Analiza proteomiczna kompleksów tworzonych przez białko hDPY30 w komórkach ludzkich

Wydział Biologii

Spectral Frame Analysis and Learning through Graph Structure

This dissertation investigates the connection between spectral analysis and frame theory. When considering the spectral properties of a frame, we present a few novel results relating to the spectral decomposition. We first show that scalable frames have the property that the inner product of the scaling coefficients and the eigenvectors must equal the inverse eigenvalues. From this, we prove a similar result when an approximate scaling is obtained. We then focus on the optimization problems inherent to the scalable frames by first showing that there is an equivalence between scaling a frame and optimization problems with a non-restrictive objective function. Various objective functions are considered, and an analysis of the solution type is presented. For linear objectives, we can encourage sparse scalings, and with barrier objective functions, we force dense solutions. We further consider frames in high dimensions, and derive various solution techniques. From here, we restrict ourselves to various frame classes, to add more specificity to the results. Using frames generated from distributions allows for the placement of probabilistic bounds on scalability. For discrete distributions (Bernoulli and Rademacher), we bound the probability of encountering an ONB, and for continuous symmetric distributions (Uniform and Gaussian), we show that symmetry is retained in the transformed domain. We also prove several hyperplane-separation results. With the theory developed, we discuss graph applications of the scalability framework. We make a connection with graph conditioning, and show the in-feasibility of the problem in the general case. After a modification, we show that any complete graph can be conditioned. We then present a modification of standard PCA (robust PCA) developed by Cand\`es, and give some background into Electron Energy-Loss Spectroscopy (EELS). We design a novel scheme for the processing of EELS through robust PCA and least-squares regression, and test this scheme on biological samples. Finally, we take the idea of robust PCA and apply the technique of kernel PCA to perform robust manifold learning. We derive the problem and present an algorithm for its solution. There is also discussion of the differences with RPCA that make theoretical guarantees difficult.

LAP1 interactome and its functional features throughout spermatogenesis

The lamina-associated polypeptide 1 (LAP1) is a type II transmembrane protein of the inner nuclear membrane encoded by the human gene TOR1AIP1. LAP1 is involved in maintaining the nuclear envelope structure and appears be involved in the positioning of lamins and chromatin. In the nuclear envelope, LAP1 is suggested to exist as a complex with A-type and B-type lamins, torsins and emerin. The presence of such complexes suggests that LAP1 may cooperate functionally with these proteins in tissues where they play a critical role. Therefore, the identification of LAP1 binding partners and the signalling pathways where LAP1 participates, is crucial for a better understanding of LAP1 functions. The work described in this thesis addresses novel human LAP1 associated proteins found through bioinformatic tools. Public databases allowed for the discovery of the LAP1 interactome, which was manually curated, identifying several functionally relevant proteins. Subsequently, the integration of multiple bioinformatic tools established novel functions to LAP1 such as DNA damage response and telomere association. In conjunction, bioinformatic results also reinforced the association of LAP1 with mitosis, and the already identified role of LAP1 in nuclear morphology. Interestingly, this association of LAP1 with the regulation of the nuclear envelope structure and mitosis progression, shares functional elements with spermatogenesis. Therefore, this work additionally described the localization of LAP1 and some of its interactors throughout the spermatogenic cycle, in mouse and human testis. The results established that the activity of LAP1 during the mouse spermatogenic cycle is most evident from stage VIII until the end of spermiogenesis, which is characteristic of manchette development. Concomitantly, some LAP1 interactors studied in this work share a similar localization, namely, PP1γ2, Lamin B1 and Lamin A/C. The results obtained from the study of LAP1 throughout different periods of the male reproductive system attributed potential new biological functions to LAP1. Thereby, this work can be the foundation of future studies regarding LAP1 and the regulation of multiple cellular processes and disease conditions.

