Pancreas is known to be an organ producing a variety of exocrine and endocrine substances, where also insulin belongs. This hormone is produced in the body almost solely by specialized β-cells of the Langerhans islets and is stored here in secretory granules. As the β-cells contain large number of these vesicles, an organism can quickly respond to the glucose stimulation. Completely processed insulin is formed in the secretory granules probably as a hexamer, where six insulin molecules are coordinated along two zinc bivalent cations. Appropriate β-cell response to higher glucose level and following insulin secretion is one of the key processes that regulate metabolism in the body. In order to study insulin production, its effects or secretion, permanent pancreatic cell lines are often used as biological models, out of primary cells from islets of Langerhans. This diploma thesis is focused on two permanent cell lines INS-1E and BRIN-BD11. We searched for the ability of the cells to produce insulin, if the hormone is fully processed, as well as zinc content, which could have a great influence on insulin's processing. Using different methods we compared these two cell lines with cells from the Langerhans islets. We succeeded in isolation of secretory granules from all three cell types and we plan to...

The cereal seed plays a crucial role in society – both in the “food as medicine” paradigm, but also in food security. It is the starch and proteins present in the seed that lend it importance in these dissimilar anthropomorphic activities. This thesis investigation first characterized the post-translational processing of the potential diabetogen, wheat globulin-3. Globulin-3-like peptides were observed primarily in the embryo. These peptides varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting. Five major polypeptide spots were sequenced by mass spectrometry, allowing for the development of a model of the post-translational events contributing to the globulin-3 processing profile. Three separate investigations of starch granules from different cereal species were performed. In the first series of experiments, pathogen-susceptible maize kernels were injected with either conidia of the fungal pathogen Fusarium graminearum or sterile water controls. Proteins in the desiccated fungal remnants on the surface of the kernels as well as in the endosperm and embryo tissues of the control and infected kernels were isolated and these proteomes were sequenced using tandem mass spectrometry. Approximately 250 maize proteins were identified. These proteins were classified into functional categories. There was an increased representation of defense proteins in the both the embryo and endosperm tissues of infected maize samples. The proteome of the fungal remnants was composed of 18 proteins. Several of these proteins were categorized as being involved in the metabolism of plant-sourced molecules, or in stress response. The second series of experiments detail the investigation of commercially prepared rice and maize starches using tandem mass spectrometry. The majority of identified proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. Markers for seed maturity and for starch mobilization were also documented. Finally, the third series of experiments investigated the non-host proteomes present in commercially-prepared starches. Non-host proteins from a variety of species, including Homarus americanus were found in the starch samples. This documentation of H. americanus proteins in these starch samples may have food safety implications with regards to shellfish allergies.

