Dissertations / Theses on the topic 'Pwwp'

L’intégration est une étape clé du cycle réplicatif du VIH-1 catalysée par l’intégrase virale, impliquant in vivo de nombreuses protéines virales et cellulaires dont LEDGF/p75. Ce cofacteur interagit directement avec l’intégrase, stimule fortement l’intégration et est indispensable à une infection productive. Dans la cellule, l’intégration cible l’ADN empaqueté dans la chromatine. Nous avons mis au point un essai d’intégration in vitro sur matrice chromatine qui a mis en évidence le rôle du domaine PWWP pour la stimulation de l'intégration dépendante de LEDGF. Nous avons de plus caractérisé les éléments structuraux de LEDGF et de ce domaine impliqués dans son interaction avec la chromatine et favorisant l’activité de LEDGF au cours de l’intégration. Ce système a permis de corréler des données in vitro avec celles obtenue sur l'effet de LEDGF au cours de l'infection par le VIH-1. Dans une seconde partie, nous avons mis au point une stratégie de ciblage de l’intégration vers des régions d’hétérochromatine. Nous avons donc construit des intégrases chimériques par fusion à l’intégrase de VIH-1 de domaines protéiques reconnaissant des motifs hétérochromatiniens. Nos résultats montrent le maintien d’une activité d’intégration de ces enzymes in vitro et in vivo. De plus, l’intégration d’un gène rapporteur apporté par ces virions subit une extinction progressive de son expression, caractéristique de l’effet négatif de variégation lié à la propagation de l’hétérochromatine environnante. Cependant, cet effet peut être levé en flanquant le transgène de séquence insulatrice. Ces résultats ouvrent des perspectives pour la réduction de la génotoxicité des vecteurs rétroviraux.

L’intégration est une étape clé du cycle réplicatif du VIH-1 catalysée par l’intégrase rétrovirale. Cette étape est la cible de nouveaux inhibiteurs incorporés dans les traitements antiviraux mais l’apparition de souches virales résistantes à ces inhibiteurs conduit à rechercher de nouvelles molécules inhibitrices de cette étape. L’intégrase du VIH-1 interagit avec de nombreux cofacteurs cellulaires dont la protéine LEDGF/p75 qui est essentielle à la réplication virale. Elle stimule les activités catalytiques de l’intégrase et participe à la sélectivité d’intégration. LEDGF/p75 interagit avec l’intégrase et la chromatine via ses domaines IBD et PWWP respectivement. Mon projet a pour but d’identifier de nouvelles molécules antivirales ciblant ces deux interactions. Mes travaux de thèse m’ont tout d’abord permis d’identifier cinq séquences peptidiques cellulaires appelées PBDs (PWWP Binding Domain) interagissant avec le domaine PWWP de LEDGF/p75. La validation de ces PBDs a été effectuée par un test de complémentation protéique dans les cellules humaines, basé sur une restauration d’activité de deux domaines inactifs de la luciférase de Gaussia princeps. Deux de ces PBDs sont très prometteurs en raison de leur capacité à fixer la protéine entière du LEDGF/p75 et de leur incapacité à interagir avec des mutants de cette protéine ne fixant plus la chromatine et n’activant plus l’intégration dans cette structure cellulaire. Dans une seconde partie, j’ai établi un essai permettant de mesurer l’interaction entre l’intégrase et LEDGF/p75 directement dans les cellules. Cet essai permettra de réaliser un criblage haut débit afin d’identifier de nouvelles molécules antivirales bloquant cette interaction. Les résultats obtenus au cours de ma thèse permettent donc de proposer de nouvelles stratégies anti-VIH-1 ciblant des interactions protéiques essentielles entre ce virus et la cellule infectée
