The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

Stress and strain-driven algorithmic formulations for finite strain viscoplasticity for hybrid and standard finite elements

This work deals with the formulation and implementation of finite deformation viscoplasticity within the framework of stress-based hybrid finite element methods. Hybrid elements, which are based on a two-field variational formulation, are much less susceptible to locking than conventional displacement-based elements. The conventional return-mapping scheme cannot be used in the context of hybrid stress methods since the stress is known, and the strain and the internal plastic variables have to be recovered using this known stress field.We discuss the formulation and implementation of the consistent tangent tensor, and the return-mapping algorithm within the context of the hybrid method. We demonstrate the efficacy of the algorithm on a wide range of problems.

Rectangular universal cellular array

A rectangular universal cellular array consisting of cells having three inputs and one output is described. This array is based on the Reed-Muller canonical expansion of a switching function. Although the total number of external input pins required in this array is the same as that of a rectangular array proposed in the literature, the number of cells is very much less.

Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.

Populational reasoning

This section focuses on systems of reasoning that imagine youth as a unified whole, one that can be researched, talked about, planned for, and managed. Even research that focuses on individuals or specific contexts depends on and reproduces ideas of youth as an identifiable population. This section interrogates the rules and scaffolding of discourses that construct the social spaces in which we problematize and study youth in society. This introduction will set the agenda by addressing four elements of this process: the first addresses the rise of some of the crucial elements of contemporary governance, the instrument and practices through which the notion of the population was able to take shape. The second examines the rise of the personage of “the child,” and how new forms of governance not only utilized this new identity for the purposes of ongoing social management, but also organized its differentiation into a growing array of new social and administrative categories. The third specifically addresses “youth,” examining its various predecessors as targets for moral concern, as well as some of the recent cultural triggers for its formation. Finally, there is an assessment of the contemporary governance of populations of youth, based as it is around its twin existence as a governmental object, a target for an almost endless array of social, educational, legal, and psychological concerns and interventions, but also as an identity, a set of practices of the self.

Connecting people and ideas from around the world: global innovation platforms for next-generation ecology and beyond

We present a case for using Global Community Innovation Platforms (GCIPs), an approach to improve innovation and knowledge exchange in international scientific communities through a common and open online infrastructure. We highlight the value of GCIPs by focusing on recent efforts targeting the ecological sciences, where GCIPs are of high relevance given the urgent need for interdisciplinary, geographical, and cross-sector collaboration to cope with growing challenges to the environment as well as the scientific community itself. Amidst the emergence of new international institutions, organizations, and meetings, GCIPs provide a stable international infrastructure for rapid and long-term coordination that can be accessed by any individual. This accessibility can be especially important for researchers early in their careers. Recent examples of early-career GCIPs complement an array of existing options for early-career scientists to improve skill sets, increase academic and social impact, and broaden career opportunities. We provide a number of examples of existing early-career initiatives that incorporate elements from the GCIPs approach, and highlight an in-depth case study from the ecological sciences: the International Network of Next-Generation Ecologists (INNGE), initiated in 2010 with support from the International Association for Ecology and 20 member institutions from six continents.

Exploring elements for innovation in the Australian water sector

This thesis explores innovation as a means to achieving an enhanced level of sustainability in the Australian water sector. A modified Delphi study uncovered sixteen key elements centred around themes of 'community acceptance' and 'innovator effectiveness', that provide insights for immediate application within the sector to address impacts of climate change, population increases and resource scarcity. This exploratory research builds a foundational understanding of the components for change and innovation within the Australian water sector, and forms the underpinning for more specific lines of enquiry.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

A new type of high-order elements based on the mesh-free interpolations

We propose a new type of high-order elements that incorporates the mesh-free Galerkin formulations into the framework of finite element method. Traditional polynomial interpolation is replaced by mesh-free interpolations in the present high-order elements, and the strain smoothing technique is used for integration of the governing equations based on smoothing cells. The properties of high-order elements, which are influenced by the basis function of mesh-free interpolations and boundary nodes, are discussed through numerical examples. It can be found that the basis function has significant influence on the computational accuracy and upper-lower bounds of energy norm, when the strain smoothing technique retains the softening phenomenon. This new type of high-order elements shows good performance when quadratic basis functions are used in the mesh-free interpolations and present elements prove advantageous in adaptive mesh and nodes refinement schemes. Furthermore, it shows less sensitive to the quality of element because it uses the mesh-free interpolations and obeys the Weakened Weak (W2) formulation as introduced in [3, 5].

Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOXI promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

Elements of Volatility at High Frequency

Volatility is central in options pricing and risk management. It reflects the uncertainty of investors and the inherent instability of the economy. Time series methods are among the most widely applied scientific methods to analyze and predict volatility. Very frequently sampled data contain much valuable information about the different elements of volatility and may ultimately reveal the reasons for time varying volatility. The use of such ultra-high-frequency data is common to all three essays of the dissertation. The dissertation belongs to the field of financial econometrics. The first essay uses wavelet methods to study the time-varying behavior of scaling laws and long-memory in the five-minute volatility series of Nokia on the Helsinki Stock Exchange around the burst of the IT-bubble. The essay is motivated by earlier findings which suggest that different scaling laws may apply to intraday time-scales and to larger time-scales, implying that the so-called annualized volatility depends on the data sampling frequency. The empirical results confirm the appearance of time varying long-memory and different scaling laws that, for a significant part, can be attributed to investor irrationality and to an intraday volatility periodicity called the New York effect. The findings have potentially important consequences for options pricing and risk management that commonly assume constant memory and scaling. The second essay investigates modelling the duration between trades in stock markets. Durations convoy information about investor intentions and provide an alternative view at volatility. Generalizations of standard autoregressive conditional duration (ACD) models are developed to meet needs observed in previous applications of the standard models. According to the empirical results based on data of actively traded stocks on the New York Stock Exchange and the Helsinki Stock Exchange the proposed generalization clearly outperforms the standard models and also performs well in comparison to another recently proposed alternative to the standard models. The distribution used to derive the generalization may also prove valuable in other areas of risk management. The third essay studies empirically the effect of decimalization on volatility and market microstructure noise. Decimalization refers to the change from fractional pricing to decimal pricing and it was carried out on the New York Stock Exchange in January, 2001. The methods used here are more accurate than in the earlier studies and put more weight on market microstructure. The main result is that decimalization decreased observed volatility by reducing noise variance especially for the highly active stocks. The results help risk management and market mechanism designing.