FUNCTIONAL AND MECHANISTIC STUDY OF DOT1L IN MOUSE EMBRYONIC HEMATOPOIESIS

DOT1 is the histone 3 lysine 79 methyltransferase with both unique structure and substrate specificity. It plays critical role in telomere silencing maintenance, transcription regulation, DNA repair, and cell cycle regulation. DOT1L in mammals is required for embryonic development. In the absence of DOT1L, mouse embryos die by E13.5. Severe anemia was observed in Dot1L mutant embryos at E10.5. In order to study a potential role for DOT1L in embryonic hematopoiesis, colony forming assays were performed using both primitive and definitive yolk sac progenitors. We found that DOT1L is required for both primitive and definitive hematopoietic development in the mouse. Interestingly, there is a specific defect in erythroid lineage development. Using flow cytometric analysis, we showed that Dot1L deficiency does not affect the emergence of the progenitor population. When Dot1L mutant progenitor cells differentiate into the erythroid lineage, they fail to progress normally through the cell cycle and undergo increased apoptosis when compared to wild type progenitors. In order to investigate the molecular mechanism of DOT1L in hematopoiesis, the expression of genes that are critical in hematopoiesis was analyzed in Dot1L mutant progenitors. Although Gata-2 expression appears to be regulated by DOT1L, the reduction of Gata-2 in yolk sac progenitors alone does not lead to an observable hematopoietic defect. Trpc6, which plays a role in EPO-mediated calcium influx, was down regulated significantly in Dot1L mutant progenitors. Based on our data and previous studies demonstrating a role for TRPC proteins in mediating EPO induced calcium influx, I hypothesize that Dot1L deficiency results in an abnormal EPO response in hematopoietic progenitor cells. This response is characterized by significantly increased calcium influx induced by EPO. This abnormal intracellular calcium levels in turn result in the observed defective erythropoiesis. In support of this hypothesis, calcium imaging analysis demonstrated that the intracellular calcium level was much higher in Dot1L mutant progenitor cells after EPO treatment in comparison with wild type cells. My study explores the cellular and biological function of Dot1L and provides more insight into the complex regulation of embryonic hematopoiesis

Expression and functional characterisation of the Rev gene product of maedi visna virus EV- 1

The ovine Maedi Visna Virus (MVV) and the Human Immunodeficiency Virus Type 1 (HIV-1) are members of the lentiviririae retrovirus subfamily. Lentiviruses possess a complex genomic organisation, encoding several genes with a regulatory or auxiliary function. Alternative splicing of the genomic-length primary transcript is used to express this complexity. mRNA expression is subject to temporal regulation in both MVV and HIV-1. In HIV-1 a highly basic protein. Rev, mediates this regulation. Rev function requires binding to a highly structured RNA target, the Rev responsive element (RRE), and may involve facilitation of RNA nucleocytoplasmic export. Rev/RRE interaction is essential for virus replication. There is little overall sequence homology between lentivirus Rev proteins. However, functionally important basic and leucine-rich motifs are conserved. These domains are found within the putative MVV Rev protein, and a predicted RRElike structure is present in a similar genomic location to that in HIV-1.To compare the mode of action of MVV Rev with that of HIV-1 Rev, a series of functional assays were planned. The rev gene of a British isolate of MVV (EV-1) was cloned and sequenced. Recombinant Rev was expressed as a fusion protein in yeast and bacterial systems. A polyclonal antiserum directed against a synthetic Rev polypeptide was generated to aid purification. Expression in yeast was characterised by a low product yield, due to the highly toxic nature of the Rev fusion protein. Alternative expression and purification protocols were unable to greatly improve the yield and purity of product. The bacterial pGEX system, in which Rev was fused to glutathione S-transferase (GSTRev), was employed as an alternative. Purification by affinity chromatography resulted in an improved yield and purity of product. Partial instability of the fusion protein may have resulted in observed contamination.Binding of GSTRev to RNA corresponding to the predicted RRE was assayed by filter binding experiments. A specific vector context and low temperature were required to generate high quality RNA. GSTRev bound with high affinity to RRE-RNA, but not to RNA corresponding to antisense RRE. Addition of a non-specific competitor RNA reduced binding to antisense, but not sense, RNA.Rev is the least well conserved protein amongst sequenced isolates of MVV. To test for the functional conservation of the Rev/RRE axis, the cross reactivity of Rev function on heterologous RREs was examined by transient transfection assay. Whilst cross-strain functional reciprocity was observed, both the EV-1 and 1514 isolate Rev proteins demonstrated greatest activity on cognate RRE. Co-divergence of the rev gene and RRE structure of each strain has therefore occurred. MVV Rev was able to function through the RRE of the closely related caprine arthritis encephalitis virus. These results may have implications for the possible development of anti-lentiviral gene therapy based on frans-dominant, inhibitory Rev molecules