Integration is an essential step of the HIV-1 retroviral replication cycle catalysed by the viral integrase. Integrase inhibitors are undergoing clinical trials, but viruses resistant to these inhibitors have already emerged. Therefore, new integrase targets need to be identified, such as cellular integrase partners. One of them, the lens epithelium-derived growth factor (LEDGF/p75), is essential for HIV-1 replication. This protein interacts with both integrase and chromatin by his IBD and PWWP domains respectively. The purpose of my project was to identify new antiviral molecules targeting these two interactions. First, I have identified five peptides sequences interacting with the PWWP domain of the LEDGF/p75 (called PWWP Binding Domains or PBD). I have confirmed these interactions in 293T cells by a Protein Complementation Assay (PCA) based on the restored activity of two inactive fragments of the Gaussia princeps luciférase. Two PBDs are very promising due to their ability to interact with the full-length LEDGF/p75 protein and their inability to interact with LEDGF/p75 PWWP mutants affected by chromatin interaction and LEDGF-dependant activation of integration. The second part of my PhD program, I was involved in the establishment of a new assay that allowed the quantification of the LEDGF/p75 – HIV-1 integrase interaction directly in cells. This assay will be used to perform a high-throughput screen to identify new inhibitors of this interaction. Results obtained during my PhD allow the proposal new anti-HIV strategies targeting essential protein-protein interactions between this virus and infected cells

Multiple imputation (MI) is a commonly used approach to impute missing data. This thesis studies missing covariates in recurrent event data, and discusses ways to include the survival outcomes in the imputation model. Some MI methods under consideration are the event indicator D combined with, respectively, the right-censored event times T, the logarithm of T and the cumulative baseline hazard H0(T). After imputation, we can then proceed to the complete data analysis. The Cox proportional hazards (PH) model and the PWP model are chosen as the analysis models, and the coefficient estimates are of substantive interest. A Monte Carlo simulation study is conducted to compare different MI methods, the relative bias and mean square error will be used in the evaluation process. Furthermore, an empirical study based on cardiovascular disease event data which contains missing values will be conducted. Overall, the results show that MI based on the Nelson-Aalen estimate of H0(T) is preferred in most circumstances.

In the context of the high unemployment and poverty rates in South Africa, this research was undertaken to explore the best practices of successful international public works programmes (PWPs) around the world. The aim was to develop a strategy for poverty alleviation and job creation with respect to the infrastructure sector of the Expanded Public Works Programme (EPWP) in South Africa. The purpose of the EPWP is to make the unemployed more employable through offering beneficiaries temporary employment and training opportunities. In the literature review, the strategy for poverty alleviation and job creation was formulated in terms of the design elements and implementation aspects of PWPs. This strategy was then used to evaluate the infrastructure sector of the EPWP. The data collection took the form of interviews with key informants who are directly involved with the infrastructure sector of the EPWP. The nature of the enquiry was qualitative, with narrative and content analysis used to explore the data. The research found that, overall, the design elements and the implementation aspects of the infrastructure sector of the EPWP are not appropriate for enabling the unemployed to become more employable on a large scale. Based on the international best practices, recommendations were then put forward as improvements which would enable the infrastructure sector of the EPWP to achieve its objectives more effectively.
Dissertation (MBA)--University of Pretoria, 2012.
