Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Mixed Mode Fracture Behaviour of Piezoelectric Materials

Piezoelectrics present an interactive electromechanical behaviour that, especially in recent years, has generated much interest since it renders these materials adapt for use in a variety of electronic and industrial applications like sensors, actuators, transducers, smart structures. Both mechanical and electric loads are generally applied on these devices and can cause high concentrations of stress, particularly in proximity of defects or inhomogeneities, such as flaws, cavities or included particles. A thorough understanding of their fracture behaviour is crucial in order to improve their performances and avoid unexpected failures. Therefore, a considerable number of research works have addressed this topic in the last decades. Most of the theoretical studies on this subject find their analytical background in the complex variable formulation of plane anisotropic elasticity. This theoretical approach bases its main origins in the pioneering works of Muskelishvili and Lekhnitskii who obtained the solution of the elastic problem in terms of independent analytic functions of complex variables. In the present work, the expressions of stresses and elastic and electric displacements are obtained as functions of complex potentials through an analytical formulation which is the application to the piezoelectric static case of an approach introduced for orthotropic materials to solve elastodynamics problems. This method can be considered an alternative to other formalisms currently used, like the Stroh’s formalism. The equilibrium equations are reduced to a first order system involving a six-dimensional vector field. After that, a similarity transformation is induced to reach three independent Cauchy-Riemann systems, so justifying the introduction of the complex variable notation. Closed form expressions of near tip stress and displacement fields are therefore obtained. In the theoretical study of cracked piezoelectric bodies, the issue of assigning consistent electric boundary conditions on the crack faces is of central importance and has been addressed by many researchers. Three different boundary conditions are commonly accepted in literature: the permeable, the impermeable and the semipermeable (“exact”) crack model. This thesis takes into considerations all the three models, comparing the results obtained and analysing the effects of the boundary condition choice on the solution. The influence of load biaxiality and of the application of a remote electric field has been studied, pointing out that both can affect to a various extent the stress fields and the angle of initial crack extension, especially when non-singular terms are retained in the expressions of the electro-elastic solution. Furthermore, two different fracture criteria are applied to the piezoelectric case, and their outcomes are compared and discussed. The work is organized as follows: Chapter 1 briefly introduces the fundamental concepts of Fracture Mechanics. Chapter 2 describes plane elasticity formalisms for an anisotropic continuum (Eshelby-Read-Shockley and Stroh) and introduces for the simplified orthotropic case the alternative formalism we want to propose. Chapter 3 outlines the Linear Theory of Piezoelectricity, its basic relations and electro-elastic equations. Chapter 4 introduces the proposed method for obtaining the expressions of stresses and elastic and electric displacements, given as functions of complex potentials. The solution is obtained in close form and non-singular terms are retained as well. Chapter 5 presents several numerical applications aimed at estimating the effect of load biaxiality, electric field, considered permittivity of the crack. Through the application of fracture criteria the influence of the above listed conditions on the response of the system and in particular on the direction of crack branching is thoroughly discussed.

The massless two-loop two-point function and zeta functions in counterterms of Feynman diagrams

The main part of this thesis describes a method of calculating the massless two-loop two-point function which allows expanding the integral up to an arbitrary order in the dimensional regularization parameter epsilon by rewriting it as a double Mellin-Barnes integral. Closing the contour and collecting the residues then transforms this integral into a form that enables us to utilize S. Weinzierl's computer library nestedsums. We could show that multiple zeta values and rational numbers are sufficient for expanding the massless two-loop two-point function to all orders in epsilon. We then use the Hopf algebra of Feynman diagrams and its antipode, to investigate the appearance of Riemann's zeta function in counterterms of Feynman diagrams in massless Yukawa theory and massless QED. The class of Feynman diagrams we consider consists of graphs built from primitive one-loop diagrams and the non-planar vertex correction, where the vertex corrections only depend on one external momentum. We showed the absence of powers of pi in the counterterms of the non-planar vertex correction and diagrams built by shuffling it with the one-loop vertex correction. We also found the invariance of some coefficients of zeta functions under a change of momentum flow through these vertex corrections.

Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice

Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells.

Decomposing Blaschke Products And Polynomials

The goal of this paper is to contribute to the understanding of complex polynomials and Blaschke products, two very important function classes in mathematics. For a polynomial, $f,$ of degree $n,$ we study when it is possible to write $f$ as a composition $f=g\circ h$, where $g$ and $h$ are polynomials, each of degree less than $n.$ A polynomial is defined to be \emph{decomposable }if such an $h$ and $g$ exist, and a polynomial is said to be \emph{indecomposable} if no such $h$ and $g$ exist. We apply the results of Rickards in \cite{key-2}. We show that $$C_{n}=\{(z_{1},z_{2},...,z_{n})\in\mathbb{C}^{n}\,|\,(z-z_{1})(z-z_{2})...(z-z_{n})\,\mbox{is decomposable}\},$$ has measure $0$ when considered a subset of $\mathbb{R}^{2n}.$ Using this we prove the stronger result that $$D_{n}=\{(z_{1},z_{2},...,z_{n})\in\mathbb{C}^{n}\,|\,\mbox{There exists\,}a\in\mathbb{C}\,\,\mbox{with}\,\,(z-z_{1})(z-z_{2})...(z-z_{n})(z-a)\,\mbox{decomposable}\},$$ also has measure zero when considered a subset of $\mathbb{R}^{2n}.$ We show that for any polynomial $p$, there exists an $a\in\mathbb{C}$ such that $p(z)(z-a)$ is indecomposable, and we also examine the case of $D_{5}$ in detail. The main work of this paper studies finite Blaschke products, analytic functions on $\overline{\mathbb{D}}$ that map $\partial\mathbb{D}$ to $\partial\mathbb{D}.$ In analogy with polynomials, we discuss when a degree $n$ Blaschke product, $B,$ can be written as a composition $C\circ D$, where $C$ and $D$ are finite Blaschke products, each of degree less than $n.$ Decomposable and indecomposable are defined analogously. Our main results are divided into two sections. First, we equate a condition on the zeros of the Blaschke product with the existence of a decomposition where the right-hand factor, $D,$ has degree $2.$ We also equate decomposability of a Blaschke product, $B,$ with the existence of a Poncelet curve, whose foci are a subset of the zeros of $B,$ such that the Poncelet curve satisfies certain tangency conditions. This result is hard to apply in general, but has a very nice geometric interpretation when we desire a composition where the right-hand factor is degree 2 or 3. Our second section of finite Blaschke product results builds off of the work of Cowen in \cite{key-3}. For a finite Blaschke product $B,$ Cowen defines the so-called monodromy group, $G_{B},$ of the finite Blaschke product. He then equates the decomposability of a finite Blaschke product, $B,$ with the existence of a nontrivial partition, $\mathcal{P},$ of the branches of $B^{-1}(z),$ such that $G_{B}$ respects $\mathcal{P}$. We present an in-depth analysis of how to calculate $G_{B}$, extending Cowen's description. These methods allow us to equate the existence of a decomposition where the left-hand factor has degree 2, with a simple condition on the critical points of the Blaschke product. In addition we are able to put a condition of the structure of $G_{B}$ for any decomposable Blaschke product satisfying certain normalization conditions. The final section of this paper discusses how one can put the results of the paper into practice to determine, if a particular Blaschke product is decomposable. We compare three major algorithms. The first is a brute force technique where one searches through the zero set of $B$ for subsets which could be the zero set of $D$, exhaustively searching for a successful decomposition $B(z)=C(D(z)).$ The second algorithm involves simply examining the cardinality of the image, under $B,$ of the set of critical points of $B.$ For a degree $n$ Blaschke product, $B,$ if this cardinality is greater than $\frac{n}{2}$, the Blaschke product is indecomposable. The final algorithm attempts to apply the geometric interpretation of decomposability given by our theorem concerning the existence of a particular Poncelet curve. The final two algorithms can be implemented easily with the use of an HTML

