Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease.

Neurodegenerative diseases such as Huntington disease are devastating disorders with no therapeutic approaches to ameliorate the underlying protein misfolding defect inherent to poly-glutamine (polyQ) proteins. Given the mounting evidence that elevated levels of protein chaperones suppress polyQ protein misfolding, the master regulator of protein chaperone gene transcription, HSF1, is an attractive target for small molecule intervention. We describe a humanized yeast-based high-throughput screen to identify small molecule activators of human HSF1. This screen is insensitive to previously characterized activators of the heat shock response that have undesirable proteotoxic activity or that inhibit Hsp90, the central chaperone for cellular signaling and proliferation. A molecule identified in this screen, HSF1A, is structurally distinct from other characterized small molecule human HSF1 activators, activates HSF1 in mammalian and fly cells, elevates protein chaperone expression, ameliorates protein misfolding and cell death in polyQ-expressing neuronal precursor cells and protects against cytotoxicity in a fly model of polyQ-mediated neurodegeneration. In addition, we show that HSF1A interacts with components of the TRiC/CCT complex, suggesting a potentially novel regulatory role for this complex in modulating HSF1 activity. These studies describe a novel approach for the identification of new classes of pharmacological interventions for protein misfolding that underlies devastating neurodegenerative disease.

Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

"Angels and Demons in Cromwellian and Restoration Ireland: Heresy and the Supernatural"

This essay explores accounts of supernatural activity in Cromwellian and Restoration Ireland. Religious life in Cromwellian Ireland was driven by expectations of the unusual—including audible voices from heaven, material encounters with angels, and spiritual encounters with demons. Some conservative Protestants linked this activity to the development and dissemination of heretical belief, while some who had such encounters were confident that it was compatible with the Cromwellian religious mainstream. Crawford Gribben explores the flexibility in the discourse of the marvelous in Ireland and the ways in which the administration contributed to it, and the alignment of the supernatural with various confessional convictions and postures, as well as theological radicalism. After the Restoration, accounts of supernatural encounters were remembered as ghost stories, not as matters for theological debate, a cultural transition linked to the development of a historiography that has continued to invest the Irish Cromwellian past with Gothic tropes.

Religion, pragmatic fundamentalism and the transformation of the Northern Ireland conflict

Since 11 September 2001, the religious dimension of conflict has been the focus of increasing attention. In The Clash of Civilizations, Huntington has identified the West in religious-cultural terms, as Christian with a dominant democratic culture emphasizing tolerance, moderation and consensus. The persistence of conflict in Northern Ireland among 'White' Protestant and Catholic Christians undermines this simplistic argument and demands a more subtle understanding of the role of religion and fundamentalism in contemporary conflict. Modernization theory - which is echoed among some theorists of globalization - had predicted the declining importance of religion as the world became industrialized and increasingly interconnected. This is echoed by those who argue that the Northern Ireland conflict is 'ethno-national' and dismiss the role of religion. On the other hand, others have claimed that the conflict is religious and stress the role of Protestant fundamentalism. This article draws on new evidence from Northern Ireland of the complex and subtle ways in which religion impacts on the conflict there, incorporating insights about the pragmatism of fundamentalist Protestants and how religious actors are contributing to conflict transformation. This analysis leads to three broader conclusions about understanding conflicts with religious dimensions. First, the complexity of religion must be understood, and this includes a willingness to recognize the adaptability of fundamentalisms to particular contexts. Second, engaging with fundamentalists and taking their grievances seriously opens up possibilities for conflict transformation. Third, governments and religious actors within civil society can play complementary roles in constructing alternative (religious) ideologies and structures as part of a process of transformation. In a world in which the impact of religion is persistent, engaging with the religious dimension is a vital part of a broader-based strategy for dealing with conflict. © 2008 Journal of Peace Research.

