Repeats as global DNA methylation marker in bovine preimplantation embryos

DNA methylation undergoes dynamic changes and is a crucial part of the epigenetic regulation during mammalian early development. To determine the DNA methylation levels in bovine embryos, we applied a bisulfite sequencing based method aimed at repetitive sequences including three retrotransposons (L1_BT, BovB, and ERV1-1-I_BT) and Satellite I. A more accurate estimate of the global DNA methylation level compared to previous methods using only one repeat sequence, like Alu, could be made by calculation of the weighted arithmetic mean of multiple repetitive sequences, considering the copy number of each repetitive sequence. Satellite I and L1_BT showed significant methylation reduction at the blastocyst stage, while BovB and ERV1-1-I_BT showed no difference. The mean methylation level of the repetitive sequences during preimplantation development was the lowest at the blastocyst stage. No methylation difference was found between embryos cultured in 5% and 20% O-2. Because mutations of CpGs negatively influence the calculation accuracy, we checked the mutation rate of the sequenced CpG sites. Satellite I and L1_BT showed a relatively low mutation rate (1.92 and 3.72% respectively) while that of ERV1-1-I_BT and BovB was higher (11.95 and 24% respectively). Therefore we suggest using a combination of repeats with low mutation rate, taking into account the proportion of each sequence, as a relatively quick marker for the global DNA methylation status of preimplantation stages and possibly also for other cell types

Retrotransposon activity and DNA methylation control in bovine preimplantation embryos

Retrotransposons, or RNA intermediated transposable elements, have been considered as ‘junk’ or ‘selfish’ DNA and dismissed as uninteresting for a long time. However, they were found active and functional in preimplantation embryos in the last decades. The impact of retrotransposon activity on the genome such as formation of new genetic elements and regulators in the genome is unneglectable. They are also suggested to participate in many activities during early embryo development, like genomic imprinting, X chromosome inactivation, cell proliferation and differentiation. In Chapter 1, the current literature on retrotransposons in mammalian genomes is reviewed. An overview of the classification and composition of retrotransposons in the genome is given, with emphasis of the bovine genome. Furthermore, the mechanisms of retrotransposition are described in detail and the activities of retrotransposons on the level of transcription, translation and mobilization are explained. The impact of retrotransposon activity on the mammalian genome is discussed, illustrating that retrotransposons influence the genome by formatting new genetic elements and working as regulators. The defense mechanisms against retrotransposon to prevent their disturbance of the genomes are also given in Chapter 1. In the latter part of Chapter 1, activation of retrotransposons in early development is discussed. Retrotransposons participate in many embryonic activities, including cell proliferation and differentiation, genomic imprinting, and X-chromosome inactivation. These activities are suggested to be mainly a consequence of DNA methylation loss during epigenetic reprogramming of preimplantation. Therefore, a description of the dynamics of DNA methylation during bovine preimplantation embryo development is included. Those facts reveal the importance of the study of the relation between DNA methylation and retrotransposon expression in embryo development. At last, a short review of using retrotransposons as marker for global DNA methylation status is given. The aims of the study are presented in Chapter 2. The general aim of the thesis was to study retrotransposon expression and their DNA methylation control in bovine preimplantation embryos. The first aim was to find out autonomous retrotransposons with consistent RNA expression, and reliable reference genes for gene expression normalization during bovine preimplantation embryo development. The second aim was to study the relation between DNA methylation and retrotransposon expression under high oxygen. The third aim was to use repetitive sequences as global DNA methylation marker in bovine preimplantation embryos. To achieve these goals, we first profiled autonomous retrotransposon expression in bovine preimplantation embryos. L1_BT, BovB and ERV1-1-I_BT were found to be expressed throughout all stages of preimplantation development (Chapter 3.1). Furthermore, reference genes were evaluated and selected for estimating retrotransposon expression in bovine embryos of different stages and under different conditions (Chapter 3.2). In Chapter 4, the relation between DNA methylation and retrotransposon expression under influence of high oxygen tension as stressor was studied. The global DNA methylation of embryos was estimated by 5-methylcytosine immunofluorescent (5-mC) staining. We found a significant increase in DNA methylation under 20% O2 at the 4-cell stage and in blastocysts. Gene expression of DNA methytransferases (DNMTs) and three retrotransposons (L1_BT, BovB and ERV1-1-I_BT) in 4-cell embryos and blastocysts was analyzed by RT-qPCR. Unexpectedly, the retrotransposon expression was not correlated with the global DNA methylation level estimated by 5-mC staining, but was negatively correlated with the expression of DNMT1. We further studied the DNA methylation level of retrotransposons during embryo development by bisulfite sequencing (Chapter 5). Using this method, a negative correlation between the methylation level (L1_BT BovB > ERV1-1-I_BT) was found. Additionally, we developed a global DNA methylation marker using a combination of repetitive sequences with low mutation rate, taking in to account the proportion of each sequence. The general discussion and the conclusions are presented in Chapter 6. The discussion focuses on the following aspects: 1) correlation of DNA methylation and retrotransposon expression; 2) influence of oxidative stress on methylation and retrotransposon expression; 3) problems in immunofluorescence staining; 4) comparison of the methylation research methods used in the thesis; 5) DNA methylation dynamics during bovine preimplantation embryo development; and 6) retrotransposons used as global DNA methylation marker