Data Quality Assessment of Ungated Flow Cytometry Data in High

Background: The recent development of semi-automated techniques for staining and analyzing flow cytometry samples has presented new challenges. Quality control and quality assessment are critical when developing new high throughput technologies and their associated information services. Our experience suggests that significant bottlenecks remain in the development of high throughput flow cytometry methods for data analysis and display. Especially, data quality control and quality assessment are crucial steps in processing and analyzing high throughput flow cytometry data. Methods: We propose a variety of graphical exploratory data analytic tools for exploring ungated flow cytometry data. We have implemented a number of specialized functions and methods in the Bioconductor package rflowcyt. We demonstrate the use of these approaches by investigating two independent sets of high throughput flow cytometry data. Results: We found that graphical representations can reveal substantial non-biological differences in samples. Empirical Cumulative Distribution Function and summary scatterplots were especially useful in the rapid identification of problems not identified by manual review. Conclusions: Graphical exploratory data analytic tools are quick and useful means of assessing data quality. We propose that the described visualizations should be used as quality assessment tools and where possible, be used for quality control.

Strengthening of laterality of verbal and visuospatial functions during childhood and adolescence

Cognitive functions in the child's brain develop in the context of complex adaptive processes, determined by genetic and environmental factors. Little is known about the cerebral representation of cognitive functions during development. In particular, knowledge about the development of right hemispheric (RH) functions is scarce. Considering the dynamics of brain development, localization and lateralization of cognitive functions must be expected to change with age. Twenty healthy subjects (8.6-20.5 years) were examined with fMRI and neuropsychological tests. All participants completed two fMRI tasks known to activate left hemispheric (LH) regions (language tasks) and two tasks known to involve predominantly RH areas (visual search tasks). A laterality index (LI) was computed to determine the asymmetry of activation. Group analysis revealed unilateral activation of the LH language circuitry during language tasks while visual search tasks induced a more widespread RH activation pattern in frontal, superior temporal, and occipital areas. Laterality of language increased between the ages of 8-20 in frontal (r = 0.392, P = 0.049) and temporal (r = 0.387, P = 0.051) areas. The asymmetry of visual search functions increased in frontal (r = -0.525, P = 0.009) and parietal (r = -0.439, P = 0.027) regions. A positive correlation was found between Verbal-IQ and the LI during a language task (r = 0.585, P = 0.028), while visuospatial skills correlated with LIs of visual search (r = -0.621, P = 0.018). To summarize, cognitive development is accompanied by changes in the functional representation of neuronal circuitries, with a strengthening of lateralization not only for LH but also for RH functions. Our data show that age and performance, independently, account for the increases of laterality with age.

PARTITIONING THE BLOCKS OF A STEINER TRIPLE SYSTEM INTO PARTIAL PARALLEL CLASSES

Does there exist a Steiner Triple System on v points, whose blocks can be partitioned into partial parallel classes of size m, where m ≤ [v⁄3], m | b and b is the number of blocks of the STS(v)? We give the answer for 9 ≤ v ≤ 43. We also show that whenever 2|b, v ≡ 3 (mod 6) we can find an STS(v) whose blocks can be partitioned into partial parallel classes of size 2, and whenever 4|b , v ≡ 3 (mod 6), there exists an STS(v) whose blocks can be partitioned into partial parallel classes of size 4.

ANOVA kernels and RKHS of zero mean functions for model-based sensitivity analysis

Given a reproducing kernel Hilbert space (H,〈.,.〉)(H,〈.,.〉) of real-valued functions and a suitable measure μμ over the source space D⊂RD⊂R, we decompose HH as the sum of a subspace of centered functions for μμ and its orthogonal in HH. This decomposition leads to a special case of ANOVA kernels, for which the functional ANOVA representation of the best predictor can be elegantly derived, either in an interpolation or regularization framework. The proposed kernels appear to be particularly convenient for analyzing the effect of each (group of) variable(s) and computing sensitivity indices without recursivity.