Gordon Institute of Business Science (GIBS)
unrestricted

O presente trabalho teve como objetivo principal identificar os fatores determinantes à adoção de estratégias de lobbying sobre a regulação contábil do setor petrolífero, bem como avaliar a qualidade das informações contábeis das empresas desse setor. Para atender a esse objetivo, recorreu-se a três estudos direcionados para cada um dos seguintes aspectos: determinantes do lobbying e características dos grupos de interesses (estudo 1); estágio atual da qualidade da informação contábil do setor petrolífero (estudo 2) e evidências empíricas às questões do Discussion Paper Extractive Activities (estudo 3). Para operacionalizar os estudos, utilizaram-se as cartas comentários enviadas ao IASB e a base de dados da Evaluate Energy®, empregando-se diversas técnicas econométricas como regressão linear simples e múltipla, regressão logística binomial e multinomial e regressão de Poisson a diversos planos amostrais contendo informações de empresas petrolíferas. Os resultados indicam que o grupo de interesse formado pelos preparadores de demonstrações financeiras do setor petrolífero possui incentivos econômicos para realizar lobbying sobre determinada regulamentação contábil no sentido de defender seus interesses como prega a teoria da regulação econômica, notadamente quanto ao fator tamanho. Tal grupo de interesse, dentro do plano das escolhas contábeis, é favorável à utilização do custo histórico como base de valor e da possibilidade de escolha entre dois modelos contábeis concorrentes (successful efforts e full cost) e desfavoráveis às mudanças que levem ao aumento do nível de divulgação das informações. Suportando essa posição, tem-se a visão dos usuários primários das demonstrações financeiras que sugerem que as informações contábeis disponibilizadas pelas empresas petrolíferas são de qualidade, ao menos sob o prisma da relevância e conservadorismo, e indicam, ainda, que o atual conjunto de informações baseado no custo histórico é relevante para as suas decisões econômicas. Levando-se em consideração o grande apoio recebido pela proposta de ampliação das divulgações obrigatórias, aí incluída a proposta do Publish What You Pay, tem-se a rejeição por parte dos participantes do mercado de capitais indicando que os benefícios decorrentes da ampliação da divulgação não serão maiores do que os existentes, considerando as regras atuais. Em contraponto, tem-se a concepção de que a informação contábil produzida pelas empresas petrolíferas é inoportuna. Nesse particular, não se vislumbram alterações nessa constatação uma vez que a proposta de um novo modelo contábil de reconhecimento apresentada no DPEA se aproxima do método full cost, enquanto que as evidências do segundo estudo apontam para direção contrária ao sugerir que o método successful efforts gera informações mais oportunas do que o método concorrente. Assim, tem-se um cenário para se acreditar na manutenção do status quo vigente.
This paper aimed to identify the determining factors for the adoption of lobbying strategies regarding the accounting regulation of the oil sector, as well as to assess the quality of the accounting information of the companies from this sector. To meet these goals, three studies directed to each of the underlined aspects were used: determinants of the lobbying and characteristics of the interest groups (study 1); current status of the quality of accounting information from the oil sector (study 2) and empirical evidence for the questions of Discussion Paper Extractive Activities (study 3). To operationalize the studies, letters and comments sent to the IASB and the database of Evaluate Energy® were deployed, through the use of several econometric techniques such as simple and multiple linear regressions, binomial and multinomial logistic regression and Poisson regression in various sampling plans featuring information of the oil companies. The results indicate that the interest group formed by preparers of financial statements for the oil sector has economic incentives to undertake lobbying for an accounting regulation, in the sense of defending their interests according to the economic regulation theory, notably when size is taken into account. This interest group, within the plane of accounting choices, is in favor of using the historical cost as a basis of value and of the possibility of choice between two competitor accounting models (successful efforts and full cost) and unfavorable to the changes that lead to the increase of the disclosure level of the information. Supporting this position, there is the view of the primary users of financial statements that suggests that the accounting information provided by oil companies is valuable, at least from the perspective of relevance and conservatism, and indicates, yet, that the current group of information based on the historical cost is relevant to their economic decisions. Considering the huge support received by the proposal to expand the required disclosures, therein included the proposal of Publish What You Pay, there is rejection by the participants of the capital market indicating that the benefit resulting from the expansion of the disclosure shall not be greater than those already in existence, considering the current rules. In contrast, there is the view that the accounting information produced by oil companies is untimely. In this, particularly, alterations in this finding are not envisaged once the proposal of the new accounting model of recognition presented at DPEA approaches the full cost method, whereas evidence of the second study points to the opposite direction by suggesting that the successful efforts method generates more appropriate information than does the current method. Therefore, there is scenario to believe in the maintenance of the present status quo.

