Alzheimer’s disease (AD) is the most prevalent form of dementia affecting the elderly, and as the aging population increases the social and economic burden of AD grows substantially. Pathological hallmarks of AD include the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs), as well as significant neuron loss. Amyloid plaques consist of aggregated amyloid beta (Aβ) peptide, which is generated from the proteolytic processing of amyloid precursor protein (APP) in addition to several other peptides. While the processing of APP has been characterized, its primary physiological function and its involvement in AD pathology are poorly understood. Developing a greater understanding of the function of APP, and the molecular and cellular functions it is involved in or other proteins it is associated with, could provide insight into its role in AD pathology. To investigate the function of APP695, the neuronal isoform of APP, we used mass spectrometry to compare changes in protein expression and phosphorylation between APP-null B103 and APP695-expressing B103-695 rat neuroblastoma cells. Mass spectrometry-based proteomics has become a powerful technique for the unbiased identification of proteins from complex mixtures. Quantitative proteomics using labeling techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), allow relative quantitation of multiple samples at once. More recently, with advances in mass spectrometer technology, label-free quantitation has become a reliable quantitative proteomics approach. Additionally, mass spectrometry can be used for the analysis of post-translational modifications, such as phosphorylation, a dynamic modification involved in the regulation of many cellular processes. Phosphoproteomics identifies site-specific phosphorylation and surrounding sequence information, which can be used for consensus motif analysis to provide further information about potential changes in kinase activity. Identifying changes in phosphorylation and kinase activity also provides information about signaling pathways and functions that may be affected by APP695 expression. Comprehensive proteomic and phosphoproteomic datasets can be used to gain insight into the molecular mechanisms that may be regulated by APP695 expression, or involved in AD progression and pathology, leading to the development of novel therapeutic and preventative strategies for AD. Proteomic and phosphoproteomic analysis of B103 and B103-695 cells identified several significant protein expression and phosphorylation changes that may be mediated by APP695-expression. Global-scale proteomic analysis identified increased expression of Ras and ƴ-synuclein in B103-695 cells, which was further validated in human AD brain tissue. Phosphoproteomic analysis showed increased phosphorylation of Histone H4 at Ser47, and led to the investigation of PCTAIRE-2 (Cdk17), and PCTAIRE-3 (Cdk18) expression, which were all shown to be increased in AD transgenic mouse tissue, culture primary rat neurons treated with Aβ, as well as mild cognitive impairment (MCI) and AD human brain tissue. Label-free quantitative proteomics was used for the analysis of human brain tissue from the cortex of individuals affected by AD, MCI, Parkinson’s disease (PD), and progressive supranuclear palsy (PSP) compared to cognitively normal, control samples. A number of differentially expressed proteins were identified in AD, MCI, PD, and PSP tissue. Bioinformatic analysis of the comprehensive proteomic datasets from AD, MCI, PD, and PSP human brain tissue identified several proteins consistent with corresponding disease pathology and neurodegeneration, such as inflammatory proteins. While some of the molecular and cellular functions were unique among neurodegenerative diseases, there also appears to be overlap of affected functions, suggesting there may be a more common mechanism of neurodegeneration.

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Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15039号 / 工博第3188号 / 新制||工||1479(附属図書館) / 27500 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 白川 昌宏 / 学位規則第4条第1項該当

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16886号 / 工博第3607号 / 新制||工||1545(附属図書館) / 29561 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 白川 昌宏 / 学位規則第4条第1項該当

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17599号 / 工博第3758号 / 新制||工||1573(附属図書館) / 30365 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 杉野目 道紀 / 学位規則第4条第1項該当

Submitted in full requirement for the Degree of Master of Management Sciences Specialising in Hospitality and Tourism, Durban University of Technology, Durban, South Africa, 2017. / Over the years the rapid growth of the tourism industry has revealed itself to be a major source of income and social improvement for many. However, with this growth comes the undoubtable carbon footprint it carries. As a result, the growth and expansion of many tourism ecolabels have surfaced in the hopes of alleviating the negative environmental impacts the tourism industry imposes. This study aimed to ascertain the impacts of tourism ecolabels on businesses. It assessed the level of success of the ecolabel within the organisation as well as ascertained the benefits and challenges associated with ecolabel certification. A quantitative research approach was used and the data was collected by means of online questionnaires that were targeted at managers of tourism businesses in South Africa. The study reveals that tourism businesses in South Africa do experience the benefits of being certified with an ecolabel in terms of the natural, socio-cultural and economic environments. However, the high costs associated with being certified, the lack of general public awareness regarding ecolabels and the absence of government support are still prevalent. Consequently, cost reduction, promotion of public awareness as well as government support are the main areas of improvement required by tourism establishments with regard to ecolabels. / M