Essays on educational production functions in England

Despite the expansion of the literature on the implications that different inputs have on the educational outcomes of students, empirical research has so far lacked the full capacity to provide unequivocal findings. Essentially, this deficiency is mainly attributed to two main factors; the lack of reliable data and the lack of full dimensionality in the theoretical model adopted to explain such data (Levaččićć and Vignoles, 2002; Knoeppel, Verstegen, and Rinehart, 2007). This dissertation aims to fill those gaps by first building a unique large dataset that covers all aspects of the educational process and second by adopting an integrated theoretical model and advanced quantitative methodological approaches to analyze it. With the fulfillment of such aim the dissertation manages to fill some of the gaps identified in the Education Economics literature related to the relationships between the cognitive and affective educational outcomes of English adolescents on one hand and three main inputs representing each of the three indentified factors in the theoretical model on the other hand controlling for other possible heterogeneities. Specifically, the thesis examines the effect of school process inputs in Chapter 2, family structure as a key family background input in Chapter 3 and finally religion and religiosity as a key adolescent’s personal input in Chapter 4.

The conformational landscape of RNA translational regulators and their potential as drug discovery targets

RNA is an underutilized target for drug discovery. Once thought to be a passive carrier of genetic information, RNA is now known to play a critical role in essentially all aspects of biology including signaling, gene regulation, catalysis, and retroviral infection. It is now well-established that RNA does not exist as a single static structure, but instead populates an ensemble of energetic minima along a free-energy landscape. Knowledge of this structural landscape has become an important goal for understanding its diverse biological functions. In this case, NMR spectroscopy has emerged as an important player in the characterization of RNA structural ensembles, with solution-state techniques accounting for almost half of deposited RNA structures in the PDB, yet the rate of RNA structure publication has been stagnant over the past decade. Several bottlenecks limit the pace of RNA structure determination by NMR: the high cost of isotopic labeling, tedious and ambiguous resonance assignment methods, and a limited database of RNA optimized pulse programs. We have addressed some of these challenges to NMR characterization of RNA structure with applications to various RNA-drug targets. These approaches will increasingly become integral to designing new therapeutics targeting RNA.

On the critical point structure of eigenfunctions belonging to the first nonzero eigenvalue of a genus two closed hyperbolic surface

We develop a method based on spectral graph theory to approximate the eigenvalues and eigenfunctions of the Laplace-Beltrami operator of a compact riemannian manifold -- The method is applied to a closed hyperbolic surface of genus two -- The results obtained agree with the ones obtained by other authors by different methods, and they serve as experimental evidence supporting the conjectured fact that the generic eigenfunctions belonging to the first nonzero eigenvalue of a closed hyperbolic surface of arbitrary genus are Morse functions having the least possible total number of critical points among all Morse functions admitted by such manifolds

Unpolarized transverse momentum dependent parton distribution and fragmentation functions at next-to-next-to-leading order

The transverse momentum dependent parton distribution/fragmentation functions (TMDs) are essential in the factorization of a number of processes like Drell-Yan scattering, vector boson production, semi-inclusive deep inelastic scattering, etc. We provide a comprehensive study of unpolarized TMDs at next-to-next-to-leading order, which includes an explicit calculation of these TMDs and an extraction of their matching coefficients onto their integrated analogues, for all flavor combinations. The obtained matching coefficients are important for any kind of phenomenology involving TMDs. In the present study each individual TMD is calculated without any reference to a specific process. We recover the known results for parton distribution functions and provide new results for the fragmentation functions. The results for the gluon transverse momentum dependent fragmentation functions are presented for the first time at one and two loops. We also discuss the structure of singularities of TMD operators and TMD matrix elements, crossing relations between TMD parton distribution functions and TMD fragmentation functions, and renormalization group equations. In addition, we consider the behavior of the matching coefficients at threshold and make a conjecture on their structure to all orders in perturbation theory.