In eukaryotes, nuclear DNA is folded with histone proteins in the form of chromatin, and this structure plays a critical role in multiple biological processes, including development, DNA damage repair, and aging. Post-translational modifications of histones, such as acetylation and methylation, are essential regulators of chromatin structure and function. Consequently, misregulation of these post-translational modifications has causal roles in numerous diseases, including multiple types of cancer. However, the mechanisms that direct the localization of histone-modifying enzymes and regulate their activities are not fully understood. This thesis focuses on the characterization of a class of proteins containing the PWWP domain. This domain is often present in chromatin proteins, and it is predicted to recognize methylated histones based on structural analysis. Here, we have demonstrated that the PWWP domain proteins in fission yeast bind to methylated histones. Additionally, we have shown that proteins with this domain form complexes with diverse histone modifying activities to regulate multiple cellular processes. Methylation of histone H4 lysine 20 (H4K20me) is essential for the activation of a DNA damage checkpoint, which blocks the progression of cell cycle to allow sufficient time for DNA damage repair. In fission yeast, only the enzyme Set9 catalyzes H4K20me, and the mechanisms that underlie the regulation of this protein are poorly characterized. Here we showed that Set9 forms a stable complex with the PWWP domain containing protein Pdp1. The PWWP domain of Pdp1 binds to H4K20me, demonstrating that the PWWP domain constitutes a novel methyl-lysine recognition motif. Moreover, the binding of PWWP domain to methylated H4K20 plays a critical role in regulating Set9 activity, thus facilitating higher degrees of H4K20 methylation. Histone H3K9 methylation is critical for heterochromatin assembly in diverse organisms. The RNAi pathway is required for the formation of pericentric heterochromatin, although the exact role that RNAi plays in heterochromatin assembly remains a topic of significant debate. We discovered that a separate PWWP domain protein, Pdp3, forms a stable complex with the H3K14 histone acetyltransferase Mst2. Interestingly eliminating the enzymatic activity of the Pdp3-Mst2 complex obviates the requirement for the RNAi machinery in pericentric heterochromatin functions. Furthermore we demonstrated that one function of RNAi during heterochromatin assembly is to exclude the Pdp3-Mst2 complex, thus maintaining low levels of RNA polymerase II localization to pericentric regions in order to retain the parental histone modification patterns for its passage through generations. Altogether, my results have firmly demonstrated that the PWWP domain is a novel class of methyl-lysine binding motifs. Moreover, in fission yeast the PWWP domain proteins form stable complexes with other chromatin proteins to regulate diverse cellular processes.

碩士
國立清華大學
生物資訊與結構生物研究所
101
Hepatoma-derived growth factor (hHDGF) stimulates cell proliferation on both sides of plasma membrane by either binding to membrane receptor as a growth factor or binding to DNA in nucleus as a transcriptional factor. Secreted hHDGF recognizes cell surface heparan sulfate to promote its internalization and the N-terminal PWWP/HATH domain have been proved to be responsible for the heparan sulfate binding. The PWWP/HATH domains are highly conserved among the HDGF-related proteins (HRPs) with high identity > 70% and all domains contain the characteristic structural motif, PWWP motif. The PWWP motifs in HRPs can be classified into two types due to the difference of the first residue. One is with sequence of PHWP (Pro-His-Trp-Pro) and the other is AHWP. In order to realize the significance of the first residue of PWWP motif in mediating protein function and structure, we chose HDGF as models and examined the protein stability and heparin binding by replacing the Pro residue to Ala (mutation P24A). As shown by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), the binding affinity of heparin (KD ≈ 1×10-6 M) was less related to the residue. However, the substitution significantly reduced PWWP/HATH domain stability as reflected in NMR H/D exchange experiment and circular dichroism (CD) melting temperature measurement. In addition, we also detected NMR dynamic parameters on the local region to illustrate the dynamic differences derived from the mutation. Nevertheless, the phenomenon well corresponds to the severe aggregation tendency previously observed in AHWP-type HRPs. In summary, using site-direct mutagenesis and solution NMR method, we identified that the first residue of PWWP motif plays the key role in mediating PWWP domain stability.