Formal Verification of P Systems

Membrane systems, also known as P systems, constitute an innovative computational paradigm inspired by the structure and dynamics of the living cell. A P system consists of a hierarchical arrangement of compartments and a finite set of multiset rewriting and communication rules, which operate in a maximally parallel manner. The organic vision of concurrent dynamics captured by membrane systems stands in antithesis with conventional formal modelling methods which focus on algebraic descriptions of distributed systems. As a consequence, verifying such models in a mathematically rigorous way is often elusive and indeed counter-intuitive when considering established approaches, which generally require sequential process representations or highly abstract theoretical frameworks. The prevalent investigations with this objective in the field of membrane computing are ambivalent and inconclusive in the wider application scope of P systems. In this thesis we directly address the formal verification of membrane systems by means of model checking. A fundamental distinction between the agnostic perspective on parallelism, advocated by process calculi, and P systems' emblematic maximally parallel execution strategy is identified. On this basis, we establish that an intuitional translation to traditional process models is inadequate for the purpose of formal verification, due to a state space growth disparity. The observation is essential for this research project: on one hand it implies the feasibility of model checking P systems, and on the other hand it underlines the suitability of this formal verification technique in the context of membrane computing. Model checking entails an exhaustive state space exploration and does not derive inferences based on the independent instructions comprising a state transition. In this respect, we define a new sequential modelling strategy which is optimal for membrane systems and targets the SPIN formal verification tool. We introduce elementary P systems, a distributed computational model which subsumes the feature diversity of the membrane computing paradigm and distils its functional vocabulary. A suite of supporting software tools which gravitate around this formalism has also been developed, comprising of 1. the eps modelling language for elementary P systems; 2. a parser for the eps specification; 3. a model simulator and 4. a translation tool which targets the Promela specification of the SPIN model checker. The formal verification approach proposed in this thesis is progressively demonstrated in four heterogeneous case studies, featuring 1. a parallel algorithm applicable to a structured model; 2. a linear time solution to an NP-complete problem; 3. an innovative implementation of the Dining Philosophers scenario (a synchronisation problem) using an elementary P system and 4. a quantitative analysis of a simple random process implemented without the support of a probabilistic model

The Symbolic Subject in Information and Communications Technologies: Michel Freitag on Technique, Science, and its Implications for Subjectivity

Grasping the novelty of contemporary technological mediation in its ubiquity, particularly within information and communications networks, typically involves recognizing the post-human status of subjects relationally embedded to technical processes that are increasingly autonomous relative to the individual. Problems with this approach become apparent once we consider both the symbolic character of human beings and the technical relation to objects as only a partial moment of a subject’s interaction with the world. Providing an answer to these concerns, Michel Freitag’s theory of the symbolic grants a sociological understanding of the role of technique within action in its historical unfolding as a distinctly productive activity in modernity. In this context, the predominance of theory over technique in the production of knowledge within modern scientific practice will slowly reverse as modern epistemology regresses into the abyss of the subject-object dualism of the enlightened individual while scientific production itself is progressively appropriated by industry as of the 19th century. This new operational understanding of scientific epistemology will reach its archetypical form in the post war field of cybernetics, where information ontology will quickly instigate further technical automation within productive labour and later influence neoliberal epistemologies in matters of governance. The latter, increasingly realized in the technocratic management of social life, will further effectuate itself in the digital infrastructure of contemporary communications systems under algorithmic governmentality in which both the normative and expressive ends of action tend to be subsumed. In this context, the reduction of language to its communication and of action to its operability will signal the ideological confusion between reality and its representation, together tending towards the unraveling of the transcendental unity of the subject and betraying the disavowed yearning for an absolute freedom liberated from worldly constraint

