Dissertations / Theses on the topic 'Proteins Analysis'

In an attempt to elucidate the transcriptional mechanisms that regulate the expression of the human α2(I) procollagen gene, cis-acting DNA-elements within the proximal promoter were identified and their corresponding trans-acting factors characterised. The fibroblast cell lines used in this study had previously been transformed with either simian virus 40 (SVWI-38) or by γ-radiation (CT-1). The SVWI-38 fibroblasts do not produce any α2(I) collagen chains, whereas the CT-1 cell line produces normal type I collagen. Previous studies suggested that trans-acting factor(s) may be responsible for the inactivation of the α2(I) procollagen gene in SVWI-38 fibroblasts (Parker et. al. (1989) J. Biol. Chem 264, 7147-7152; Parker et. al. (1992) Nucleic Acids Res. 20, 5825-5830). In this study, the SVWI-38 proximal promoter (-350 to +54) was sequenced and shown to be normal, thereby ruling out any possibility that mutations within this region was responsible for inactivation of the gene.

The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery. 

Computer and Information Science
Ph.D.
Information fusion aims to develop intelligent approaches of integrating information from complementary sources, such that a more comprehensive basis is obtained for data analysis and knowledge discovery. Our Protein Biological Unit (ProtBuD) database is the first database that integrated the biological unit information from the Protein Data Bank (PDB), Protein Quaternary Server (PQS) and Protein Interfaces, Surfaces and Assemblies (PISA) server, and compared the three biological units side-by-side. The statistical analyses show that the inconsistency within these databases and between them is significant. In order to improve the inconsistency, we studied interfaces across different PDB entries in a protein family using an assumption that interfaces shared by different crystal forms are likely to be biologically relevant. A novel computational method is proposed to achieve this goal. First, redundant data were removed by clustering similar crystal structures, and a representative entry was used for each cluster. Then a modified k-d tree algorithm was applied to facilitate the computation of identifying interfaces from crystals. The interface similarity functions were derived from Gaussian distributions fit to the data. Hierarchical clustering was used to cluster interfaces to define the likely biological interfaces by the number of crystal forms in a cluster. Benchmark data sets were used to determine whether the existence or lack of existence of interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. The probability that a common interface is biological is given. An interface shared in two different crystal forms by divergent proteins is very likely to be biologically important. The interface data not only provide new interaction templates for computational modeling, but also provide more accurate data for training sets and testing sets in data-mining research to predict protein-protein interactions. In summary, we developed a framework which is based on databases where different biological unit information is integrated and new interface data are stored. In order for users from the biology community to use the data, a stand-alone software program, a web site with a user-friendly graphical interface, and a web service are provided.
Temple University--Theses

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1992.
Cover title: Novel approaches to protein purification and analysis: counter-current electrophoretic filtering: spectral enhancement. Spine title: Protein purification and analysis. Includes bibliographical references.

Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.