Environmental variables, habitat discontinuity and life history shaping the genetic structure of Pomatoschistus marmoratus

Coastal lagoons are semi-isolated ecosystems exposed to wide fluctuations of environmental conditions and showing habitat fragmentation. These features may play an important role in separating species into different populations, even at small spatial scales. In this study, we evaluate the concordance between mitochondrial (previous published data) and nuclear data analyzing the genetic variability of Pomatoschistus marmoratus in five localities, inside and outside the Mar Menor coastal lagoon (SE Spain) using eight microsatellites. High genetic diversity and similar levels of allele richness were observed across all loci and localities, although significant genic and genotypic differentiation was found between populations inside and outside the lagoon. In contrast to the FST values obtained from previous mitochondrial DNA analyses (control region), the microsatellite data exhibited significant differentiation among samples inside the Mar Menor and between lagoonal and marine samples. This pattern was corroborated using Cavalli-Sforza genetic distances. The habitat fragmentation inside the coastal lagoon and among lagoon and marine localities could be acting as a barrier to gene flow and contributing to the observed genetic structure. Our results from generalized additive models point a significant link between extreme lagoonal environmental conditions (mainly maximum salinity) and P. marmoratus genetic composition. Thereby, these environmental features could be also acting on genetic structure of coastal lagoon populations of P. marmoratus favoring their genetic divergence. The mating strategy of P. marmoratus could be also influencing our results obtained from mitochondrial and nuclear DNA. Therefore, a special consideration must be done in the selection of the DNA markers depending on the reproductive strategy of the species.

Connectivity patterns inferred from the genetic structure of white seabream (Diplodus sargus L.)

Themarine environment seems, at first sight, to be a homogeneousmediumlacking barriers to species dispersal. Nevertheless, populations of marine species show varying levels of gene flow and population differentiation, so barriers to gene flow can often be detected. Weaimto elucidate the role of oceanographical factors ingenerating connectivity among populations shaping the phylogeographical patterns in the marine realm, which is not only a topic of considerable interest for understanding the evolution ofmarine biodiversity but also formanagement and conservation of marine life. For this proposal,we investigate the genetic structure and connectivity between continental and insular populations ofwhite seabreamin North East Atlantic (NEA) and Mediterranean Sea (MS) aswell as the influence of historical and contemporary factors in this scenario using mitochondrial (cytochrome b) and nuclear (a set of 9 microsatellite) molecular markers. Azores population appeared genetically differentiated in a single cluster using Structure analysis. This result was corroborated by Principal Component Analysis (PCA) and Monmonier algorithm which suggested a boundary to gene flow, isolating this locality. Azorean population also shows the highest significant values of FST and genetic distances for both molecular markers (microsatellites and mtDNA). We suggest that the breakdown of effective genetic exchange between Azores and the others' samples could be explained simultaneously by hydrographic (deep water) and hydrodynamic (isolating current regimes) factors acting as barriers to the free dispersal of white seabream(adults and larvae) and by historical factors which could be favoured for the survival of Azorean white seabream population at the last glaciation. Mediterranean islands show similar genetic diversity to the neighbouring continental samples and nonsignificant genetic differences. Proximity to continental coasts and the current system could promote an optimal larval dispersion among Mediterranean islands (Mallorca and Castellamare) and coasts with high gene flow.

GENETIC VARIATION, LOCAL ADAPTATION AND POPULATION STRUCTURE IN NORTH AMERICAN RED OAK SPECIES, QUERCUS RUBRA L. AND Q. ELLIPSOIDALIS E. J. HILL

