Anterior Gradient-2 protein (AGR2) has recently been linked to the onset of several pathologies including asthma and inflammatory bowel disease. Most interestingly, it has been discovered to influence the transformation of cells and metastatic growth essential to cancer development, and has subsequently been linked to the development of resistance to anti-cancer therapeutics. AGR2 protein is overexpressed in a diverse range of human cancer types, and has been detected secreted into the extracellular milieu. Thus, AGR2 protein represents a compelling pro-oncogenic signalling intermediate in tumour emergence and endurance. This thesis presents an interdisciplinary approach including structural biology, cell biology and synthetic biology, and clinical studies to shed more light on the role of AGR2 in cancer development. Synthetic cell based reagents were developed to define the dominant pathways that are reprogrammed in a cell as a result of AGR2 synthesis. A cell panel was engineered incorporating the AGR2 (and mutants thereof) allele into the AGR2-null A375 cell line. These tools were then coupled to quantitative proteomics (SILAC) to unravel the mechanism whereby introduction of AGR2 alters cell phenotype, allowing identification of dominant pathways affected by AGR2 signalling. Using pathway analysis tools, the dominant pathway suppressed by wt-AGR2 expression highlighted the p53-signalling axis. DNA damage induced p53 stabilisation and p21 induction by cisplatin treatment confirmed the influence of AGR2 gene expression. Further data analysis identified the outlying protein expression changes identified by SILAC was the anti-viral cell cycle regulator TSG101 (tumour susceptibility gene 101), and confirmed by immunoblotting. Transfection and silencing studies of TSG101 confirmed that TSG101 attenuates p53 function. These data provide a mechanism to explain the most dominant pathways reprogrammed by AGR2 expression, incorporating ER stress response, proliferation markers and p53 pathway attenuation. Further advances were made in analysis of the function, regulation, and drugability of AGR2 protein. Assays were devised to define the subunit structure of AGR2 as a dimer unit; subsequent functional studies defined intrinsically disordered motifs that regulate stability of the dimer. A two-site sandwich microtiter assay (2SMTA) was designed to screen for self-peptides and mutations that regulate oligomer stability. These assays were used to identify the first biochemical property of AGR2 being that the dimer unit is required for maximal binding to the AAA+ protein, and well characterised AGR2 interactor, Reptin. In addition, based on this dimeric structure, a novel solution based dimerisation assay was developed to identify natural products that are able to disrupt the dimer suggesting that AGR2 itself can be targeted in principle with small molecules for therapeutic purposes.

No description available.

No description available.

The superfamily Sauropodomorpha comprised some of the largest terrestrial herbivorous vertebrates that have ever walked on this planet. The study of these fascinating beings has been at the forefront of palaeontological research throughout the years. Especially in the last 20 years, due to the advent of modern techniques and technologies, significant steps have been made towards our understanding of their physiology. One of the sauropodomorphs’ central morphological characteristics is the expression of postcranial skeletal pneumaticity (PSP), a condition that is also expressed in the other superfamily of the order Saurischia, the Theropoda, as well as in their extant relatives, the Avians. This condition remodels the vertebrae and, occasionally the girdles and appendicular elements, producing perforations, depressions, excavations, cavities and internal chambers. This is a result of bone invasion and resorption from the development of the lung-air sac diverticula of the respiratory system. The various forms of this expression have been addressed across all subfamilies of sauropodomorphs mostly from an evolutionary aspect and, recently, from a developmental scope too. The resulting hypotheses state that PSP may have acted as a mechanism for weight reduction, allowing for sauropods to attain large sizes without having to suffer analogous gravitational constraints from equally attained masses. Any possible associations, though, between the expression of pneumaticity and body size have not been put under test. Through this study, a method of quantification and categorisation of PSP, and therefore, classification of the sauropodomorphs which express it, is created from the data retrieved from 61 taxa across all subfamilies, permitting us not only to trace any correlation between PSP and metric size data (body mass and body length) but also to visualise the evolution of PSP throughout Sauropodomorpha. This classification scheme from highest to lowest expression of PSP, ‘Alpha’, ‘Beta’, ‘Gamma’, ‘Delta’, and ‘Epsilon’ stems from the numerical estimate of pneumaticity in terms of a percentage, called the Pneumaticity Degree Index (PDI%). The revised scheme, Pneumaticity Degree Index (PDI%), takes account of the number of vertebral elements that are pneumatized in a single vertebra, the nature of pneumaticity traits, as well as the intensity of pneumatization in different body regions of interest (e.g. vertebral column), resulting in an integral and comprehensive measure of PSP. The proposed method ranks each pneumaticity trait with a value from 1 to 5 with 1 (100%) representing the most invasive unambiguous trait (e.g. camellae) and 5 (~ 10%) the least invasive and most ambiguous trait (i.e. fossa). By adding and dividing by their number all of the observed traits of every available vertebra of a region we retrieve a decimal numeric outcome and this outcome is translated to a percentage. The total average pneumatization of any taxon is calculated by retrieving the total average of the pneumaticity from all available body regions. In this study, only vertebral and pelvic elements were used for the retrieval of pneumaticity data since they are the most frequently exhibiting pneumatic traits in comparison to pectoral and appendicular elements. Results show that the total average PDI% range of basal Sauropodomorpha is 0%-59%, of the non-neosauropod Eusauropoda is 23%-90%, of Diplodocoidea is 45%-73%, of Macronaria is 45%-92% and of Somphospondyli is 46%-94%. The most pneumatized vertebral landmarks are the centrum, neural arch and neural spine. The vertebral regions most commonly pneumatized are the cervicals and dorsals. No genus or subfamily of sauropodomorphs ever reaches 100% pneumatization in all vertebral regions. Furthermore, PSP is not always positively correlated with mass, rather its expression is mostly correlated with length. Taxa with low or high masses may exhibit either low or high PDI’s. In addition, increasing progression of the extent and expression of PSP occurs mostly on a subfamilial level and less throughout the entire lineage. Finally, modelling calculations result in an Ornstein-Ulenbeck with an early burst progress of the expression of pneumatisation in sauropodomorphs. After that, the expression proceeds relatively steadily throughout the entire superfamily. The biotic causes of PSP are still unclear, as it could be an artefact of inheritance and genetic drift throughout speciation events. The pneumatization degree index (PDI%) is a means of quantifying and categorising pneumatization in any archosaurian taxon that is faster and cheaper, though less accurate, than CT scanning. It is more precise than the Pneumaticity Index since it integrates a wider selection of the qualities and osteological characteristics of pneumaticity we want to measure, provided that the bone under study is at least 50% free of matrix.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