博士
國立成功大學
生物科技與產業科學系
106
Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor for gene regulation Li-Ying Chen Chun-Jung Chen Department of Biotechnology and Bioindustry Sciences College of Bioscience and Biotechnology SUMMARY Human Hepatoma-derived growth factor (human HDGF) is highly expressed in the tumour cell lines and is related to various cancers. The expression yield of HDGF could be increased by rare codon mutation for E. coli, whereas the stability of HDGF and the PWWP domain can be optimized by the addition of ligand, protease inhibitor or chelator agent. The apo PWWP domain contains four β-strands, two α-helices, a flexible loop2, and the conserved PWWP motif locates on the loop1. The HDGF PWWP domain undergoes domain swapping to transform its overall conformation from monomeric globular folding into an extended dimeric structure upon 10-bp SMYD1 (SET-MYND domain) binding dramatically. The flexible loop2 functions as a hinge loop with the partially built structure in the apo PWWP domain, refolds into a visible and rigid α-helix in the DNA complex notably. The swapped PWWP domain interacts with the minor groove of 10-bp SMYD1 via residues Lys19, Gly22, Arg79 and Lys80 with variable characters on loops 1 and 4 at the swapped C/N terminus, and the structure becomes more stable and rigid than the apo form. Together with physiological assays, these novel structural findings may provide new insights into the mechanism of DNA binding and the functional process of HDGF. Key words: HDGF, PWWP domain, SMYD1, domain swapping INTRODUCTION Human HDGF belongs to the HDGF-related protein (HRP) family and is highly expressed in the tumour cell lines, developing heart and the normal tissue ubiquitously with angiogenic and mitogenic activities (Yang and Everett, 2009). HDGF participates in different cellular processes, such as cardiovascular differentiation (Everett, 2002), the formation of vascular lesion (Everett et al., 2000) and astrocyte proliferation (Crossin et al., 1997). The over- expressed HDGF is related to several kinds of cancers such as hepatocellular carcinoma (Chen et al., 2015), non-small cell lung cancer (Iwasaki et al., 2005), etc. Human HDGF comprises the chromatin-associated N-terminal PWWP domain which is capable of binding the nonspecific DNA, specific SMYD1 promoter and histone (Yang and Everett, 2007). Some of the proteins in eukaryotes contain the highly conserved PWWP domains from unicellular organisms to human species and most of them belong to chromatin-associated proteins (Rondelet et al., 2016). The variable C-terminus of HDGF which participates in various cellular processes is in charge of translocation and gene regulation. (Kishima et al., 2002; Wang et al., 2011). To date, the conserved PWWP domain and complexes with histone-related peptides structures have been solved (Qiu et al., 2002; Sue et al., 2004; Vezzoli et al., 2010; Wu et al., 2011). However, the interaction between DNA and HDGF remains unclear because of a lack of comparatively of essential knowledge about its exact structure in protein-DNA complex. In the thesis, we report the first crystal structures of the human HDGF PWWP domain with a 10-bp SMYD1 in complex and its unbound apo form. Our studies provide new insights into the PWWP-DNA interaction which could facilitate study about the role of the PWWP domain in nucleosomal context. MATERIALS AND METHODS The DNA sequence of HDGF has been mutated on the rare codon from the library of human fetal brain cDNA (Stratagene, La Jolla, CA) (Hu et al., 2003). The constructs of HDGF and the PWWP domain have constructed into artificial vectors between NdeI and EcoRI restriction sites for further production of the recombinant N-terminal His-tagged fusion protein. The HDGF and the PWWP domain constructs were transformed and over-expressed in Escherichia coli (E. coli) BL21 (DE3) and BL21-Codon Plus ® -RIL, respectively. Overexpression of HDGF and the PWWP domain were induced with 0.5 mM and 1 mM IPTG (isopropyl β-D-thio-galactopyranoside) for overnight at 37 °C, respectively. The His- tag fusion proteins were purified from the supernatant after sonication based on Ni 2+ -NTA agarose column (GE healthcare). The endogenous DNA from E. coli was then removed from the purified proteins were then removed by the anion-exchange chromatography (Hitrap Q) owing to the distinct pI values between DNA and