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

Contribution of Ectodomain Mutations in Epidermal Growth Factor Receptor to Signaling in Glioblastoma Multiforme

CONTRIBUTION OF ECTODOMAIN MUTATIONS IN EPIDERMAL GROWTH FACTOR RECEPTOR TO SIGNALING IN GLIOBLASTOMA MULTIFORME Publication No._________ Marta Rojas, M.S. Supervisory Professor: Oliver Bögler, Ph.D. The Cancer Genome Atlas (TCGA) has conducted a comprehensive analysis of a large tumor cohort and has cataloged genetic alterations involving primary sequence variations and copy number aberrations of genes involved in key signaling pathways in glioblastoma (GBM). This dataset revealed missense ectodomain point mutations in epidermal growth factor receptor (EGFR), but the biological and clinical significance of these mutations is not well defined in the context of gliomas. In our study, we focused on understanding and defining the molecular mechanisms underlying the functions of EGFR ectodomain mutants. Using proteomic approaches to broadly analyze cell signaling, including antibody array and mass spectrometry-based methods, we found a differential spectrum of tyrosine phosphorylation across the EGFR ectodomain mutations that enabled us to stratify them into three main groups that correlate with either wild type EGFR (EGFR) or the long-studied mutant, EGFRvIII. Interestingly, one mutant shared characteristics of both groups suggesting a continuum of behaviors along which different mutants fall. Surprisingly, no substantial differences were seen in activation of classical downstream signaling pathways such as Akt and S6 pathways between these classes of mutants. Importantly, we demonstrated that ectodomain mutations lead to differential tumor growth capabilities in both in vitro (anchorage independent colony formation) and in vivo conditions (xenografts). Our data from the biological characterization allowed us to categorize the mutants into three main groups: the first group typified by EGFRvIII are mutations with a more aggressive phenotype including R108K and A289T; a second group characterized by a less aggressive phenotype exemplified by EGFR and the T263P mutation; and a third group which shared characteristics from both groups and is exemplified by the mutation A289D. In addition, we treated cells overexpressing the mutants with various agents employed in the clinic including temozolomide, cisplatin and tarceva. We found that cells overexpressing the mutants in general displayed resistance to the treatments. Our findings yield insights that help with the molecular characterization of these mutants. In addition, our results from the drug studies might be valuable in explaining differential responses to specific treatments in GBM patients.

Improving executive functions in 5- and 6-year-olds: Evaluation of a small group intervention in prekindergarten and kindergarten children

Research suggests a central role of executive functions for children's cognitive and social development during preschool years, especially in promoting school readiness. Interventions aiming to improve executive functions are therefore being called for. The present study examined the effect of a small group intervention implemented in kindergarten settings focusing on basic components of executive functions, i.e., working memory, interference control and cognitive flexibility. A total of 135 children enrolled in Swiss prekindergarten (5-year-olds) and kindergarten (6-year-olds) were involved. Results revealed that the small group intervention promoted gains in all three included components of executive functions: prekindergarten children substantially improved their working memory and cognitive flexibility processes, whereas significant training effects were found for the kindergarten children in interference control. Implications of these findings for early intervention programs and for tailoring preschool curricula are discussed, particularly with respect to children's school readiness. Copyright © 2011 John Wiley & Sons, Ltd.

On the signature of positive braids and adjacency for torus knots

The objects of study in this thesis are knots. More precisely, positive braid knots, which include algebraic knots and torus knots. In the first part of this thesis, we compare two classical knot invariants - the genus g and the signature σ - for positive braid knots. Our main result on positive braid knots establishes a linear lower bound for the signature in terms of the genus. In the second part of the thesis, a positive braid approach is applied to the study of the local behavior of polynomial functions from the complex affine plane to the complex numbers. After endowing polynomial function germs with a suitable topology, the adjacency problem arises: for a fixed germ f, what classes of germs g can be found arbitrarily close to f? We introduce two purely topological notions of adjacency for knots and discuss connections to algebraic notions of adjacency and the adjacency problem.