Male butterflies have some certain pathways to prevent the female butterfly which has mated with them from mating with other butterflies. Research shows that some proteins from the male butterflies may play an important role in this mechanism. To investigate how the pathway works, the proteins contained in the spermatophore which is injected into the female butterfly by the male one during mating are very important. In this study, the butterfly spermatophore proteins were mainly studied. The proper procedures to obtain the spermatophore from the female body were developed. Since capillary electrophoresis (CE) has many advantages for separation and detection of low amounts of sample, it was used in this study in order to separate the proteins obtained from spermatophores. Finally, Mass Spectrometry (MS) was performed to analyze the proteins after separation in order to identify the proteins. This thesis mainly introduced the following points: 1. The method for obtaining spermatophores from butterfly bodies and the procedures for extracting proteins from spermatophores. 2. Optimized methods and conditions of Capillary Zone Electrophoresis (CZE) for butterfly spermatophore proteins. Several CZE methods and sets of conditions were compared in order to find the optimized ones. 3. Protein samples were applied to Mass Spectrometry (MS) to try to analyze and identify them. Moreover, the effect of using ZiptipsTM for sample pretreatment was discussed.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 131-145).
Proteins are essential to organisms and play a central role in almost every biological process. The analysis of the conformational dynamics and mechanics of proteins using numerical methods, such as normal mode analysis (NMA), provides insight into their functional mechanisms. However, despite the fact that much effort has been focused on improving NMA over the last few decades, the analysis of large-scale protein motions is still infeasible due to computational limitations. In this work, first, we identify the usefulness and effectiveness of the subspace iteration (SSI) procedure, otherwise widely used in structural engineering, for the analysis of proteins. We also develop a novel technique for the selection of iteration vectors in protein NMA, which significantly increases the effectiveness of the method. The SSI procedure also lends itself naturally to efficient NMA of multiple neighboring macromolecular conformations, as demonstrated in a conformational change pathway analysis of adenylate kinase. Next, we present a new algorithm to account for the effects of solvent-damping on slow protein conformational dynamics. The algorithm proves to be an effective approach to calculating the diffusion coefficients of proteins with various molecular weights, as well as their Langevin modes and corresponding relaxation times, as demonstrated for the small molecule crambin. Finally, the structure of Homo sapiens fascin-1, an actin-binding protein that is present predominantly in filopodia, is examined and described in detail. Application of a sequence conservation analysis to the protein indicates highly conserved surface patches near the putative actin-binding domains of fascin. A novel conformational dynamics analysis suggests that these domains are coupled via an allosteric mechanism that may have important functional implications for F-actin bundling by fascin.
by Reza Sharifi Sedeh.
Ph.D.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
Defensinas de planta, componentes inatos do sistema imune das plantas, são peptídeos antifúngicos, catiônicos, com estrutura primária rica em cisteína. Evidência dada pela literatura demonstrou que trechos de esfingolipídios complexos na membrana dos fungos, contendo manosildiinositolfosforilceramida e glicosilceramida, são sítios de ligação seletivos para as defensinas de planta isoladas de Dahlia merckii e Raphanus sativus, respectivamente. Entretanto, desconhece-se se as defensinas de planta interagem direta ou indiretamente com alvos intracelulares dos fungos. A fim de identificar interações físicas e diretas do tipo proteína- proteína, um sistema de duplo-híbrido, em levedura, baseado no fator de transcrição GAL4, foi construído utilizando-se como isca, a defensina da planta Pisum sativum, Psd1 (Pisum sativum defensin 1). Proteínas alvos, capazes de interagirem com o peptídeo Psd1, foram detectadas através do rastreamento de uma biblioteca de cDNA do fungo Neurospora crassa. Do resultado deste rastreamento, nove dentre quinze candidatos, selecionados pelo método do duplo-híbrido, foram identificados como proteínas nucleares da N. crassa. Um clone, detectado com alta freqüência neste rastreamento, apresentou homologia de seqüência com a proteína ciclina F, relacionada com o controle do ciclo celular. O ensaio de co-purificação utilizando a proteína conjugada a glutationa S-transferase (GST) validou in vitro o resultado obtido pelo sistema duplohíbrido. Análise por microscopia de fluorescência da Psd1, conjugada a FITC, e, dos núcleos do fungo Fusarium solani, marcados com DAPI, demonstrou in vivo a co-localização da defensina de planta Psd1 com os núcleos do fungo. Para pesquisar o modo de ação da Psd1 ao nível do ciclo celular, utilizou-se o modelo multicelular da retina de ratos neonatais, em desenvolvimento. Neste modelo, a migração nuclear intercinética, correlacionada com as transições de fase de S para M do ciclo celular, foi observada na presença da Psd1. Verificouse que Psd1 impediu a migração nuclear em neuroblastos, parando o ciclo celular na transição de S para G2. Estes resultados revelaram modos de ação da defensina de planta Psd1 sobre a fisiologia nuclear.
Plant defensins, innate components of the plant immune system, are cationic, antifungal peptides, with a cysteine- rich primary structure. Evidence from the literature demonstrated that fungus membrane patches containing complex sphingolipids, mannosyldiinositolphosphorylceramide and glucosylceramides, are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. However, whether the plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify direct physical protein-protein interactions, a GAL4-based yeast two-hybrid system was constructed, using the plant peptide, Pisum sativum defensin 1 (Psd1), as the bait protein. Target proteins, capable of interacting with the bait Psd1, were detected by screening a Neurospora crassa cDNA library. In this screening, nine out of fifteen two-hybrid candidates were identified as N. crassa nuclear proteins. One clone, detected with high frequency in the screening, presented sequence similarity to a N. crassa cyclin F, related to the cell cycle control. The GST pull- down co purification assay corroborated this two-hybrid result in vitro. Fluorescence microscopy analysis of FITC- conjugated Psd1 and DAPI-stained Fusarium solani nuclei demonstrated in vivo the co-localization of the plant peptide Psd1 and the fungus nuclei. We used the developing retina of neonatal rats as a multicellular model to study Psd1 mode of action at the cell cycle level. In this model, we observed in vivo the interkinetic nuclear migration, correlated to the transitions from S to M-phase of the cell cycle, in the presence of the Psd1 peptide. It was shown that Psd1 impaired nuclear migration of neuroblasts by arresting the cell cycle at the S to G2- phase transition. These results revealed modes of action of the plant defensin Psd1 upon the nuclear physiology.