proteins. The protein with various lengths designed SMYD1 complexes were further collected by size-exclusion chromatography (Superdex-200). Crystallization trials were performed using several crystal-screening kits with 96-well plates (JET Biofil) based on the hanging-drop vapor-diffusion method. The apo PWWP domain crystals appeared under the condition containing sodium chloride (0.2 M) and polyethylene glycol (PEG) 4000 (25%, w/v) and Tris (0.1 M, pH 8.5). The crystals of PWWP-SMYD1 complex were obtained in a condition containing sodium phosphate dibasic (0.09 M), sodium nitrate (0.09 M), PEG 1000 (12.5%), PEG 4000 (12.5%, w/v) and Tris; Bicine (0.1 M, pH 8.5). The initial phase of the apo PWWP domain has been solved by the HDGF2 PWWP domain (PDB entry: 3QBY) with one PWWP domain in asymmetric unit, and the structure was refined to 3.3 Å resolution, whereas 10-bp SMYD1 in asymmetric unit was determined at 2.84 Å . All structures were determined by the molecular replacement method with the program, Molrep (Vagin and Teplyakov, 1997). RESULTS AND DISCUSSION We have improved the expression yield of human HDGF by rare codon mutation for structural studies, confirmed that the binding ability of both NCL and DNA for HDGF is through its N-terminal PWWP domain and the addition of SMYD1 interferes the binding capability of NCL for HDGF, and determined the first crystal structures of human HDGF PWWP domain with 10-bp SMYD1 complex and its apo form, respectively. The apo PWWP domain reveals a monomeric structure with four β-strands, two α-helices and a flexible loop2 with diminished electron density. However, the PWWP domain undertakes domain swapping to alter markedly its secondary structures and transform the overall conformation through a globular monomer into an extended dimer with newly formed αC upon DNA binding. The flexible loop2 in the apo PWWP domain is replaced by newly formed αC upon DNA binding in the PWWP-SMYD1 complex and functions as hinge loop which participates in domain swapping. (Fig. 1). The PWWP domain interacts with the minor groove of DNA through the residues with variable characters, Lys19, Gly22, Arg79 and Lys80, at DNA binding loops: 1 and 4 from two chains at C/N terminus of the swapped dimer. Together with physiological assays, these novel structural findings may provide new insights into the mechanism of DNA binding and the functional process of HDGF. The part of potential DNA binding residues in the structures monitored from NMR chemical shift perturbation (PDB entries: 2B8A, 2M16, 2GFU) are consistent with the DNA-binding area in the HDGF PWWP-SMYD1 complex, suggesting that these PWWP domains may react to DNA through loops: 1 and 4. Figure 1. The density at hinge loop region in the apo PWWP domain and PWWP- SMYD1 complex and schematic representation of the dimeric swapped PWWP. (A) The incomplete density causes the difficulty of structure trace from Ala36 to Lys44 (blue mesh, 2F o -F c at 1.5 σ). (B) The well determined and defined αC structure from Asp31 to Lys44 could be covered within the continuous density (blue mesh, 2F o -F c at 1.5 σ). (C) The involved swapped region is shown in green and the other regions are in red in one monomer (left panel). The other molecule of a dimer is shown in gray (right panel). The DNA-binding loops are shown in blue, whereas the residues of the PWWP motif are indicated in stick. CONCLUSION In this study, we have increased the expression yield of HDGF for structural studies successfully, confirmed that the binding capability of both DNA and NCL for HDGF through its N-terminal PWWP domain and addition of SMYD1 interferes the binding capability of NCL for HDGF, and determined the first crystal structures of human HDGF PWWP domain with 10-bp SMYD1 complex and its apo form at 2.84 Å and 3.3 Å resolution, respectively. The PWWP domain undertakes domain swapping to alter markedly its overall conformation through a globular monomer into an extended dimer upon DNA binding. The PWWP-DNA complex is more stable and rigid compared to the apo form. The PWWP domain interacts with the minor groove of DNA through the residues with variable characters at DNA binding loops: 1 and 4 from two chains at C/N terminus of the swapped dimer. Together with physiological assays, these novel structural findings may provide new insights into the mechanism of DNA binding and the functional process of HDGF.