Characterisation of Transcriptional Regulation of the Human Telomerase RNA Gene

The human telomerase core enzyme consists of an essential structural RNA (hTERC) with a template domain for telomeric DNA synthesis and of a catalytic protein (hTERT) with reverse transcriptase activity. Expression of the hTERC and hTERT are essential for telomerase activity. Variation in telomerase activity is correlated with cellular senescence and tumour progression. Recent studies indicate that the regulation of telomerase activity is multifactorial in mammalian cells. The primary mode of control of hTERT appears to be transcriptional regulation but very little is known about the molecular mechanisms involved in the regulation of hTERC transcription. In this study, I have cloned and characterised the genomic sequences and promoter of the hTERC gene to provide evidence that transcriptional mechanisms are involved in hTERC gene regulation. Transient transfection with a series of 5'-deletion mutants demonstrated that between -5.0 kb and -51 by of the hTERC gene is responsible for high promoter activity, the minimal promoter region was defined as 176 by (-107 to +69 bp). With the aid of in vitro DNase I footprinting, electrophoretic mobility shift assays (EMSAs) and mutagenesis analysis, four Sp1 binding sites and one CCAAT-box bound by the transcription factor NF-Y were identified to be involved in regulation of hTERC transcription. Co-transfection experiments showed that Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. Mutation of the CCAAT-box or the coexpression of a dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by pRb and Sp1, suggesting that NF-Y binding is a prerequisite for pRb and Sp1 to activate the hTERC promoter. The different transcriptional regulators appear to act in a species-specific manner. Whilst Sp1 and Sp3 act on the human, bovine and mouse TERC promoters, pRb activates only the human and bovine promoter and NF-Y is important for the human TERC gene. The hTERC gene is expressed during embryogenesis and then down-regulated during normal development but is re-expressed in tumour cells, the hTERC promoter activity was therefore further investigated and a higher promoter activity in immortal cells than in two mortal cell strains (WI38 and IMR90) was shown. In conclusion, hTERC promoter contains sequence elements that allow interactions with several different transcription factors. The interplay between NF-Y, pRb, Sp1 and Sp3 within the architecture of the hTERC promoter may combine to enable a wide variety of cell types from mortal to immortal to regulate hTERC expression through transcriptional control

The Balance between the Data Protection Law Regime and Modern Technologies: Collision or Collaboration? − A Comparative Study of Regulatory Instruments in the EU and Taiwan

The aim of this thesis is to discuss and evaluate how to strike a balance between the benefits and the risks of biometric and Radio-frequency Identification (RFID) technologies within a data protection regime. This presents a problem because of the lack of an applicable theoretical framework and clear guidelines and principles for legal regulations to deal with such technologies. The theory chosen here is the Principle of Generic Consistency (PGC), which has been justified as the basic principle of human rights in any given community. This thesis then elaborates on specific applications of the PGC in relation to various issues by defining relevant privacy concepts and describing how they are analysed to allow the identification, evaluation, and comparison of competing rights and interests in a specific conflict. Probing and evaluating current regulation of technologies at stake in Europe and Taiwan, it is argued that the right to benefit from advances in science and technology and the right to privacy are bound to come into conflict. However, it is problematic to suggest that the balancing of competing rights is a zero-sum trade-off. Instead, in line with the broad concept of privacy, it is contended that there is the possibility for the two sets of values to support each other. In this case, the thesis suggests a co-operative framework, which relies on a consistent approach to maintain valid consent, precautionary and preventive measures to tackle the risks of developing such technologies, and an independent institutional framework for personal data protection. Lastly, the thesis proposes a PGC-derived regulatory framework and model for Taiwan. As the Formosan hydra-headed bureaucracy model generates inconsistent data protection consequences, it is suggested that an institutional framework comprising an independent regulatory body might be able to assist the success of the co-operative model more effectively

Modeling adaptive dynamics in microbial populations with applications to the evolution of cellular resource allocation trade-offs