Les ARNms des cellules eucaryotes sont liés à des protéines de liaison aux ARNs (RBPs) et empaquetés au sein d’assemblages macro-moléculaires appelés granules RNP. Dans les cellules neuronales, les granules RNP de transport sont impliqués dans le transport d’ARNms spécifiques jusqu’aux axones et dendrites, ainsi que dans leur traduction locale en réponse à des signaux externes. Bien que peu de choses soient connues sur l’assemblage et la régulation de ces granules in vivo, des résultats récents ont indiqué que la présence de domaines de type prion (PLDs) dans les RBPs facilite les interactions protéines-protéines et protéines-ARN, favorisant ainsi la condensation de complexes solubles en granules RNP. La RBP conservée Imp est un composant central de granules RNP qui sont transportés dans les axones lors du remodelage neuronal chez la drosophile. De plus, la fonction de Imp est nécessaire au remodelage des axones lors de la maturation du système nerveux de drosophile. Une analyse de la séquence de la protéine Imp a révélé qu’en plus de quatre domaines de liaison aux ARNs, Imp contient un domaine C-terminal désordonné enrichi en Glutamines et Serines, deux propriétés caractéristiques des domaines PLDs. Lors de ma thèse, j’ai étudié la fonction de ce PLD dans le contexte de l’assemblage et du transport des granules RNP. J’ai observé en culture de cellules que les granules Imp s’assemblent en absence de PLD, bien que leur nombre et leur taille soient augmentés. Des protéines présentant une séquence PLD mélangée, au contraire, s’accumulent dans des granules au nombre et à la taille normale, indiquant que l’état désordonné de ce domaine, et non sa séquence primaire, est essentiel à l’homéostasie des granules. De plus, des expériences de FRAP réalisées en culture de cellule et in vivo ont révélé que le domaine PLD de Imp favorise la dynamique des granules. In vivo, ce domaine est nécessaire et suffisant à l’accumulation axonale de Imp. Comme montré par une analyse en temps réel, l’absence de domaine PLD aboutit également à une diminution du nombre de granules axonaux motiles. Fonctionnellement, le domaine PLD de Imp est essentiel au remodelage neuronal car des protéines sans ce domaine ne sont pas capables de supprimer les défauts de repousse axonale observés après inactivation de imp. Enfin, la génération d’un variant de Imp dans lequel le domaine PLD a été déplacé en N-terminus a montré que les fonctions du PLD dans le transport des granules et dans leur assemblage sont découplées, et que la modulation des propriétés des granules Imp médiée par le domaine PLD n’est pas nécessaire au remodelage neuronal in vivo. En conclusion, mes résultats ont montré que le domaine PLD de Imp n’est pas nécessaire à l’assemblage des granules RNP Imp, mais régule leur nombre et leur dynamique. De plus, mon travail a mis en évidence une fonction inattendue pour un domaine PLD dans le transport axonal et le remodelage des neurones lors de la maturation du système nerveux. / Eukaryotic mRNAs are bound by RNA Binding Proteins (RBP) and packaged into diverse range of macromolecular assemblies named RNP granules. In neurons, transport RNP granules are implicated in the transport of specific mRNAs to axons or dendrites, and in their local translation in response to external cues. Although little is known about the assembly and regulation of these granules in vivo, growing evidence indicates that the presence of Prion Like domains (PLD) within RBPs favours multivalent protein–protein and protein-RNA interactions, promoting the transition of soluble complexes into RNP granules. The conserved RBP Imp is as a core component of RNP granules that are actively transported to axons upon neuronal remodelling in Drosophila. Furthermore, Imp function was shown to be required for axonal remodelling during Drosophila nervous system maturation. Analyses of the domain architecture of the Imp protein revealed that, in addition to four RNA binding domains (RBD), Imp contains a Cterminal domain showing a striking enrichment in Glutamines and Serines, which is one of the characteristics of a PLD. During my PhD, I explored the function of the PLD in the context of granule assembly and transport. In cultured cells, I observed that Imp granules assembled in the absence of the PLD, however their number and size were increased. Proteins with scrambled PLD sequence accumulated in granules of normal size and number, implying that the degree of disorder of this domain, and not its sequence, is essential for granule homeostasis. Moreover, FRAP experiments, performed on cultured cells and in vivo, revealed that Imp PLD is important to maintain the turnover of these granules. In vivo, this domain is both necessary and sufficient for efficient transport of Imp granules to axons. These defects are associated with a reduction on the number of motile granules in axons. Furthermore, mutant forms lacking the PLD do not rescue the axon remodelling defects observed upon imp loss of function. Finally, a swapping experiment in which I moved Imp PLD from the C-terminus to the N-terminus of the protein revealed that the functions of Imp PLD in granule transport and homeostasis are uncoupled, and that PLD-dependent modulation of Imp granule properties is dispensable in vivo. Together, my results show that Imp PLD of is not required for the assembly of RNP granules, but rather regulates granule number and dynamics. Furthermore, my work uncovered an unexpected in vivo function for a PLD in axonal transport and remodelling during nervous system maturation.

Aerobic granular sludge (AGS) technology is a next-generation technology for the biological treatment of wastewater. The advantages of AGS in terms of small footprint, low operation and capital cost and high effluent quality makes it a strong candidate for replacing conventional biological wastewater treatment based on activated sludge (CAS) process, and potentially become the standard for biological wastewater treatment in the future. Saline wastewater is generated from many industrial processes as well as from the use of sea water as a secondary quality water for non-potable use such as toilet flushing to mitigate shortage of fresh water in some coastal cities. Salt is known to inhibit biological wastewater treatment processes in terms of organic and nutrient removal. In the first part of my dissertation, I conducted three lab-scale experiments to 1) evaluate the effect of salt on granulation and nutrient removal in AGS (330 days); 2) develop engineering strategies to mitigate the adverse effect of salt on nutrient removal of AGS (164 days); and 3) compare the effect of salt on the stoichiometry and kinetics of different phosphate accumulating organisms (PAO) clades (PAOI and PAOII) and to determine the effect of potassium and sodium ions on the activities of different PAO clades (225 days). Like other artificial microbial ecosystems (e.g. CAS plant and anaerobic digester), a firm understanding of the microbial ecology of AGS system is essential for process design and optimization. The second part of my dissertation reported the first microbial ecology study of a full-scale AGS plant with the aim of addressing the role of regional (i.e. immigration) versus local factors in shaping the microbial community assembly of different-sized microbial aggregates in AGS. The microbial communities in a full-scale AGS plant in Garmerwolde, The Netherlands, was characterized periodically over 180 days using Illumina sequencing of 16S ribosomal RNA amplicons of the V3-V4 regions. Overall, the discovery of this PhD study sheds light on the application of AGS for the treatment of saline wastewater and deepens our understanding on the microbial ecology of AGS systems, which is essential for process design and optimization.