Abstract Failure to maintain an appropriate balance between cell death and proliferation is partly due to derangements in the regulation of apoptosis. In this work, apoptosis and the expression of apoptosis regulating proteins were studied by 3' - end labeling of fragmented apoptotic DNA (TUNEL) and immunohistochemistry in a set of 147 tissue samples consisting of 44 biopsies of normal and dysplastic bronchial epithelium, and 103 non-small cell lung carcinomas. The quantity of apoptotic cells and bodies, apoptotic index (AI%), is defined as a percentage of apoptotic cells in the entire tumor cell population. Changes in the apoptotic activity were already seen in the metaplasia-dysplasia-carcinoma sequence of the lung, where the AI% increased gradually until moderate epithelial dysplasia but started to decrease after that. Thus, the AI% for invasive NSCLC (1.20 for squamous cell carcinoma and 1.24 for adenocarcinoma) was slightly lower than in premalignant bronchial epithelium (mean 1.50), but clearly higher than in normal tissue (0.20 for normal bronchial epithelium and 0.24 for lung interstitial cells). 53% of SQCCs and 50% of ACs showed p53 positive nuclei indicative of mutated p53 protein. The immunostaining of bcl-2, bax and mcl-1 revealed diffuse, cytoplasmic staining and was present in most tissues studied. No statistically significant associations between the extent of apoptosis and the expression of p53, bcl-2, bax, or mcl-1 could be found, although . The immunostaining for caspases 3, 6 and 8 was restricted to the tumor areas, reflecting increased apoptotic activity in them. The AI% was significantly higher in NSCLCs in which the single-cell staining pattern for caspase-8 was dominant (P = 0.017), whereas the expression of caspases 3 and 6 had no association with apoptosis. The number of apoptotic cells was significantly higher in NSCLC tumors with a high number of CD3+ and CD8+ T-lymphocytes (P = 0.01) and B-cells (P = 0.05). By multivariate analysis, enhanced apoptosis in NSCLC showed a 1.9-fold risk (95% CI 1.04–3.60; P = 0.04) and p53 positivity a 2.3-fold risk (95% CI 1.30–4.10; P = 0.005) for a shortened survival. Both factors appeared as independent prognostic variables. Apoptosis is clearly enhanced in premalignant and malignant lung tissue in comparison with normal tissue. Furthermore, the expression of the apoptosis-regulating genes is different in tumor tissue from that in normal tissue, and some of the changes in their expression can be seen even in the premalignant lesions of the bronchial epithelium. The expression of caspases seen only in tumor tissue implies the activation of the apoptotic mechanisms and, thus, the lowered treshold of tumor cells to undergo apoptosis. Even in the advanced stages of the disease, the immune defense is effective and the cytotoxic action of activated CD8+ T-cells clearly involves apoptosis. Based on these results it is concluded that alterations in the apoptotic activity and changes in the expression of apoptosis-regulating genes are associated with malignant transformation and growth in lung tissue.

Labeling proteins with chemical tools is important for examining natural systems, discovering therapeutic agents and developing protein constructs. These methods offer simple but reliable chemistry to the study of peptides and proteins and thus have gained popularity among chemists and biologists. Despite the fact that the number of successful examples has been largely increased over the past decade, there is still an ongoing need for new reagents with better accessibility and reactivity. Diazonium ions are known to selectively react with tyrosine residues for more than a century. But the harsh condition required for diazotization makes it difficult to use this strategy in biological applications. To address this, bench-stable triazabutadienes are made to release diazonium ions upon mild acidification or photoirradiation. Based on our previous study, imidazole N-alkyl substituted triazabutadienes were synthesized and tested for diazonium ion-releasing rates. Surprisingly, the imidazole N-tert-butyl substituted triazabutadiene showed the fastest rate in neutral and basic aqueous solutions. A subsequent NMR study revealed that this rapid release of diazonium ions might be ascribed to the lack of intramolecular π-interactions. In addition, triazabutadienes can be rendered more basic upon photo-isomerization. A water-soluble triazabutadiene was shown to adjust the pH of aqueous solutions. These findings open up new opportunities in protein labeling with unprecedented ease. Moreover, a boronic acid-based photocrosslinker was synthesized to detect protein-protein interactions of glycoproteins. By incorporating benzophenone with a boronic acid and a terminal alkyne, this photocrosslinker is designed to capture the glycoprotein-substrate complex using the combination of photochemistry and bioorthogonal reactions. In conclusion, this dissertation demonstrates progress in developing new probes for protein labeling and protein-protein interactions. These newly developed strategies offer convenient alternatives to those wishing to explore protein activities.