Adaptive evolution is the process by which natural selection, acting on variation within a population, promotes the survival of individuals that are more successful at reproducing and contributing to future generations. Evolutionary processes in microbes occur at the intersection of population genetics, natural selection, and underlying mechanistic constraints, to give rise to the repertoire of adaptation observed in nature. Understanding microbial adaptive evolution is of critical importance for human health for example, through the emergence of pathogenicity and antibiotic resistance. Moreover, the stability and function of natural and artificial ecosystems is contingent on the evolving interactions between microbes, and between microbes and the environment. We present a modelling framework, based on the theory of adaptive dynamics, to investigate how cellular resource allocation trade-offs affect the adaptation process. We used resource-consumer theory, which explicitly models the interactions between cells and their environment, together with matrix models of structured populations, to implement phenotype-determined cellular strategies of resource allocation between mutually exclusive processes. We then analyse the outcome of competitions between different phenotypes across environmental and competitive conditions. We applied our methods to the evolution of strategies (phenotypes) for resource allocation between two competing cellular process in microbial populations growing in chemostat-like environments. We calculated the adaptively stable strategies for several models and showed how state-structured population models can be mapped to simpler chemostat models on invariant manifolds. We then extended our analysis to the case where a limiting nutrient may be utilized using two alternative metabolic pathways. We described how the total population fitness of a metabolic strategy can be constructed from the individual decisions of its constituent members. We developed numerical methods to simulate and analyse general models of adaptive dynamics using principles from graph theory and discrete Markov processes. The methods were used to explore the evolution of nutrient use strategies for microbial populations growing on two and three substitutable nutrients. We highlight the importance of the ancestral phenotype in channelling the adaptation process, which, together with the choice of the mutational kernel, influences the adaptively stable strategies and modes of co-existence. In a related finding, we show how some phenotypes are adaptively stable only in the presence of a competitor lineage that modifies the environment in a manner that permits another phenotype to invade. Our methods also reveal instances where historical contingency and chance have an important effect on determining the stable nutrient use strategies. Finally, we demonstrate the existence of adaptively stable periodic solutions whereby, under some conditions, phenotype successions are cyclical. Our work builds on the foundation of adaptive dynamics theory to provide a general framework for analysing models of microbial adaptation. We focused on understanding the implications of underlying constraints and cellular resource allocation trade-offs in the context of adaptive evolution

Africa's development : the imperatives of indigenous knowledge and values

In post-colonial Africa, conceptions of the nature and purposes of development as well as the theories and strategies for achieving them have remained a territory traversed predominantly by non-African social scientists. In this context, social scientists studying Africa's development proclaimed, at the dawn of the 1990s, a "paradigmatic crisis" and embarked on a quest for new paradigms . In advancing this quest, a number of "homegrown" development strategies have emerged. This work argues that these are mere adaptations and reconstructions of dominant Eurocentic paradigms that exaggerate the value of economic goods and wealth creation founded on a competitive marketplace by making them immutable features of development. Yet the ethic of competition theoretically condones a trajectory of killing in the quest for wealth accumulation. In this way, internalist epistemologies perpetuate epistemicide and valuecide in Africa's strides towards development. The stranglehold of internalist epistemologies has resulted in the impasse of rationality. By this we mean that Reason, apotheosized since the Enlightenment, has advanced humanity out of barbarism to "civilization" but has now placed humanity on the brink of unredeemable barbarism. Reason, through its manifestations in the philosophy of Mutual Assured Destruction and global warming, has condemned humanity to willful but avoidable suicide. Since the subjects and objects of development must be one and the same, development is necessarily culture-derived and culture-driven, with the preservation and improvement of human dignity and welfare as its ultimate aims. Accordingly, we defend the thesis that it is necessary for a framework meant for Africa's development to be founded on indigenous knowledge and values, if it is to succeed. And at this moment of impasse reached by Reason, an African ethics-based development paradigm, predicated on humaneness and "life is mutual aid", can restore Reason to sober rationality and liberate Africa's development efforts from the intoxicating prison of profit making. Hence the institutions and frameworks devoted to Africa's development, such as the Constitution and Strategic Plan of the African Union as well as NEPAD, must incorporate salient features of the philosophic ethic emanating from the knowledge and ontological systems of indigenous Africa into visions of the African future.Philosophy, Practical and Systematic TheologyD. Phil. (Philosophy