Forest trees, like oaks, rely on high levels of genetic variation to adapt to varying environmental conditions. Thus, genetic variation and its distribution are important for the long-term survival and adaptability of oak populations. Climate change is projected to lead to increased drought and fire events as well as a northward migration of tree species, including oaks. Additionally, decline in oak regeneration has become increasingly concerning since it may lead to decreased gene flow and increased inbreeding levels. This will in turn lead to lowered levels of genetic diversity, negatively affecting the growth and survival of populations. At the same time, populations at the species’ distribution edge, like those in this study, could possess important stores of genetic diversity and adaptive potential, while also being vulnerable to climatic or anthropogenic changes. A survey of the level and distribution of genetic variation and identification of potentially adaptive genes is needed since adaptive genetic variation is essential for their long-term survival. Oaks possess a remarkable characteristic in that they maintain their species identity and specific environmental adaptations despite their propensity to hybridize. Thus, in the face of interspecific gene flow, some areas of the genome remain differentiated due to selection. This characteristic allows the study of local environmental adaptation through genetic variation analyses. Furthermore, using genic markers with known putative functions makes it possible to link those differentiated markers to potential adaptive traits (e.g., flowering time, drought stress tolerance). Demographic processes like gene flow and genetic drift also play an important role in how genes (including adaptive genes) are maintained or spread. These processes are influenced by disturbances, both natural and anthropogenic. An examination of how genetic variation is geographically distributed can display how these genetic processes and geographical disturbances influence genetic variation patterns. For example, the spatial clustering of closely related trees could promote inbreeding with associated negative effects (inbreeding depression), if gene flow is limited. In turn this can have negative consequences for a species’ ability to adapt to changing environmental conditions. In contrast, interspecific hybridization may also allow the transfer of genes between species that increase their adaptive potential in a changing environment. I have studied the ecologically divergent, interfertile red oaks, Quercus rubra and Q. ellipsoidalis, to identify genes with potential roles in adaptation to abiotic stress through traits such as drought tolerance and flowering time, and to assess the level and distribution of genetic variation. I found evidence for moderate gene flow between the two species and low interspecific genetic differences at most genetic markers (Lind and Gailing 2013). However, the screening of genic markers with potential roles in phenology and drought tolerance led to the identification of a CONSTANS-like (COL) gene, a candidate gene for flowering time and growth. This marker, located in the coding region of the gene, was highly differentiated between the two species in multiple geographical areas, despite interspecific gene flow, and may play a role in reproductive isolation and adaptive divergence between the two species (Lind-Riehl et al. 2014). Since climate change could result in a northward migration of trees species like oaks, this gene could be important in maintaining species identity despite increased contact zones between species (e.g., increased gene flow). Finally I examined differences in spatial genetic structure (SGS) and genetic variation between species and populations subjected to different management strategies and natural disturbances. Diverse management activities combined with various natural disturbances as well as species specific life history traits influenced SGS patterns and inbreeding levels (Lind-Riehl and Gailing submitted).

Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.

The Herpesvirus Nuclear Egress Complex Component, UL31, is Recruited to Sites of DNA Damage Through Poly(ADP-RIBOSE) Binding

Thesis (Master, Biomedical & Molecular Sciences) -- Queen's University, 2016-08-23 15:03:30.807

“Acid-spike” effect in spurs/tracks of the low/high linear energy transfer radiolysis of water : potential implications for radiobiology and nuclear industry

Résumé : Les ions hydronium (H3O + ) sont formés, à temps courts, dans les grappes ou le long des trajectoires de la radiolyse de l'eau par des rayonnements ionisants à faible transfert d’énergie linéaire (TEL) ou à TEL élevé. Cette formation in situ de H3O + rend la région des grappes/trajectoires du rayonnement temporairement plus acide que le milieu environnant. Bien que des preuves expérimentales de l’acidité d’une grappe aient déjà été signalées, il n'y a que des informations fragmentaires quant à son ampleur et sa dépendance en temps. Dans ce travail, nous déterminons les concentrations en H3O + et les valeurs de pH correspondantes en fonction du temps à partir des rendements de H3O + calculés à l’aide de simulations Monte Carlo de la chimie intervenant dans les trajectoires. Quatre ions incidents de différents TEL ont été sélectionnés et deux modèles de grappe/trajectoire ont été utilisés : 1) un modèle de grappe isolée "sphérique" (faible TEL) et 2) un modèle de trajectoire "cylindrique" (TEL élevé). Dans tous les cas étudiés, un effet de pH acide brusque transitoire, que nous appelons un effet de "pic acide", est observé immédiatement après l’irradiation. Cet effet ne semble pas avoir été exploré dans l'eau ou un milieu cellulaire soumis à un rayonnement ionisant, en particulier à haut TEL. À cet égard, ce travail soulève des questions sur les implications possibles de cet effet en radiobiologie, dont certaines sont évoquées brièvement. Nos calculs ont ensuite été étendus à l’étude de l'influence de la température, de 25 à 350 °C, sur la formation in situ d’ions H3O + et l’effet de pic acide qui intervient à temps courts lors de la radiolyse de l’eau à faible TEL. Les résultats montrent une augmentation marquée de la réponse de pic acide à hautes températures. Comme de nombreux processus intervenant dans le cœur d’un réacteur nucléaire refroidi à l'eau dépendent de façon critique du pH, la question ici est de savoir si ces fortes variations d’acidité, même si elles sont hautement localisées et transitoires, contribuent à la corrosion et l’endommagement des matériaux.