The Climate of War: Violence, Warfare and Climatic Reductionism

National security agencies and other interested parties now often regard conflict as the inevitable consequence of climate change. This inclination to reduce war to the vicissitudes of climate is not new however. Here I examine some of the earlier ways in which violence was attributed to climatic conditions, particularly in the United States, and trace links between these older advocates of climatic determinism and the recent writings of those insisting that climate change will usher in a grim world of chronic warfare. It ends by drawing attention to the writings of some critics who are troubled by the ease with which climatic reductionism is capturing the public imagination.

A Woodcut and Its Wanderings in Seventeenth-Century England

This essay traces the career of a distinctive woodcut picture that appears on dozens of seventeenth-century ballad broadsheets. Christopher Marsh argues that woodcuts have often been neglected by scholars and that they deserve careful attention. The common habit of redeploying old pictures on new ballads may, for example, have encouraged consumers to build associations between individual woodcuts and particular characteristics or themes. In order to understand the visual aesthetic of early-modern balladry, it is therefore necessary to think in fresh and creative ways about the effects of the repetition of pictures on cognition.

Deciphering the molecular role of DJ-1 in the etiology of Parkinson’s and Huntingtons’s disease

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

The Day Roads Became Rivers: A GIS-Based Assessment of Flash Floods in Worcester

In the last 4 years Worcester, UK has been hit by several intense convective rainstorms, which caused flash floods outside of existing surface drainage networks. This paper addresses two questions related to such events: Firstly to what extent can the occurrence of flash flood flow accumulation can be determined using only commonly available data and tools, assuming the rainfall events caused mainly surface runoff due to their tropical intensity and the relatively impermeable urban catchment surface? Secondly, are the flood in-cidents in Worcester aggravated by roads serving as preferential flow paths under these conditions? The as-sessment results indicated that roads do not have an influence on the flow path of flash flood rainfall in Worcester. Flow accumulation calculated with a 10m DEM, corresponds well with reported flood incidents. This basic assessment method can be used to inform the implementation of non structural flood mitigation and public awareness.

Business Models for Cloud Computing: Experiences from Developing Modeling & Simulation as a Service Applications in Industry

The potential of cloud computing is gaining significant interest in Modeling & Simulation (M&S). The underlying concept of using computing power as a utility is very attractive to users that can access state-of-the-art hardware and software without capital investment. Moreover, the cloud computing characteristics of rapid elasticity and the ability to scale up or down according to workload make it very attractive to numerous applications including M&S. Research and development work typically focuses on the implementation of cloud-based systems supporting M&S as a Service (MSaaS). Such systems are typically composed of a supply chain of technology services. How is the payment collected from the end-user and distributed to the stakeholders in the supply chain? We discuss the business aspects of developing a cloud platform for various M&S applications. Business models from the perspectives of the stakeholders involved in providing and using MSaaS and cloud computing are investigated and presented.