碩士
國立清華大學
生物資訊與結構生物研究所
99
中文摘要 肝癌衍生生長因子(HDGF)從肝癌細胞株的培養液發現後，目前已經在很多組織中被發現，細胞外提供的HDGF能藉由與細胞膜上的受器或硫酸乙醯肝素(heparan sulfate)結合後進入細胞質，且可被運送到細胞核內促進細胞分裂及增生，具有生長因子的功能；也能進入細胞核與DNA結合，具有轉錄因子的功能。分泌型hHDGF的N端PWWP/HATH domain含有100個胺基酸，過去研究已經證實會和肝素 (heparin)結合。HDGF相關蛋白(HRPs)在N端皆具有高度保留的PWWP/HATH domain，且序列相似性高於70%，我們利用核磁共振(NMR)化學位移擾動(chemical-shift perturbation)的方法，找出肝素可能結合的14個正電胺基酸位置，利用點突變的方法將正電胺基酸(Lys/Arg)突變成不帶電的丙胺酸(Alanine)，此14個點突變Ala分子結合表面電漿共振(SPR)技術發現K19、K21、K61、K70、K72、R79應為影響肝素結合的胺基酸位置。為了了解HRPs家族與肝素的結合能力，分別使用恆溫滴定微卡計(ITC)及表面電漿共振(SPR)，竟發現肝素在序列相似性這麼高的HRPs PWWP/HATH domain中有不同的結合能力，HRP-4是結合能力最強的蛋白質，而LEDGF是結合能力最差的蛋白質，兩者的結合能力(KD)相差高於20倍。在此正電胺基酸具有高度保留的PWWP/HATH domain卻有如此大的不同，推測在序列上局部的變異可能會影響結構穩定或調節肝素多醣鏈的分子排列。

In this thesis, we study the discrete logarithm problem in the context of TFNP - the complexity class of search problems with a syntactically guaranteed existence of a solution for all instances. Our main results show that suitable variants of the discrete logarithm problem, which we call Index and DLog, are complete for the classes PPP and PWPP, respectively. Additionally, our reductions provide new structural insights into PWPP by establishing two new PWPP-complete problems. First, the problem Dove, a relaxation of the PPP-complete problem Pigeon. Dove is the first PWPP-complete problem not defined in terms of an explicitly shrinking function. Second, the problem Claw, a total search problem capturing the computational complexity of breaking claw-free permuta- tions. In the context of TFNP, the PWPP-completeness of Claw matches the known intrinsic relationship between collision-resistant hash functions and claw-free permuta- tions established in the cryptographic literature. 1

We improved two beamforming-based methodologies for seismological analysis. The first one is a new Three-Dimensional Phase-Weighted Relative Back Projection (3-D PWBP) method to improve the spatial resolution of Back Projection results. We exploit both phase and amplitude of the seismogram signal to enhance the distinction of correlated signals. Also, we implement a 3-D velocity model to provide more accurate travel times. We vindicate these refinements with several synthetic tests and an analysis of the 1997 Mw 7.2 Zirkuh (Iran) earthquake, which we show ruptured mainly unilaterally southwards at a rupture speed of ∼3.0 km/s along its ∼125 km- long, mostly single-stranded surface rupture. Then, we apply the new method to the more complex case of the 2016 Mw 7.8 Kaikoura (New Zealand) earthquake, which we demonstrate is divided into two major stages separated by a gap of ∼8 s and ∼30–40 km. The overall rupture speed is ∼1.7 km/s and the overall duration is ∼84 s, considerably shorter than some earlier estimates. We see no clear evidence for continuous failure of the subduction interface that underlies the known, surface-rupturing crustal faults, though we cannot rule out its involvement in the second major stage in the northern part of the rupture area. The late (∼80 s) peak in relative energy is likely a high-frequency stopping phase, and the rupture appears to terminate southwest of the offshore Needles fault. The second methodology is a novel workflow for earthquake detection and location, named Seismicity-Scanning based on Navigated Automatic Phase-picking (S-SNAP). By taking a cocktail approach that combines