Le cervelet est une structure cérébrale impliquée dans de multiples fonctions motrices mais aussi cognitives et dont le développement postnatal est sous le contrôle de divers types de facteurs dont les neuropeptides. Les peptides capables d’agir sur le développement du cortex cérébelleux présentent généralement un profil d’expression particulier, avec chez le rongeur un pic d’expression au cours des 2 premières semaines postnatales. L’objectif de cette étude était d’identifier d’autres peptides présentant ce même type d’expression et de caractériser leurs potentiels effets au cours du développement du cortex cérébelleux, et plus particulièrement dans la mise en place des neurones en grain qui sont les plus abondants de cette structure. Pour cela, des cervelets de rats âgés de 8 à 90 jours ont été analysés par spectrométrie de masse. Parmi les 33 peptides identifiés, 4 présentent le profil recherché et nous avons choisi d’étudier l’un d’entre eux, la nociceptine. La mesure de l’expression du gène de la nociceptine et de son récepteur montre un profil d’expression similaire à celui observé en peptidomique. De plus, ces 2 gènes sont retrouvés principalement exprimés dans la couche granulaire interne du cortex cérébelleux par microdissection et qPcr. La recherche de la fonction de la nociceptine montre qu’elle exerce un effet neurotrophique en augmentant la survie et la différenciation des neurones en grain, sans affecter la motilité de ces cellules. Des tests préliminaires réalisés in vivo indiquent que la nociceptine est aussi capable de bloquer la toxicité induite par l’alcool. La dernière partie de l’étude avait pour but d’identifier de nouveaux neuropeptides exprimés dans le cervelet en utilisant une approche par séquençage de novo. L’application de filtres comme la récurrence des séquences peptidiques ou leur régulation au cours du développement a permis de ne retenir que 6 séquences pour la suite de l’analyse. Des études génomiques permettront de restreindre encore ce nombre afin de focaliser les tests d’activité biologique sur la ou les cibles qui ont la plus grande probabilité de correspondre à des peptides biologiquement actifs. / The cerebellum is a structure involved in many motor and cognitive functions whose development occurs after birth under the control of various factors, including neuropeptides. Peptides acting on cerebellar cortex development often exhibit a specific pattern of expression with in rodents a high expression over the 2 first postnatal weeks which then decreases at adulthood. The aim of this study was to identify additional peptides with such expression profile and to characterize their putative functions in the development of the cerebellar cortex and more particularly, in the establishment of cerebellar granule neurons which are the most abundant cells of the cerebellum. To address this, cerebella of rats aged from 8 to 90 days-old were analyzed by mass spectrometry. Among the 33 peptides identified in the cerebellum, 4 had the particular expression profile we were looking for. We choose to study further one of them, i.e. the nociceptin, and confirmed peptidomic results by measuring the expression of its gene precursor and of its receptor. Combining laser microdissection and qPCR approaches revealed that both nociceptin and its receptor genes were expressed in the internal granular layer of the cerebellar cortex. Functional studies showed that nociceptin exerts a neurotrophic effect on granule neurons by increasing their survival and differentiation, but had no effect on their motility. Preliminary in vivo experiments indicate that nociceptin can also counteract ethanol-induced toxicity. The last part of the present study aimed to identify new neuropeptides expressed in the rat cerebellum by using de novo sequencing. The large amount of peptide sequences initially found was then reduced to only 6 candidates for further analysis, by using filters such as recurrence of the sequences and their differential expression in between the four developmental stages considered. Additional genomic studies will help to decrease even further this number, in order to focus the biological tests on the targets which are most likely to code for biological active peptides.

