify the primary molecular target of Cory or Cory B, which may turn out to be potential therapeutic targets for treating PD. Keywords: Parkinson’s disease, Cory, Cory B, autophagy, phosphoproteomic, neuroprotection.

Plant autophagy is a crucial evolutionary conserved process for recycling cytoplasmic material under stress conditions or during development. The autophagic pathway is negatively regulated by TOR kinase, a versatile molecule modulating a wide range of cellular processes. In mammals, TOR kinase may be activated by phosphatidic acid, a vital signalling lipid. This thesis aims to prove the possible involvement of phospholipids in plant autophagy. I analysed the rate of primary root inhibition in knock-out mutants coding phospholipases in A. thaliana with induced autophagy, measured activity of lipid metabolising enzymes in wild type and atg10 mutant and observed autophagosome formation in selected mutants. Autophagosomes were labelled by fluorescent protein in vivo and by indirect immunolabelling in fixed samples. Using advanced stereological approach, I optimized a method for obtaining an unbiased estimate of autophagosome number in plant root cells.

Posidonia oceanica (L.) Delile is an endemic marine plant of the Mediterranean Sea of great ecological importance. Although the literature tells of a millennial relationship between P. oceanica and humans and despite the tradition has handed down the use of this seagrass as a natural herbal remedy, the potential benefits of P. oceanica for human health are poorly documented. The research group of Prof. Degl'Innocenti of the Department of Biomedical, Experimental and Clinical Sciences "Mario Serio" (University of Florence) was among the first to explore the bioactivities of a hydroalcoholic extract from P. oceanica leaves (POE), shedding light on its efficacy and non-toxic nature in cell-based experimental models. The possibility of working with a mixture of compounds of marine origin able to act synergistically without leaving signs of cellular toxicity has aroused my interest in continuing to explore the bioactive properties of POE with particular reference to mechanisms and molecular interactions and targeted signalling pathways underlying its activity, with the future prospective of exploring its potential benefits, hitherto unexplored, for human health. During my research activity, I have achieved excellent results related to the ability of POE to inhibit the migration of cancer cells (such as human fibrosarcoma HT1080 cells and human neuroblastoma SH-SY5Y cells) without any effects on cell viability. A more in-depth investigation of the mechanism of action of POE revealed that the phytocomplex controlled cell migration process through the activation of a transient autophagic flux. During my research, nanotechnology was also investigated as an innovative system for the nano-delivery of phytocomplexes as well. In particular, Soluplus® polymeric nanomicelles proved to be an exceptional delivery system capable of increasing the aqueous solubility of POE and bio-enhancing its anti-migratory ability on SH-SY5Y cells. In light of a tradition handed down in Anatolia on the antidiabetic role of P. oceanica, confirmed by an in vivo study in animal models, my research activity investigated the properties of POE as an antioxidant, anti-inflammatory and anti-glycation agent. In fact, it is well known that oxidative stress and inflammation are among the factors characterizing the pathophysiology of diabetes, while protein glycation is a crucial event responsible for the onset of diabetics complications under chronic hyperglycemia. The results obtained with my research help to give a more complete picture of the POE molecular mechanisms of action by shedding light on its potential benefits in the management of chronic diseases such as diabetes and related complications, but also of other inflammatory-related diseases, including cancer. So, overall my mainly cell-based experimental research used a marine resource of extreme environmental importance to explore it in terms of bioactivity from a biochemical point of view, highlighting that the P. oceanica phytocomplex is undoubtedly a promising resource of effective and non-toxic bioactive compounds. My research also established POE bioactivities also in in vivo animal models, opening new research perspectives concerning the development of alternative and complementary POE-based therapeutic strategies against various diseases. This research activity carried out during the three years of the PhD is disclosed in papers published in international peer-reviewed scientific journals.