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Ítem Acceso Abierto Structural characterization of a-synuclein aggregates seeded by patient material(2018-12-14) Strohäker, Timo; Zweckstetter, Markus; Fernandez, ClaudioNeurodegenerative diseases share a common underlying pathologic hallmark, the appearance of insoluble protein aggregates in diverse tissues of the nervous system. For many neurodegenerative diseases a common temporal and spatial spreading of the pathology is proposed in analogy to prion disease and discussed under the term “prion-like”. For many diseases the major component of the insoluble protein aggregates is known and aggregation into higher molecular weight amyloid fibrils with intermolecular b-sheet rich cores can be studied in vitro. The aggregation process involves the templated misfolding and aggregation of native monomeric proteins, involving severe conformational changes. An important family of neurodegenerative diseases is caused by the misfolding and aggregation of the protein a-synuclein, the so-called synucleinopathies. asynuclein, which in vivo forms disease-specifically the main component of intracellular inclusions such as Lewy bodies in neurons and cytoplasmatic inclusions in glial cells, undergoes in vitro dramatic conformational changes from a monomeric intrinsically disordered state over transient oligomeric b-sheet rich species into highly ordered asynuclein fibrils. a-synuclein pathology in patients is diverse and there are clinically distinct disease entities with defined pathologic phenotypes among those Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are the best characterized. Similar to prion diseases, key differences within the broad clinical representation of synucleinopathies are thought to be structurally encoded by distinct protein aggregate conformations, referred to as a-synuclein polymorphs. The aim of the study was to amplify a-synuclein aggregates from brain extracts of patients thoroughly diagnosed on the basis of the molecular pathology as well as the clinical symptoms as PD, DLB and MSA, using the established protocol of protein misfolding cyclic amplification (PMCA). A combination of hydrogen-deuterium (HD) exchange coupled to nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) and the specific binding of fluorescent probes to amyloid fibrils was chosen to obtain single-residue resolution of the conformational properties of brain-extract seeded a-synuclein fibrils. The same approach was also applied to two well-characterized in vitro a-synuclein polymorphs, their aggregation was performed in the absence of brain extract seeds following published aggregation procedures and they acted as internal references for benchmarking the methodological approach. On the other hand, the availability of a high-resolution cryo-electron VII microscopy model of the fibrillar core for one of the in vitro a-synuclein polymorphs obtained under high salt conditions, allowed direct correlation of the residue-specific conformational restraints to a structural model, both for in vitro polymorphs of asynuclein as well as brain-extract amplified a-synuclein fibrils of PD, MSA and DLB. Distinct highly ordered conformational features of in vitro a-synuclein fibrils were successfully reproduced, detecting solvent-protected residues with high precision and in agreement with published data. In contrast, a-synuclein fibrils amplified from brain extracts were more flexible and differed structurally from in vitro fibrils. Hydrogendeuterium exchange coupled to NMR spectroscopy identified a common solventprotected core shared among all patient brain derived a-synuclein fibrils for the synucleinopathies PD, MSA and DLB. The solvent-protected fibrillar core was formed by the most hydrophobic residues of a-synuclein. Outside the common core structure, a-synuclein fibrils derived from brain extracts differed disease-specifically in the conformation. Residue-specific conformational differences in core-flanking residues of a-synuclein as well as in defined N-terminal regions were observed. This study establishes a strong correlation between a-synuclein aggregate structure and the disease phenotype for the synucleinopathies Parkinson’s disease, Dementia with Lewy bodies and multiple system atrophy and the data provide further insight in “prion-like” features of neurodegenerative diseases in general and synucleinopathies in particular. The work presented here is a step forward towards the understanding of a-synuclein pathology and hopefully contributes to improved disease diagnosis and treatment of synucleinopathies.Ítem Acceso Abierto Functional study of potential sHSPs in Arabidopsis and tomato under environmental stress.(2019-03-26) Escobar, Mariela Raquel; Valle, Estela; Feussner, IvoSmall heat shock proteins (sHSPs) respond to many environmental stresses, stabilizing early unfolding protein intermediates and avoiding their irreversible aggregation. In plants, organellar sHSPs are a unique cellular feature. The primary structure of sHSPs includes a N-terminal sequence of variable sequence and length, a conserved domain known as α-crystallin domain (ACD) and a non-conserved C-terminal sequence. The ACD represents the conserved characteristic present in all sHSPs, although there are other proteins that contain an ACD but are not sHSPs. sHSPs belong to a big superfamily, and the functional and physiological relevance of the different sHSPs remains largely unknown. The objective of this study was to understand the role of mitochondrial sHSPs in Arabidopsis thaliana and Solanum lycopersicum under environmental stresses and to characterize putative bidirectional promoters driving the expression of ACD proteins with head-to-head orientation. This work covers aspects from the genomic organization and function of sHSPs-M in Arabidopsis to the role of sHSPs-M in chilling stress of tomato fruit. To perform all the experiments, Arabidopsis and tomato mutants using artificial microRNA technology were generated and analyzed in their proteome, metabolome, and lipidome. In the first part of this work, the functional characterization of head-to-head oriented genes encoding ACD proteins and the correspondent intergenic regions was performed. Four different bidirectional promoters in the A. thaliana genome, including the one of At5g51440 that encodes a mitochondrial sHSP (sHSP23.5), were successfully identified and characterized. The data suggest that the bidirectional promoter contained in the pair At5g51430-At5g51440 is strongly heat induced in one direction but not in the other. The promoter of At1g06460-At1g06470 showed comparable high activity in both directions and thus has a great