TCC(graduação) - Universidade Federal de Santa Catarina. Centro Tecnológico. Ciências da Computação. / Submitted by José Victor Feijo de Araujo null (victor.feijo@ufsc.br) on 2017-12-12T00:28:08Z No. of bitstreams: 1 TCC_JOSE_VICTOR_FEIJÓ.pdf: 18256303 bytes, checksum: 6f566a4daec3603fa7cc31bf1d8da5c8 (MD5) / Approved for entry into archive by Renato Cislaghi (renato.cislaghi@ufsc.br) on 2017-12-13T21:10:36Z (GMT) No. of bitstreams: 1 TCC_JOSE_VICTOR_FEIJÓ.pdf: 18256303 bytes, checksum: 6f566a4daec3603fa7cc31bf1d8da5c8 (MD5) / Made available in DSpace on 2017-12-13T21:10:36Z (GMT). No. of bitstreams: 1 TCC_JOSE_VICTOR_FEIJÓ.pdf: 18256303 bytes, checksum: 6f566a4daec3603fa7cc31bf1d8da5c8 (MD5) / A proposta sugerida por este trabalho foi de analisar o impacto de um modelo de classificação, seguido de técnicas de PDI e OCR para extração de texto em cupons fiscais, classificando-os em subgrupos. Técnicas selecionadas de PDI foram aplicadas para cada grupo com suas devidas características, por fim extraindo texto dessas imagens através de um algoritmo de OCR. Foi realizado um estudo sobre os algoritmos clássicos de classificação na área de aprendizado de máquinas, com foco nos algoritmos de “clusterização” e sua correlação com a classificação de imagens em um modelo de aprendizado não supervisionado. Também foi feita uma análise sobre as características das imagens de cupons fiscais e das possíveis técnicas de PDI que podem ser aplicadas. Em relação ao OCR, também foi realizado um estudo para verificar possíveis soluções na extração de texto e entender seu comportamento, possibilitando desta maneira implementar a arquitetura proposta. Sendo assim, foram desenvolvidos métodos para classificar as imagens em clusters utilizando algoritmos de “clusterização”. Também foram propostas três técnicas de PDI, a primeira aplicando uma série de realces, a segunda uma binarização adaptativa e a terceira técnica utilizando a compressão de dados JPEG. Essas imagens foram enviadas para o serviço de OCR do Google Vision, onde foi possível extrair o texto das imagens em formato de blocos. Os resultados do modelo desenvolvido foram avaliados comparando a taxa de acerto do OCR com os valores de texto reais presentes nos cupons fiscais, onde foi possível analisar a precisão de cada técnica proposta e da arquitetura como um todo. Foram obtidos resultados positivos utilizando o modelo desenvolvido, melhorando a extração do valor total da compra em aproximadamente 6%. Além disso, os resultados da compressão JPEG melhoraram também a extração de outros dados do cupom fiscal, como por exemplo o CNPJ e a data da compra.