Caractérisation structurale de Miz-1 dans le cadre de la répression génique causée par le complexe c-Myc/Miz-1

Résumé : c-Myc est un facteur de transcription (FT) dont les niveaux cellulaires sont dérégulés dans la majorité des cancers chez l’homme. En hétérodimère avec son partenaire obligatoire Max, c-Myc lie préférentiellement les séquences E-Box (CACGTG) et cause l’expression de gènes impliqués dans la biosynthèse des protéines et des ARNs, dans le métabolisme et dans la prolifération cellulaire. Il est maintenant bien connu que c-Myc exerce aussi son potentiel mitogène en liant et inhibant différents FTs impliqués dans l’expression de gènes cytostatiques. Entre autres, c-Myc est en mesure d’inhiber Miz-1, un FT comportant 13 doigts de zinc de type Cys2-His2 (ZFs) impliqué dans l’expression de plusieurs gènes régulateurs du cycle cellulaire comprenant les inhibiteurs de CDK p15[indice supérieur INK4], p21[indice supérieur CIP1] et p57[indice supérieur KIP2]. Plus récemment, il fut démontré qu’en contrepartie, Miz-1 est aussi en mesure de renverser les fonctions activatrices de c-Myc et de prévenir la prolifération de cellules cancéreuses dépendantes de c-Myc. Ces différentes observations ont mené à la suggestion de l’hypothèse intéressante que la balance des niveaux de Miz-1 et c-Myc pourrait dicter le destin de la cellule et a permis d’établir Miz-1 comme nouvelle cible potentielle pour le développement d’agents anti-cancéreux. Malgré le fait que ces deux protéines semblent centrales à la régulation du cycle cellulaire, les mécanismes moléculaires leur permettant de s’inhiber mutuellement ainsi que les déterminants moléculaires permettant leur association spécifique demeurent assez peu documentés pour le moment. De plus, la biologie structurale de Miz-1 demeure à être explorée puisque qu’aucune structure de ses 13 ZFs, essentiels à sa liaison à l’ADN, n’a été déterminée pour l’instant. Les travaux réalisés dans le cadre cette thèse visent la caractérisation structurale et biophysique de Miz-1 dans le contexte de la répression génique causée par le complexe c-Myc/Miz-1. Nous présentons des résultats d’éxpériences in vitro démontrant que Miz-1 interagit avec c-Myc via un domaine contenu entre ses ZFs 12 et 13. De plus, nous démontrons que Miz-1 et Max sont en compétition pour la liaison de c-Myc. Ces résultats suggèrent pour la permière fois que Miz-1 inhibe les activités de c-Myc en prévenant son interaction avec son partenaire obligatoire Max. De plus, ils laissent présager que que Miz-1 pourrait servir de référence pour le développement d’inhibiteurs peptidiques de c-Myc. Finalement, nous avons réalisé la caractérisation structurale et dynamique des ZFs 1 à 4 et 8 à 10 de Miz-1 et avons évalué leur potentiel de liaison à l’ADN. Les résultats obtenus, couplés à des analyses bio-informatiques, nous permettent de suggérer un modèle détaillé pour la liaison spécifique de Miz-1 à son ADN consensus récemment identifié.