potential to be used in genetic engineering. The other two promoters showed greater strength in one side and can be considered as asymmetric bidirectional promoters. This functional study of the promoters revealed the biotechnological potential of them because they can be induced specifically in a certain condition (such as high temperature) in one or two directions when it is required. In the second part, functional characterization of the mitochondrial sHSPs under stress conditions and during A. thaliana development is presented. Three gene paralogues were found in Arabidopsis (At5g51440, At4g25200, and At1g52560), and artificial microRNA were used to generate knock-down mutants (single, double and triple amiR). The single and double amiRs (for sHSP23.5 and sHSP23.6) did not show evidently affected phenotype, probably because of functional compensation or redundancy of the mitochondrial sHSPs. On the other hand, the triple amiR23.5/23.6/26.5 mutants showed an altered phenotype in the vegetative and reproductive stages. They have reduced leaves areas, but not number of epidermal cells per leaf, chlorotic leaves, shorter root, and reduced seed yield when compared to Col-0 plants. Plants of triple amiR were considerably small due to the alteration in the process of cell expansion but not in the cellular proliferation, which indicates a profound alteration in the plant developmental program. Proteomic analysis of the amiR mutants revealed significant upregulation of various metabolism-related proteins and alterations in the abundance of several proteins that are involved in translation and in the ribosome functioning and structure. Triple amiR mutant exhibited a higher number of proteins with differential abundance related to these processes compared to the other single and double amiR23.5/23.6 mutants. Such a wide change in ribosome-related proteins suggests a possible alteration in the proper ribosome function. The data exposed in this work provide evidence of the important roles that sHSPs-M may play, not only in the heat response but also in the plant development of Arabidopsis. Results demonstrate that a functional compensation might be responsible for the phenotype in mutants lacking single sHSPs-M. However, the reduction of the three sHSPs-M caused a profound disruption in the mitochondria and ribosome functionality that severely affected the energy metabolism and the overall cell homeostasis, leading to alterations in the correct plant development. In the last part of this work, the functional consequences of the down-regulation of sHSP23.8 in tomato fruit were investigated and analyzed in their phenotype and in their susceptibility to chilling injury. Pre-chilled fruit of amiR23.8 mutant showed higher loss of water and increased ion leakage of pericarp tissue compared to WT fruit. The amiR23.8 fruit deterioration indicates that it is highly susceptible to cold stress and developed chilling injury symptoms. The lipidome of fruit after chilling of amiR23.8 showed altered amounts of glycerolipids, and the level of saturated lipids in amiR23.8 decreased, but not lower than the level in WT under normal conditions. The opposite was found in the relative percentage of unsaturated lipids, having amiR23.8 fruit significantly lower levels in normal conditions and after chilling. The results presented here indicate a differential degradation of extraplastidic and plastidic lipids in amiR23.8 fruit, and alterations in the remodeling of the lipidome after cold stress, which may lead to higher sensitivity to chilling injury. The results discussed here indicate that sHSP23.8 may play an important role in the protection mechanisms against chilling stress in tomato fruit.Ítem Acceso Abierto Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach(2018-06-11) Quintana, Ingrid M.; Magni, Christian; Stülke, JörgCyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes such as cell wall homeostasis, biofilm formation, antibiotics and heat resistance, among others. In Lactococcus lactis and Enterococcus faecalis, Lactic Acid Bacteria used not only as research models but also as a cell factory in biotechnological processes, the only reported interaction partner of c-di-AMP is the pyruvate carboxylase enzyme, PyrCarb. Nevertheless, in the last year investigations directed its main role towards potassium metabolism. In this thesis, KupA and KupB, two potassium transporters encoded in L. lactis IL1403 genome, are described for the first time. According to an in silico analysis, these proteins, which belong to the Kup/HAK/KT family, are highly conserved in this species, being therefore a strain independent potassium uptake system. In addition, evidence shows that both proteins are able to uptake this cation with high affinity, and we demonstrate that KupA as well as KupB bind to and are down-regulated by c-di-AMP. On the other hand, different strains derived from L. lactis IL1403 were developed aiming to modify intracellular pools of c-di-AMP in a stable system. One strategy for the reduction of c-di-AMP levels was the obtaining of ΔgdpP mutants via homologous recombination. Maintenance of this second messenger levels close to wild type ones, suggested the presence of another c-di-AMP degrading enzyme. A first description of a putative enzyme with this activity, encoded by yheB gene was done by BNPP assay. In addition, by use of a pH inducible vector, construction of strain L. lactis LL03 with concentrations of this second messenger above 15 times basal levels was possible. This system was therefore selected for further investigations on the development of a vaccine prototype against Chagas disease. On the other hand, L. lactis is a promising candidate for the development of mucosal vaccines with more than 20 years of experimental research. Moreover, c-di-AMP has been reported as a strong mucosal adjuvant promoting both humoral and cellular immune responses. Altogether, in this thesis the development of a recombinant L. lactis strain is reported, able to produce both an antigen as well as an adjuvant in order to develop a novel vaccine prototype against the Trypanosoma cruzi parasite, the causal agent of Chagas disease. This is a tropical disease originated in a specific area of South America but currently spreading in four continents. Finally, an initial approach was done on c-di-AMP metabolism in E. faecalis. The presence of a Kup transporter was also corroborated in this species, and some basic characteristics of the c-di-AMP degradative pathway were explored via a ΔgdpP mutant construction. Finally, the impact of GdpP on the virulence of E. faecalis was analyzed by use of the infection model Galleria mellonella.