Source-Scanning, Kurtosis-based Phase-picking and the Maximum Intersection location technique into a single integrated workflow, this new method is capable of delineating complex spatiotemporal distributions of seismicity. It is automatic, efficiently providing earthquake locations with high comprehensiveness and accuracy. We apply S-SNAP to a dataset recorded by a dense local seismic array during a hydraulic fracturing operation to test this novel approach and to demonstrate the effectiveness of S-SNAP in comparison to existing methods. Overall, S-SNAP found nearly four times as many high-quality events as a template-matching based catalogue. All events in the previous catalogue are identi- fied with similar epicenter, depth and magnitude, while no false detections are found by visual inspection.
Graduate

Tese de mestrado em Bioestatística, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012
O enfarte agudo do miocárdio constitui atualmente uma das principais causas de morte em todo o mundo. Este trabalho tem como objetivo a modelação do tempo até à ocorrência de múltiplos enfartes como complicações de uma síndrome coronária aguda e a determinação dos seus fatores de risco de entre as variáveis registadas na admissão hospitalar. Podendo existir várias ocorrências do mesmo acontecimento para o mesmo indivíduo, a utilização do modelo de Cox para tempos independentes não é adequada. De entre os diversos modelos de sobrevivência para acontecimentos múltiplos, optou-se por considerar o modelo de regressão PWP desenvolvido por Prentice, Williams e Peterson (1981) para a avaliação da influência dos diversos fatores no tempo de vida dos indivíduos. Este modelo revelou-se o mais adequado nesta situação de acontecimentos ordenados com risco condicional, uma vez que o risco de sofrer cada novo enfarte é diferente do risco associado ao enfarte anterior, sendo assumido que o doente apenas está em risco de sofrer o enfarte de ordem k quando tiver sofrido o enfarte de ordem k-1. O modelo será aplicado a dados reais respeitantes a um estudo envolvendo doentes hospitalizados com síndrome coronária aguda na Unidade de Cuidados Intensivos de Cardiologia dos Hospitais da Universidade de Coimbra.
Nowadays, acute myocardial infarction (AMI) is a major cause of death in developed countries. The aim of this work is to study the time to recurrent infarctions as complications of an acute coronary syndrome and to determine their risk factors among information collected at patient's admission in the hospital. Since, in this case, the event of interest may occur several times for the same individual, the use of Cox proportional hazards model is not appropriate. Among the various multiple event time models, it was considered the PWP regression model proposed by Prentice, Williams and Peterson (1981) to evaluate the influence of different covariates in survival. This model proved to be the most suitable for ordered multiple events with conditional risk, whereas the risk of each new AMI is different from the risk of the previous one, being assumed that a patient can only be at risk for the kth event after experiencing the (k-1)th event. The model will be applied to real data from a study involving hospitalized patients with acute coronary syndrome in the Cardiology Intensive Care Unit of the University Hospital of Coimbra.