The functional significance of EEG microstates-Associations with modalities of thinking

The momentary, global functional state of the brain is reflected by its electric field configuration. Cluster analytical approaches consistently extracted four head-surface brain electric field configurations that optimally explain the variance of their changes across time in spontaneous EEG recordings. These four configurations are referred to as EEG microstate classes A, B, C, and D and have been associated with verbal/phonological, visual, attention reorientation, and subjective interoceptive-autonomic processing, respectively. The present study tested these associations via an intra-individual and inter-individual analysis approach. The intra-individual approach tested the effect of task-induced increased modality-specific processing on EEG microstate parameters. The inter-individual approach tested the effect of personal modality-specific parameters on EEG microstate parameters. We obtained multichannel EEG from 61 healthy, right-handed, male students during four eyes-closed conditions: object-visualization, spatial-visualization, verbalization (6 runs each), and resting (7 runs). After each run, we assessed participants' degrees of object-visual, spatial-visual, and verbal thinking using subjective reports. Before and after the recording, we assessed modality-specific cognitive abilities and styles using nine cognitive tests and two questionnaires. The EEG of all participants, conditions, and runs was clustered into four classes of EEG microstates (A, B, C, and D). RMANOVAs, ANOVAs and post-hoc paired t-tests compared microstate parameters between conditions. TANOVAs compared microstate class topographies between conditions. Differences were localized using eLORETA. Pearson correlations assessed interrelationships between personal modality-specific parameters and EEG microstate parameters during no-task resting. As hypothesized, verbal as opposed to visual conditions consistently affected the duration, occurrence, and coverage of microstate classes A and B. Contrary to associations suggested by previous reports, parameters were increased for class A during visualization, and class B during verbalization. In line with previous reports, microstate D parameters were increased during no-task resting compared to the three internal, goal-directed tasks. Topographic differences between conditions concerned particular sub-regions of components of the metabolic default mode network. Modality-specific personal parameters did not consistently correlate with microstate parameters except verbal cognitive style which correlated negatively with microstate class A duration and positively with class C occurrence. This is the first study that aimed to induce EEG microstate class parameter changes based on their hypothesized functional significance. Beyond, the associations of microstate classes A and B with visual and verbal processing, respectively and microstate class D with interoceptive-autonomic processing, our results suggest that a finely-tuned interplay between all four EEG microstate classes is necessary for the continuous formation of visual and verbal thoughts, as well as interoceptive-autonomic processing. Our results point to the possibility that the EEG microstate classes may represent the head-surface measured activity of intra-cortical sources primarily exhibiting inhibitory functions. However, additional studies are needed to verify and elaborate on this hypothesis.

The effects of qualitatively different acute physical activity interventions in real-world settings on executive functions in preadolescent children

There is growing evidence indicating a positive effect of acute physical activity on cognitive performance in children. Most of the evidence originates, however, from studies in highly controlled laboratory settings. The aim of the present study was to investigate whether the same effects can be found in more real-world settings. We examined the effects of qualitatively different acute physical activity interventions on the three core dimensions of executive functions (updating, inhibition, shifting). In an experimental between-subject design, 219 ten to twelve year-olds were assigned to one of four conditions which varied systematically in physical activation and cognitive engagement. Executive functions were measured before and immediately after the intervention. Contrary to the hypothesis, no effects of acute physical activity with and without cognitive engagement were found on executive functions in the overall sample. Only children with higher fitness and/or higher academic achievement benefitted from the interventions in terms of their updating performance. Thus, the results indicate that it may be more difficult to attain positive effects through acute physical activity in real-world settings than in laboratory settings and that physiological and cognitive requirements may have to be adjusted to individual capacity to make an intervention effective.