Thesis (Ph.D. in Biophysics & Genetics, Program in Molecular Biology) -- University of Colorado Denver, 2007.
Typescript. Includes bibliographical references (leaves 90-98). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Oregon, 2004.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 142-152). Also available for download via the World Wide Web; free to University of Oregon users.

A transcription factor (TF) is a protein that binds to specific DNA sequences as part of the initiation stage of transcription. Various methods of finding these transcription factor binding sites (TFBS) have been developed. In vivo technologies analyze DNA binding regions known to have bound to a TF in a living cell. Most widely used in vivo methods at the moment are chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and DNase I hypersensitive sites sequencing. In vitro methods derive TFBS based on experiments with TFs and DNA usually in artificial settings or computationally. An example is the Protein Binding Microarray which uses artificially constructed DNA sequences to determine the short sequences that are most likely to bind to a TF. The major drawback of this approach is that binding of TFs in vivo is also dependent on other factors such as chromatin accessibility and the presence of cofactors. Therefore TFBS derived from the PBM technique might not resemble the true DNA binding sequences. In this work, we use PBM data from the UniPROBE motif database, ChIP-seq data and DNase I hypersensitive sites data. Using the Spearman’s rank correlation and area under receiver operating characteristic curve, we compare the enrichment scores which the PBM approach assigns to its identified sequences and the frequency of these sequences in likely binding regions and the human genome as a whole. We also use central motif enrichment analysis (CentriMo) to compare the enrichment of UniPROBE motifs with in vivo derived motifs (from the JASPAR CORE database) in their respective TF ChIP-seq peak region. CentriMo is applied to 14 TF ChIP-seq peak regions from different cell lines. We aim to establish if there is a relationship between the occurrences of UniPROBE 8-mer patterns in likely binding regions and their enrichment score and how well the in vitro derived motifs match in vivo binding specificity. We did not come out with a particular trend showing failure of the PBM approach to predict in vivo binding specificity. Our results show Ets1, Hnf4a and Tcf3 show prediction failure by the PBM technique in terms of our Spearman’s rank correlation for ChIP-seq data and central motif enrichment analysis. However, the PBM technique also matched the in vivo binding specificities of FoxA2, Pou2f2 and Mafk. Failure of the PBM approach was found to be a result of variability in the TF’s binding specificity, the presence of cofactors, narrow binding specificity and the presence ubiquitous binding patterns.