PANcreatic DERived factor (PANDER, FAM3B) is a member of a superfamily of FAM3 proteins that are uniquely structured and strongly expressed from the endocrine pancreas and co-secreted with insulin. Unique animal models available to our lab have indicated that PANDER can induce a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased. The complexity of the biological networks involved with this process warranted the logical approach of employing quantitative mass spectrometry based proteomic analysis using stable isotope labeling of amino acids in cell culture (SILAC) to identify the global proteome differences between the PANDER transgenic (TG) overexpressing murine model to matched wild-type mice under three metabolic states (fasted, fed and insulin stimulated). Additionally, this technique was used to compare the hepatic proteome of mice on a high fat diet to elucidate early and late mechanisms of disease progression. The “spike-in” process was employed by equal addition of lysate obtained from livers of heavy L-Lysine (13C6, 97%) fed mice to the mice liver protein lysate (PANTG and WT) for relative quantitative analysis. Upon acquisition of the dataset by use of liquid chromatography tandem mass spectrometry (LC-MS/MS, LTQ Orbitrap), geometric means and Uniprot Protein identification numbers were uploaded to Ingenuity Pathway Analysis (IPA) to reveal the effect of PANDER on hepatic signaling. IPA identified lipid metabolism and fatty acid synthesis as top cellular functions differentially altered in all metabolic states. Several molecules with a role in lipid metabolism were identified and include FASN, ApoA1, ApoA4, SCD1, CD36, CYP7A1 and ACC. Furthermore, central to the differentially expressed proteins was the revealed activation of the liver X receptor (LXR) pathway. In summary, our SILAC proteomic approach has elucidated numerous previously unidentified PANDER induced molecules and pathways resulting in increased hepatic lipogenesis. In addition, we have demonstrated strong utility of this approach in comprehensively phenotyping animal models of hepatic insulin resistance. PANDER may potentially propagate pro-hepatic lipogenic effects by LXR activation in contrast to increased LXRα expression. This can be evaluated through the use of LXR agonists (T0901317) antagonists (GSK 2033). LXR activity can be measured by luciferase assays using an LXRE response plasmid. Our central hypothesis is that PANDER induces activation of LXR and is measured and predicted in our line of experiments. In general, PANDER induced LXR activation will be enhanced by T0901317 and diminish effects of GSK 2033 along with direct correlation of downstream metabolic effects such as increased hepatic lipogenesis and fatty acid metabolism. Taken together, PANDER strongly impacts hepatic lipid metabolism and may induce a SHIR phenotype via the LXR pathway. Additionally, phosphoproteomic analysis uncovered large-scale differences in protein phosphorylation states as PANDER impacts insulin signaling. A notable finding was the increased phosphorylation of glycogen synthase (GSK), possibly responsible for the decreased hepatic glycogen content in the PANTG mouse. In an effort to map out critical molecules involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis, the same proteomic approach was carried out, providing a unique dataset of differentially expressed hepatic proteins due to a high at diet.

Orientador: Maria Irma Hadler Coudry / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Estudos da Linguagem / Made available in DSpace on 2018-08-18T14:48:04Z (GMT). No. of bitstreams: 1 Antonio_GiovanaDragoneRosseto_M.pdf: 25623579 bytes, checksum: 9f9d0525db03c5082327dc36d7287434 (MD5) Previous issue date: 2011 / Resumo: Diante de um contexto de patologização excessiva, em que há um grande número de patologias e diagnósticos relacionados a questões escolares, sobretudo referentes ao processo de aquisição e uso da leitura e escrita, este trabalho busca discutir, com base na Neurolinguística Discursiva, a forma como tais diagnósticos têm sido feitos e as consequências que trazem para a vida das crianças. Apesar de serem muitas as "doenças" que surgem para justificar o fracasso escolar ou o comportamento inadequado de algumas crianças, este trabalho se aterá à discussão sobre a dislexia. Nosso objetivo, portanto, é discutir o estatuto de doença que é atribuído à dislexia, analisando, para isso, o discurso autorizado proferido sobre ela (sobretudo o da Associação Brasileira de Dislexia - ABD) e a concepção de linguagem na qual este discurso se baseia, que funciona como um dispositivo capaz de controlar os sujeitos, de acordo com Agamben (2004). Além disso, comparamos as propostas de material e atividades da Associação Brasileira de Dislexia com o trabalho realizado no Centro de Convivência de Linguagens (CCazinho/IEL/Unicamp) / Abstract: In the face of a background of excessive labelling in which there are a great number of diseases and diagnostics related to school problems, mostly concerning the processes of reading and writing acquisition, this masters dissertation discusses, based on the Discoursive Neurolinguistics, how these diagnostics are being given and the consequences that they bring to children's lives. In spite of the many "diseases" that are emerging to justify school problems and failures or to justify an inappropriate behavior from some children, this text will focus on the debates on dyslexia. Therefore, our goal is to discuss how dyslexia came up as a disease, analyzing for that matter the authorized discourse on it (mostly from the Associação Brasileira de Dislexia - ABD) and the conception of language in which this discourse is based on that work as an apparatus capable of controlling the subject, according to Agamben (2004). Besides, we compare the activities and material from the Associação Brasileira de Dislexia with the work done at the Centro de Convivência de Linguagens (CCazinho/IEL/Unicamp) / Mestrado / Linguistica / Mestre em Linguística