New Paradigm of Defibrillation: Towards Painless Therapy

Sudden cardiac death: SCD) causes approximately 300,000 - 400,000 deaths a year in the United States. It usually starts as ventricular tachycardia: VT) and then degenerates into ventricular fibrillation: VF). Implantable cardioverter defibrillator: ICD) therapy is the only reliable treatment of VT/VF and has been shown to effectively reduce mortality by many clinical trials. However, high-voltage ICD shocks could result in myocardial dysfunction and damage. The majority of patients receiving ICD therapy have a history of coronary disease; their hearts develop myocardium infarction, which could provide a substrate for reentrant tachy-arrhythmias. Other than lethal ventricular tachycardia, atrial fibrillation: AF) became the most common arrhythmia by affecting 2.2 to 5.6 millions of Americans. The complications of AF include an increased rate of mortality, heart failure, stroke, etc. In this dissertation, we explore mechanisms of sustained ventricular and atrial tachyarrhythmias and the mechanisms of defibrillation using the conventional high-voltage single shock. Through the use of novel fluorescent optical mapping techniques and several animal models of ventricular and atrial arrhythmias, we develop and validate several novel low-voltage defibrillation therapies for atrial and ventricular arrhythmias. Several important previous studies on mechanisms of arrhythmia maintenance and termination using mathematical and experimental models are overviewed in Chapter 2. A study on multiple monophasic shocks improving electrotherapy of ventricular tachycardia in rabbit model of chronic infarction is presented in Chapter 3. Ventricular arrhythmias and low-voltage defibrillation therapy are studied in a more clinically-relevent in vivo canine model of healing myocardial infarction in Chapter 4. Finally, Chapter 5 presents a novel multi-stage low-energy defibrillation therapy for atrial fibrillation in in vivo canine hearts

GeoAI: Where machine learning and big data converge in GIScience

In this paper GeoAI is introduced as an emergent spatial analytical framework for data-intensive GIScience. As the new fuel of geospatial research, GeoAI leverages recent breakthroughs in machine learning and advanced computing to achieve scalable processing and intelligent analysis of geospatial big data. The three-pillar view of GeoAI, its two methodological threads (data-driven and knowledge-driven), as well as their geospatial applications are highlighted. The paper concludes with discussion of remaining challenges and future research directions of GeoAI

Changes in Higher Education Policies: A Case Study of Projects in China

Many measures have been taken to promote the development of higher education by the Chinese government since 1978. This study examined the projects launched by the Chinese government to improve the quality of higher education and elevate the status of targeted institutions to world-class universities. The policies reviewed include Project 211, Project 985, and the Double First-rate University Plan. By examining the implementation of these projects, this study aimed to understand if the policy goals sought by the government were achieved. The conceptual framework used to analyze the projects included Lowi’s theory of distribution and McDonnell and Elmore’s typology of policy instruments. This study found that the projects demonstrated characteristics unique to China and that they focused on efficiency, quality, and equality

Factors behind rural student drop-out rates in North China: a qualitative study

To promote the development of compulsory education and educational equality in China, Two Exemptions and One Subsidy (TEOS) policy which initially aimed to help students from poor background finish their compulsory education was implemented by Chinese government step by step. This study examined the fact there were students in rural areas still dropping out under the policy of TEOS using a qualitative approach. Interviews were made and information was collected from different perspectives, dropouts, parents as well as teachers. The analysis from this study suggests that it was time to rethink grade retention removed in the revised compulsory education law. Furthermore, programs intended to help low achievers and parents in rural areas is suggested. Ultimately, a comprehensive evaluation should be adopted when assessing students and capabilities of teachers should be built to enhance the teaching in rural areas