<p>Urea has been widely used as a crop fertilizer to increase crop yield. The low nutrient use efficiency (NUE) of urea, however, is a challenge. Coated fertilizers are considered a solution not only for enhancing the NUE but also for alleviating soil and water pollution. In this paper, the physical properties of coated fertilizers were analyzed, including their particle size distribution, fracture force, thermal behavior, envelope density, and apparent density (regular fertilizer: pure urea and the Anderson 12-6-6; slow release fertilizer: Osmocote 14-14-14, the Anderson 18-6-12; controlled release fertilizer: Environmentally smart nitrogen (ESN), Florikan 14-14-14, Everris 17-3-6). The granules’ closed and open pore number, pore volume, and total porosity were analyzed using X-ray micro-tomography (XRCT). The results demonstrated that pure urea and Florikan have a similar median particle size, around 4 mm, while ESN and Osmocote have a similar median particle size of around 3 mm. Finally, Everris, the Andersons 18-6-12, and the Andersons 12-6-6, have a similar median particle size of roughly 2.5 mm. The fracture pressure of ESN (4.58±0.98 MPa) and the NPK combination fertilizers (Florikan: 9.40±1.46 MPa and Osmocote: 8.94±2.09 MPa) were higher than pure urea. The envelope and apparent density of pure urea (envelope: 1.22±0.02 kg/m³ and apparent: 1.27±0.01 kg/m³) and ESN (envelope: 1.26±0.03 kg/m³ and apparent: 1.27±0.00 kg/m³) are similar, while all NPK fertilizers have a significantly higher density (envelope: 1.68–1.87 kg/m³ and apparent: 1.83–2.09 kg/m³). ESN had higher internal pore space and a higher total pore volume than pure urea, while NPK combination fertilizer showed lesser pores and significantly smaller pore volumes. The physical properties were also significantly different when comparing urea and NPK compound fertilizers, mainly because of the differences in their nutrient coatings and manufacturing methods. The coating of the urea increases the granule strength but does not alter the thermal properties; however, the overall porosity of the granules is influenced by the coating. In this thesis core, different binders were used to alter the internal structure of the urea granule to control the dissolution behavior and to make it a slow-release fertilizer. The layered agglomeration technique was used to manufacture the granules. The core of the granule was made by granulating technical urea powder in a drum granulator, with corn starch as the binder. A second layer of urea was added to the core by drum granulation in order to obtain a nutrient release pattern that matches with the crop demand. Corn starch, PEG 4000, and corn starch hydrogel were used as binders for the second layer. The density, thermal properties, strength, and internal porosity were measured to compare with market urea and coated slow-release fertilizer granules. All the dissolution rates of the double layer granules were slower than for market urea. Among these granule types, the dissolution rate curve of the granule with starch hydrogel in the second layer better matched the crop demand curve than those of the other two types of granules. Moreover, the strength of the double layer granules with hydrogel was the greatest of the three double layer granules. So, overall, the double layer granule manufactured with corn starch in the core and starch hydrogel in the second layer performed the best. Although the pattern of dissolution of the double layer granule was similar to the crop nitrate demand curve, a soil-based study is needed to verify the nitrate release characteristics.<br></p><ul> </ul>

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RACK1 (Receptor for activated C kinase 1) is an evolutionary conserved protein which has essential role in most studied eukaryotic organisms, except for yeast. Although RACK1 was originally described as a binding partner of protein kinase C, later studies re- vealed its significant role in other cellular signalizations such as MAPK, Src or FAK. Thanks to this, RACK1 participates in the regulation of key cellular processes including migration, apoptosis or translation. As a binding partner of a small ribosomal subunit, RACK1 contributes to transla- tion regulation by integrating signals from different cellular pathways and several transla- tional components such as PKC and eIF6. Moreover, RACK1 has a role in translation regu- lation during stress. Under stress conditions there is a global reduction of translation, in- creased expression of specific mRNAs important for cellular stress response and formation of cytosolic foci called stress granules (SGs). SGs play an important role in protection of mRNAs and translation components against degradation. SGs also function in prevention of apoptosis. RACK1 has been identified as one of many components of SGs and its localization into SGs leads to inhibition of RACK1-mediated pro-apoptotic pathways. Aim of this diploma thesis was to elucidate the role of...

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18627号 / 生博第318号 / 新制||生||42(附属図書館) / 31527 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 米原 伸, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM