Evaluation of Stem-Loop Reverse Transcription and Poly-A Tail Extension in MicroRNA Analysis of Body Fluids

MicroRNA has been demonstrated to be a viable tool for body fluid identification purposes in forensic casework. Stem-loop reverse transcription (slRT) is regularly used for cDNA synthesis from mature miRNA, along with poly-A tail extension. Both have been used in a forensic context, but no direct comparison has been carried out. It has also not been shown whether poly-A tail extension can be used upon DNA extracts, as previously shown with slRT. Blood and saliva samples were collected and underwent DNA extraction with or without on-column DNA digestion. All samples were then aliquoted and underwent slRT and poly-A tail extension separately. qPCR was then conducted targeting microRNA markers hsa-miR-451 and hsa-miR-205. It was shown that the DNA digestion step did not affect the ability to differentiate between blood and saliva. It was also shown that this differentiation was possible using poly-A tail extension, and that poly-A tail extension exhibited more amplification than slRT. So whilst the choice of slRT and poly-A tail extension for the purpose of forensic body fluid identification is not critical, it may be best to use poly-A tail extension, particularly where there are low traces of sample

Studies of soil copper in relation to its availability to plants

The adsorption of copper by various soil materials and intact soils was examined at equilibrium solution copper concentrations within the range found in natural soil solutions. The distribution of copper between solution and solid phases was measured by means of labelling with radioactive copper-6 4. In the majority of soils specific adsorption of copper by oxides and organic materials is considered likely to control solution copper concentrations.Although the adsorbed copper is strongly held, experiments have shown that a large proportion of it remains isotopically exchangeable and can still therefore be regarded as 'plant available'. Solution concentrations of copper were relatively unaffected by both the background concentration of major cations and by changes in pE within the ionic strength and pH range found in normal agricultural soils.The relative abundance of different forms of copper in pasture and arable soils was compared by means of a fractionation scheme. The principle difference found was that the pasture soils contained higher concentrations of soluble copper complexes. A specific ion electrode was used successfully to differentiate between ionic and complexed copper in simple systems but suffered from interference by other ions when used with natural soil solutions.Soil samples, both with and without added copper were incubated moist at a temperature of 20°G for a period of ten months. Duplicates were stored dry. Regular analysis showed that the amount of copper extractable with EBTA decreased with time, especially in the moist soils. Fixation of copper by soil oxides appeared to be responsible. Additional changes were observed in both calcium chloride extraetable and isotopically exchangeable copper which were not reflected by changes in the amount of copper extracted by EDTA.Plant uptake studies with red clover under glass demonstrated that significant increases in plant copper content can be achieved by adding copper to a variety of soils. The size of the response varied considerably from soil to soil but no clear relationship was observed between the copper content of the controls or the response to copper additions and individual soil properties.A second glasshouse trial, using barley, clover and ryegrass, indicated that there appears to be no advantage in using Cu-EDTA rather than CuSO₄ as a copper fertilizer. Clover responded to copper additions more readily than ryegrass and both plants responded far better when the copper fertilizer was mixed into the soil rather than applied to the surface.The existence of interactions between copper and other trace elements was also demonstrated in pot trials. However, the nature and importance of such interactions seems to depend on the particular conditions under which an experiment is carried out

Influenza A virus Genomic Reassortment and Packaging

Influenza A viruses (IAV) are a major human and environmental pathogen. IAV successfully infects a diverse host range and adaptation of new viral strains to humans may cause pandemic events with high morbidity and mortality. As a member of the Orthomyxoviridae family, IAV inherently possesses a segmented genome, which enables a process of segment transmission between viruses following cellular co-infection, a process termed reassortment. The high rate of IAV mutation and continued co-circulation of diverse viral strains in divergent host species leads to the persistent prospect for emergence of new IAV with pandemic potential. Therefore, it is of great importance to understand the viral and host factors that restrict and promote the generation of emergent virus strains, their potential for pathogenesis, and discover novel mechanistic countermeasures against IAV, including improved vaccination and targeted therapeutic strategies. Human and avian IAV co-circulate and occasionally co-infect the same host, leading to the potential for generation of novel genome constellations following reassortment. The specific host and viral molecular determinants that allow replication of reassortant progeny virus are not well defined. Here, I show that the viral genetic context and host cell in which reassortment occurs determine the potential for genetic diversity derived from multiple distantly related strains. Importantly, we identify single gene reassortants between a North American avian strain and the 2009 pandemic H1N1 virus that are capable of causing disease in mammals and replicate in a human cell line as well as induce the production of several pro-inflammatory cytokines linked to severe disease outcomes. Additionally, utilizing a different viral genetic background, I show that the reassortment potential is regulated by species and cell type specific differences in viral replication due to augmented viral polymerase function dependent on the identity of a single amino acid in the PA protein. Together, these studies provide evidence that context- dependent compatibility between both viral and host factors determine the possibility for generation of novel reassortant genome constellations and regulate their potential for replication and transmission in new host species. Reassortment between IAV strains is likely dictated by the functional compatibility of vRNA segments bound by IAV nucleoprotein during genome packaging. I hypothesized that nucleoprotein (NP) scaffolds specific RNA elements that are required for genome packaging and interaction between viral RNA (vRNA) genome segments. Therefore, I sought to determine the functional consequences of genome architecture on genome packaging and for the first time determine the nucleotide-resolution landscape of NP-vRNA interactions in infected cells. We utilized Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) coupled to next-generation sequencing to determine the specific interaction sites of vRNA bound by NP. We then interrogated the functional importance of regions of vRNA bound or unbound by NP and identified a number of potentially structured RNA features required for efficient genome packaging and virus propagation. These studies provide a framework for understanding the multifactorial restrictions of IAV reassortment and potential for generation of novel genome constellations with pandemic potential. Finally, these studies expand our understanding of how viral and host determinants shape the possible evolutionary trajectories of IAV through reassortment and required genetic elements needed for genome assembly

Persons, property and morality : a defence of political libertarianism

Bibliography: p.191-197.This dissertation adopts as its starting point the beliefs that moral truths can be known and that political philosophy is a branch of ethics. The author identifies three variants of libertarianism on the basis of their different treatments of the right to private property, which all three consider to be the cornerstone of political libertarianism. The author evaluates the arguments of Robert Nozick, Murray Rothbard, John Hospers and Ayn Rand for the moral foundations of libertarianism and finds them to be methodologically inadequate. None is able to furnish libertarianism with the moral foundations it requires. Following the example of Jan Narveson in his recent defence of the libertarian idea, the author adopts as the correct metaphysic of morality the method of hypothetical contract. The contractarian method is capable of determining both the nature and the extent of moral obligation. From application of the method of hypothetical contract, the author concurs with the above-mentioned authors that morality is a system of rights and duties, i.e. deontological in character, and that persons are indeed bearers of moral, non-conventional rights. One of these rights is the negative right to equal social liberty. The author differs, however, in finding that contractarianism favours also a positive right to basic, standard welfare. Recognition of this latter right commits the author to a form of moderate or Lockean libertarianism that endorses the in-principle justice of coercive redistribution to meet persons' basic welfare. Consequently, the orthodox libertarianism advocated by Nozick, Rothbard, Hospers, Rand and Narveson which recognises only negative moral rights is rejected by the author. All of the libertarians cited accept in one form or another John Locke's labour theory of appropriation. However, the author eschews the standard reading of Locke they are wedded to. The standard reading premises the labour theory on a person's ownership of himself. This reading is rejected on the grounds that the idea of self-ownership is insufficiently determinate to act as a sure basis for establishing property rights in things one has mixed one's labour with. A reconstructed defence of the moral right to private property through labouring which avoids this difficulty is given. That defence is premised not on self-ownership but on the right to equal social liberty. Save for the requirement to meet basic welfare there are no limits to the extent of acquisition. The author argues that, despite his avowals to the contrary, Nozick in fact endorses a positive right to welfare, and that this positive right is one that is co-extensive with the right to basic welfare established by the method of hypothetical contract. Two arguments are given. The first argument draws on Nozick's Lockean proviso that an act of appropriation not worsen the position of others. The second is based upon the application to an envisaged society of libertarian-rights bearers of Nozick's clause that permits the violation of rights in order to avoid catastrophic moral horror. This latter argument the author believes to be successful against any libertarianism that is wedded to absolute property rights. Redistribution to meet the demands of basic welfare necessitates taxation. Taxation is to be levied proportionately and not progressively, and is to be coupled with a system of private social insurance. None of the three variants of libertarianism identified, and which the author maintains sustain redistribution as a matter of justice, is ostensibly committed to redistribution more extensive than required to meet persons' basic welfare~ Ernest Loevinsohn's argument to the effect that libertarians are - by the very principle they defend as libertarians - committed to more far-reaching welfare and redistribution is examined and rejected. Because Loevinsohn's argument is directed against a consequentialist defence of libertarianism and not a deontological version it is misplaced. Furthermore, it fails to establish the conclusion Loevinsohn supposes it

Pathophysiology of Skeletal Disease

These studies have resulted in substantial advances in the field of osteoporosis and osteoarthritis research. Crucial contributions to pivotal osteoporosis GWAS identified 518 bone mineral density and 13 fracture loci that now account for over 20% of the population variance in bone density. We identified over 300 novel eBMD loci and validated numerous genes, including (i) transferrin receptor-2 as a novel regulator of bone mass acting via BMP/p38MAPK/Wnt signalling, (ii) over 100 genes that determine bone mass, quality and strength comprising enzymes, ion and amino acid transporters, cell cycle regulators, transcription factors and modulators of non-canonical Wnt signalling, and (iii) age-specific and sexually dimorphic genetic effects on bone mineral density. Recent discoveries include (i) identification of a new cell type with a unique transcriptome, termed the “osteomorph”. Bone resorbing multinucleated osteoclasts undergo cycles of cell fission and fusion, recycling via osteomorphs in the bone marrow to regulate osteoclast motility and dynamic bone remodelling in vivo, (ii) elucidation of a transcriptome map of genes expressed in osteocytes, the master regulatory cells in bone. Osteocyte signature genes correlate closely with loci identified in human GWAS and in the nosology of monogenic skeletal disorders, establishing the cellular pathogenesis of various skeletal diseases, and (iii) development of novel imaging methods in osteoarthritis disease models and generation of the first multi ‘omic molecular QTL map of human disease to accelerate causative gene discovery in osteoarthritis. This multidisciplinary and international approach is transformative and has resulted in a comprehensive atlas of human and murine genetic influences on bone and joint disease that offer novel insights into the pathophysiology of osteoporosis and osteoarthritis with exciting opportunities for biomarker discovery and drug development. This body of work has resulted in invitations to contribute seminal chapters in major international textbooks, including (i) Genetics of Bone Biology and Skeletal Disease (2018) and (ii) Osteoporosis (2020), and Plenary Lectures to the (i) 21st World Congress on Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (IOFESCEO WCO), (ii) ASBMR Bone Turnover Markers Annual Meeting, and (iii) 9th Congress of the Romanian Society of Osteoporosis and Musculoskeletal Diseases (all in 2021). My work was recognised by the European Calcified Tissue Society Steven Boonen Clinical Research Award (2018), and I was elected Fellow of the Academy of Medical Sciences (2019), Member of Academia Europaea (2021) and Fellow of the Association of Physicians of Great Britain & Ireland (2021).Open Acces

Actions of thyroid hormones in bone

Hormony tarczycy są niezbędne dla prawidłowego rozwoju układu kostnego i uzyskania szczytowej masy kostnej. Niedoczynność tarczycy u dzieci prowadzi do ograniczenia (opóźnienia) tempa wzrostu spowodowanego opóźnieniem rozwoju kości, podczas gdy nadczynność tarczycy przyspiesza dojrzewanie układu kostnego. U dorosłych hormony tarczycy regulują obrót kostny i gęstość mineralną układu kostnego. Stan eutyreozy jest więc istotny dla utrzymania optymalnej jakości układu kostnego. Wyniki badań populacyjnych wykazały, że zarówno niedoczynność, jak i nadczynność tarczycy są związane ze zwiększonym ryzykiem złamań kości. Mimo to mechanizm działania trijodotyroniny (T3) w kości nie jest do końca poznany. W badaniach zmutowanych myszy wykazano, że działanie T3 na kość zachodzi poprzez interakcje hormonu z receptorem a (TRα). W okresie młodości i wzrastania T3 wywiera na kość efekt anaboliczny i odpowiada za szczytowy przyrost kości. W późniejszym okresie życia T3 wywiera efekt kataboliczny, zwiększając resorpcję wapna i obrót kostny. W ostatnim okresie wyniki niektórych badań mogą wskazywać, że TSH ma bezpośredni wpływ na komórki kości, ale problem ten jest bardzo trudny do ostatecznego ustalenia w warunkach in vivo, wobec tego, że stężenie TSH i hormonu tarczycy jest utrzymywane w odwrotnym wzajemnym stosunku (sprzężeniu zwrotnym) przez układ podwzgórze-przysadka-tarczyca. Obecna wiedza w tym zakresie wynika z doświadczeń myszami, u których zmutowano geny kodujące TRα, TRβ i receptor TSH. Jednak nie można kategorycznie ustalić, czy wpływ tych mutacji na szkielet zależy od zmienionej ekspresji T3 w komórkach kośćca, czy jest następstwem tych mutacji na czynność innych układów endokrynnych, które regulują rozwój szkieletu i masę kostną. Blokowanie sygnału od hormonu tarczycy w poszczególnych tkankach i układach będzie konieczne, aby rozwiązać te niejasności. Przeprowadzenie takich badań pozwoli zidentyfikować kluczowe mechanizmy działania T3 na komórki kości i określić nowe cele w leczeniu osteoporozy.Thyroid hormones are required for skeletal development and establishment of peak bone mass. Hypothyroidism in children results in growth retardation with delayed skeletal development, whereas thyrotoxicosis accelerates bone maturation. In adults, T3 regulates bone turnover and bone mineral density, and normal euthyroid status is essential to maintain optimal bone strength. Population studies indicate that hypothyroidism and hyperthyroidism are both associated with an increased risk of fracture. Nevertheless, the mechanism of T3 action in bone is incompletely understood. Studies in mutant mice have demonstrated that T3 action in bone is mediated principally by T3 receptor α (TRα). T3 exerts anabolic actions during growth to stimulate peak bone mass acrrual, but has catabolic effects on the adult skeleton that increase bone turnover. Recent studies have also suggested that TSH may have direct actions in bone cells, but such effects are difficult to resolve in vivo because thyroid hormone and TSH concentrations are maintained in an inverse relationship by the hypothalamic-pituitary-thyroid axis. Current understanding is based on studies in mice that harbor germline mutations in the genes encoding TRα, TRβ or the TSH receptor and it is not clear whether the skeletal effects of these mutations result from disruption of primary T3 actions in bone cells or whether they are secondary to systemic effects on other endocrine pathways that regulate skeletal development and bone mass. Tissue-specific disruption of thyroid hormone signalling in bone cells will be required to address this issue. Such studies are likely to identify key components of the T3 signalling pathway that may represent suitable drug targets for treatment of osteoporosis