Magnaporthe grisea is a notorious pathogenic fungus that causes rice blast disease worldwide. Proteins secreted by the fungus are likely candidates for being effectors that are potentially recognized by determinants of resistance or susceptibility in host plants. However, knowledge of the role of secreted proteins of M. grisea is still limited. In this study, I identified 29 proteins that were secreted into culture filtrates from M. grisea strains expressing candidate proteins. I confirmed secretion of these proteins and tested them for elicitor activity on plants. Among them, I studied two groups: cell wall degrading enzymes (CWDEs) and small cysteine-rich proteins. Cysteine-rich proteins have been shown in other systems to function as elicitors. Initially, I expressed and purified proteins in M. grisea to obtain proteins by a homologous expression system. Although this was effective for a number of proteins, the need for greater amounts of protein led me to express several proteins in the Pichia pastoris system. Several candidate proteins were purified and found to induce symptoms on rice and maize. Hypothetical proteins MG10424.4 and MG09998.4 were both found to have elicitor activity. Lipase MG07016.4 did not induce response of plants and we concluded that the lipase activity of MG07016.4 does not function as an elicitor. I also purified a small cysteine-rich protein, which belongs to the group of cluster 180 proteins in M. grisea, MG10732.4 from P. pastoris. It is able to cause yellowing symptoms and hydrogen peroxide production in plants and it might contain elicitor activity.

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Budded virions of AcMNPV can enter a variety of non-host cells, a characteristic likely due to the presence of GP64, an envelope protein found on a small subset of baculoviruses. Results show that AcMNPV's tropism for vertebrate cells can be restricted - a prerequisite for using AcMNPV for targeted in vivo gene delivery - by replacing the gp64 gene with SeF from SeMNPV. Unlike the relatively well characterized GP64 protein, the significance and function of the F homolog (Ac23, a pathogenicity factor), is poorly understood. How Ac23 might contribute to the faster speed of kill was examined by comparing occlusion bodies and occlusion-derived virions (ODV) of Ac23null mutant viruses with control viruses at the ultrastructural level. The results show that Ac23null mutant produces a significantly higher percentage of ODVs with single or lower number of nucleocapsids than controls, suggesting Ac23 may play a role in multicapsid envelopment of ODVs.
xiii, 101 leaves : ill. (some col.) ; 28 cm. --