The Versal Adaptive Compute Acceleration Platform (Versal ACAP) is a system-on-chip (SoC) developed by AMD Xilinx. To help protect the programmable logic from soft errors, the configuration needs to be constantly checked and repaired through a process called scrubbing. This thesis provides a methodology for scrubbing the configuration over JTAG. The scrubber uses two platform device image (PDI) files, one to read the configuration and one to send corrected frames. The methodology is characterized to determine the time it takes to completely scrub the configuration. The designed scrubber was able to scrub the VM1802 in 11.5 seconds, or 41.6 Mbits/second, when the JTAG interface was operated at 50MHz.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study aimed to analyze the perspective of sustainability in Institutional Development Plans (PDIs), identifying the dimensions that are basing the Mission and Vision of Federal Institutions of Higher Education (IFES). The qualitative and quantitative research had 28 institutions that had a clearly defined mission and vision as units of analysis. The first step was to collect data from a desk study, in which information about the PDI published on the websites of IFES (Bardin, 2011) were observed. In the second step was carried out a qualitative study using the technique of content analysis and the process of progressive/analog systematization, inductively-constructive approach. In the third stage there was a non-parametric quantitative analysis based on descriptive statistics, chi-square tests and test T. The main findings showed that 28 IFES have not updated the IDP, as Brazilian law establishes. All 28 IFES have analyzed this social dimension in PDI, standing out as the main key words highlighted in the Mission and Vision: "professional, knowledge, reference, scientific and production." Regarding the profile, most IFES included in the study belongs to the southeastern region of the country have an average of 30,000 students and an average budget in the range of 400 million. Of IFES analyzed, 03 are not in the current document. In the quantitative phase, it was found that the region variable significantly associated with the national political dimension, and that , among others , realized the alignment with the guideline of improving the quality of teaching , the PNE. In general, it was found that sustainability is being addressed in the IDP incipient, seeking to meet the objectives of the NAP policies and some of the MEC , with a focus on teaching, research and extension. / Este estudo teve como objetivo analisar a perspectiva da sustentabilidade em Planos de Desenvolvimento Institucional (PDI), identificando as dimensões que estão embasando a Missão e Visão de Instituições Federais de Ensino Superior (IFES). A pesquisa qualitativa e quantitativa teve como unidades de análise 28 instituições que apresentavam a Missão e a Visão claramente definidas. A primeira etapa consistiu na coleta de dados de um estudo documental, no qual foram observados informações sobre o PDI divulgadas nos sites das IFES (BARDIN, 2011). Na segunda etapa procedeu-se a um estudo qualitativo por meio da técnica de análise de conteúdo e o processo de sistematização progressivo/analógico, com abordagem indutivo-construtiva. Na terceira etapa realizou-se uma análise quantitativa com base na estatística descritiva simples, testes não paramétricos Qui-quadrado e Teste T. As principais evidências apontaram que 28 IFES não possuem o PDI atualizado, conforme a estabelece legislação brasileira. Todas as 28 IFES analisadas possuem a dimensão social presente no PDI, destacando-se como principais palavras chaves em evidência na Missão e na Visão: profissional, conhecimento, referência, científico e produção . Quanto ao perfil, a maioria das IFES incluídas na pesquisa pertence à região sudeste do país, possuem em média 30 mil estudantes e um orçamento médio na faixa de 400 milhões. Das IFES analisadas, 03 não estão com o documento vigente. Na etapa quantitativa, verificou-se que a variável região apresentou associação significativa com a dimensão política nacional, e que, dentre outras, percebeu-se o alinhamento com a diretriz de melhoria da qualidade do ensino, do PNE. Em linhas gerais, verificou-se que a sustentabilidade vem sendo tratada no PDI de maneira incipiente, buscando atender aos objetivos do PNE e algumas políticas do MEC, com foco no ensino, pesquisa e extensão.

This research has investigated the gene expression of protein disulfide isomerase (PDI; EC 5.3.4.1) and thioredoxin (Trx) in the marine macroalga Ulva fasciata Delile in response to hypersaline (90‰) for 1 h. 90‰ induced H2O2 accumulation, reflecting the occurrence of oxidative stress. The contents of free and protein-bound SH were increased by 90‰. Trx transcripts increased in response to 90‰. PDI transcripts and enzyme activities increased in response to 90‰. H2O2 accumulation under 90‰ condition was increased by putrescine (Put) but decreased by spermidine (Spd) and spermine (Spm). By treatment of spermidine and spermine, the contents of free SH was increased and the contents of protein-bound SH decreased, showing that spermidine and spermine can increase free SH against oxidative stress. The gene expression and activity of PDI were further increased by Spd and Spm. Overall, the gene expression of PDI and Trx were responded to 90‰ for 1 hour and were adjusted protein¡VSH in polyamines treatment.