The human rights dilemma : an east-west encounter : Germany and China in 1996 /

Conflicts over human rights in relations between East Asia and the West have increased since the end of the Cold War. Western governments express concern about human rights standards in East Asian countries. In the East, these expressions have been perceived as interference in internal affairs. Due to dramatic economic development, East Asian nations recently have gained in pride and self-confidence as global actors. Such development is observed with suspicion in the West. Concerned about the decline of global U.S. influence, some American scholars have re-invented the notion of "culture" to point at an alleged East Asian threat. Also East Asian statesmen use the cultural argument by claiming the existence of so-called 'Asian values', which they allege are the key to Eastern economic success. This thesis argues that issues of human rights in East-West relations are not only a consequence of well-intended concern by Western governments regarding the human rights and welfare of the citizens of East Asian nations, but are in fact dominated by and used as a pawn in interplay with more complicated questions of global power and economic relations between East and West. The thesis reviews the relevance of culture in East-West relations. In the West, particularly Samuel P. Huntington with his prediction of the Clash of Civilizations stands out. Singapore's Lee Kuan Yew has been very vocal on the Eastern side. Whereas the West tries to cope with its decrease of global influence, after hundreds of years under Western hegemonism, the East believes in an Asian way of development without interference form the West. Most of this dispute revolves around the issue of human rights. The West claims the universality of rights which in fact emphasizes political and civil rights. Western countries critizise poor human rights standards in East Asia. The East, in return, accuses the West of hypocritical policies that seek global dominance. East Asian governments assert that due to a different stage of development they have to stress first their rights to development in order to assure stability. In particular, China argues this way. The country's leadership, however, shows concern about human rights and has already improved its human rights record over the past years. This thesis analyses the dispute over human rights in a case study on Germany and China. Both countries have a mutual interest in trade relations which has conflicted with Germany's criticism of China's problematic human rights record. In 1996, the two countries clashed after the German parliament passed a resolution condemning China's treatment of Tibet. This caused a lot of damage to the Chinese-German relationship which in the course of the year went back to normality. In the light of these frictions a German human rights policy that focuses on unspectacular grass-roots support of China, for example in strengthening China's legal system, would be preferable. Such co-operation must be based on mutual respect.

Polyglutamine monomer structure and its implications for molecular self-assembly

Polyglutamine is a naturally occurring peptide found within several proteins in neuronal cells of the brain, and its aggregation has been implicated in several neurodegenerative diseases, including Huntington's disease. The resulting aggregates have been demonstrated to possess ~-sheet structure, and aggregation has been shown to start with a single misfolded peptide. The current project sought to computationally examine the structural tendencies of three mutant poly glutamine peptides that were studied experimentally, and found to aggregate with varying efficiencies. Low-energy structures were generated for each peptide by simulated annealing, and were analyzed quantitatively by various geometry- and energy-based methods. According to the results, the experimentally-observed inhibition of aggregation appears to be due to localized conformational restraint placed on the peptide backbone by inserted prolines, which in tum confines the peptide to native coil structure, discouraging transition towards the ~sheet structure required for aggregation. Such knowledge could prove quite useful to the design of future treatments for Huntington's and other related diseases.

The true history regarding alleged connection of the order of ancient, free and accepted Masons: with the abduction and murder of William Morgan in western New York, in 1826... (1886)

Cover title: Masonic light on the abduction and murder of Wm. Morgan.

Homogeneity and heterogeneity of political traditions in the remaking of world order

Deux décennies après la chute de l'URSS (1991), ce mémoire propose une réévaluation de la thèse de Francis Fukuyama sur la Fin de l'Histoire, élaborée en 1989, qui postule qu'avec la chute de l'URSS aucune idéologie ne peut rivaliser avec la démocratie libérale capitaliste; et de la thèse de Samuel P. Huntington sur le Choc des civilisations, élaborée en 1993, qui pose l'existence d'un nombre fini de civilisations homogènes et antagonistes. Pourtant, lorsque confrontées à une étude approfondie des séquences historiques, ces deux théories apparaissent pour le moins relatives. Deux questions ont été traitées: l'interaction entre Idéologie et Conditions historiques, et la thèse de l'homogénéité intracivilisationnelle et de l'hétérogénéité antagoniste intercivilisationnelle. Sans les invalider complètement, cette recherche conclut toutefois que ces deux théories doivent être nuancées; elles se situent aux deux extrémités du spectre des relations internationales. La recherche effectuée a montré que les idéologies et leur poids relatif sont tributaires d'un contexte, contrairement à Fukuyama qui les pose dans l'absolu. De plus, l'étude de la Chine maoïste et particulièrement de la pensée de Mao Zedong montre que les traditions politiques locales sont plus hétérogènes qu'il n'y paraît au premier abord, ce qui relativise la thèse de Huntington. En conclusion, les rapports entre États sont plus dynamiques que ne le laissent penser les thèses de Fukuyama et de Huntington.