Der Abbau des Chlorophyll (Chl) ist ein Prozess, der typischerweise während der Blattseneszenz und der Reifung von Früchten und Samen stattfindet. Eine Störung dieses koordinierten Prozesses unter Frostbedingungen verzögert den Chl-Abbau und ist ein grosses Hindernis bei der Herstellung von hochwertigem Rapsöl. Der Abbau von Chl zu farblosen Kataboliten erfolgt in einer Serie von enzymatischen Schritten und wird durch die Chlorophyllase begonnen (Chlase). Es wurde vorgeschlagen, dass ein wasserlösliches Chl Protein (WSCP) den Transport des Chl von der Thylakoidmembran zum Wirkort der Chlase übernimmt. Weiterhin wurde angenommen, dass die Steigerungen der Genexpressionen dieser frühen Schritte den Prozess des Chl-Abbaus beschleunigen. In der vorliegenden Arbeit werden die Auswirkungen der Überexpression der Chlase aus Citrus clementii (CcCHLASE) und von WSCP aus Blumenkohl (Cau-WSCP) in transgenen Tabakpflanzen analysiert. Dazu wurde die cDNA Sequenz der CcCHLASE in E. coli exprimiert und mittels in vitro Experimenten die Hydrolysierung von Chl durch die Chlase bestätigt. Anschließend wurden CcCHLASE exprimierende Tabakmutanten generiert und drei T1-Linien wurden unter verschiedenen Stress- und Seneszenzbedingungen untersucht. Die Chlase überexprimierenden Linien zeigten unter allen getesteten Bedingungen einen im Vergleich zum Wildtyp erhöhten Chlide a Gehalt. Trotzdem unterschied sich die Menge an Endkataboliten in diesen Mutanten nicht vom Wildtyp. Andererseits zeigten WSCP überexprimierende Linien zwar keine erhöhten Chlide a Gehalte jedoch erhöhte Protochlorophyllid-(Pchlide)-Level. Das deutet auf eine Rolle des WSCP als Speichermolekül für Chlorophyllvorstufen hin. Die photoprotektive Funktion des WSCP wurde zusätzlich in WSCP überexprimierenden Linien bestätigt. Diese zeigen im Vergleich zu Wildtyp-Tabakpflanzen auch bei hohen Lichtintensitäten von 700 – 900 µmol Photonen m-2 s-1 verringerte Gehalte an Zeaxanthin und reduzierte Peroxidaseaktivitäten.
Chlorophyll (Chl) catabolism is characteristically seen during leaf senescence, fruit ripening and seed maturation. Disruption of this coordinated process under frost conditions delays Chl breakdown and is a great concern in rapeseed oil production. The present work addresses this problem by studying the effect of enhanced Chl catabolism in genetically modified tobacco plants. Chl is catabolised to colourless catabolites through a series of enzymatic reactions initiated by Chlorophyllase (Chlase). A water soluble chlorophyll protein (WSCP) has been proposed to transport Chl from thylakoid membranes to the site of action of Chlase. It was assumed that enhancing the gene expression of these early events in Chl catabolism would increase the Chl breakdown process. The present work analysed the overexpression of Chlase from Citrus clementii (CcCHLASE) and WSCP gene from cauliflower (Cau-WSCP) in modified tobacco plants. Initially, the cDNA sequence of CcCHLASE was expressed in E. coli and in vitro tests confirmed the hydrolytic activity of Chlase on Chl. Subsequently, tobacco plants overexpressing CcCHLASE were generated and three T1 lines were analysed at various stress and senescence conditions. The in vivo production of Chlorophyllide (Chlide) indicated the extent of increased Chl breakdown. The Chlase overexpressor lines showed higher Chlide a steady state levels under all tested conditions in comparison to the WT tobacco plants. However, the end catabolites did not show much difference from WT plants. On the other hand, WSCP overexpressor lines did not show any increase in Chlide a levels, but demonstrated an increased protochlorophyllide (Pchlide) levels. This suggested the role of WSCP as a storage molecule of Chl precursors. Additionally, photoprotective function of WSCP was confirmed in WSCP overexpressors, by lower zeaxanthin levels and peroxidase activity even at high light intensities of 700 – 900 µmol photons m-2 s-1 in comparison to the WT tobacco plants.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム
Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第21790号
工博第4607号
新制||工||1718(附属図書館)
京都大学大学院工学研究科分子工学専攻
(主査)教授 白川 昌宏, 教授 梶 弘典, 教授 森 泰生
学位規則第4条第1項該当

The general aim of the activities conducted in the framework of my PhD were to learn modern techniques for analyzing proteins by mass spectrometry (MS) and then to apply these techniques and approaches to the analysis of allergenic proteins contained in foods. My research activities were conducted at the Laboratory of Protein Chemistry of CRIBI, University of Padua, where previously I have conducted the research for my Thesis for the Doctor degree in Pharmaceutical Biotechnologies. During the fist year of my PhD I have concluded the Thesis project on the amyloid aggregation of -lactalbumin, a model protein utilized for investigating molecular aspects of protein amyloidogenesis. The results of this research were quite interesting and indeed they have been published in an international journal. During the first two years I acquired a solid knowledge on several aspects of the MS methodology and I was able to learn the theory and practice of several modern techniques and approaches in this ambit. The specific aim was to analyze allergenic proteins contained in complex matrices as foods and to this aim several proteins were extracted and purified from several food samples. The research has been focused mostly on the allergenic proteins from milk and eggs, known to cause widespread allergies. The proteins of interest were analyzed by using several chromatographic and electrophoretic techniques and also by means of HPLC connected to a tandem MS electrospray instrument. I was able to show that MS techniques can be used to identify allergenic proteins even when contained in very complex mixtures. Therefore, these MS techniques perhaps can be used as an alternative to the immunochemical methods nowadays in use for detecting allergens. I have also analyzed the chemical modifications that allergenic proteins suffer during several industrial treatments of foods, including heat treatment. During the third year of my PhD I spent a six months period at the Biochemistry Laboratory of the Imperial College in London, being involved in a project aimed to study in a large scale the proteins of the mosquito Anopheles gambiae. The MS analyses were focused on the proteins responsible of the mating behaviour of A. gambiae, hoping to identify a target for controlling the behaviour of this vector of the malaria disease. Summing up, besides the publication dealing with amyloid aggregates of beta- lactalbumin, this PhD Thesis is composed by a major part dealing with MS analysis of allergenic proteins and by a minor one dealing with MS analysis of proteins from A. gambiae.

MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes. Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergen’s structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data. Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays. Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins. In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (α-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked. Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named “targeted proteomics” was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample. The procedure involves specific labelling and isolation of the specific peptide, named “proteotypic”. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The “targeted proteomics” procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research. Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis. The molecular properties of the complex formed by α-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation. Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).
Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi d’elezione applicati per l’identificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattività e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare l’interazione con gli anticorpi specifici, invalidando i risultati. Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per l’identificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, l’identificazione delle proteine mediante MS si basa sull’analisi della sequenza amminoacidica di quest’ultime, mentre i saggi immunochimici sono strettamente dipendenti dall’integrità della struttura tridimensionale della proteina antigenica. Al fine di testare la validità dell’approccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte α-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualità di questi anticorpi, in termini di specificità di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattività. Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico. Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata l’applicazione di una tecnica detta “targeted chromatography”. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso l’analisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti “prototipici”. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dell’anello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando l’aumento di idrofobicità e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinità utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di ”targeted proteomics” è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per l’identificazione di una proteina allergenica contenuta in un prodotto dolciario. È stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente l’analisi di fingerprinting/MS che è solitamente utilizzata per l’identificazione di proteine nei protocolli di proteomica. Altre attività di ricerca. Durante il periodo di dottorato, ho avuto l’opportunità di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato. Sono state investigate le proprietà molecolari del complesso formato da α-lattalbumina con l’acido oleico. Il complesso appare interessante poiché ha mostrato avere tossicità cellulare diretta selettivamente contro cellule tumorali. È stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che l’acido oleico interagisce anche con altre proteine, come l’apomioglobina. La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola l’acido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dell’acido grasso e conseguentemente della sua proprietà citotossica. È in preparazione un articolo riguardante questi risultati. L’enterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attività antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticità e una ridotta stabilità alla denaturazione termica, ma mantiene la sua attività antibatterica., mentre i frammenti presentano un’attività ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per l’attività antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008).

This thesis describes an immunochemical and immunocytochemical study of the bovine brain S-lOOb protein. The two major forms of the S-100 isoproteins (S-lOOa and S-lOOb) were purified to apparent homogeneity from bovine brain. A polyclonal rabbit antiserum to the S-lOOb protein was prepared. The antiserum was characterized by solid phase immunochemical methods. The S-lOOb derived antiserum displayed a high degree of specificity for S-lOOb, but also crossreacted with the purified S-lOOa protein. The characteristics of the immunochemical reactivity of the antiserum towards these two isoproteins suggests the antiserum has specificity for the 6-subunit of the S-100 proteins. An immunohistochemical analysis of the cellular localization of S-lOOb immunoreactivity was undertaken. In the adult rat brain only the astrocytes were S-lOOb immunoreactive. This conclusion is supported by the morphological characteristics of the immunolabelled cells, as well as the observed co-localization of the immunoreactivities for S-lOOb and the Glial Fibrillary Acidic protein (GFAP), the major protein of the astrocyte intermediate filaments. These two antigens were always found to coexist. The immunolabelling of rat brain astrocytes by the S-lOOb derived antiserum stained the entire cell, yielding more complete morphological detail than is possible with the GFAP immunohistochemistry, which only labels the filamentous glial processes. It is concluded that S-lOOb immunohistochemistry could be of general utility in the investigation of astrocyte morphology. The present results also determine that the as yet unknown biological function(s) of the S-100 proteins must be related to a property of astrocytes.
Medicine, Faculty of
Graduate