Characterisation and functional analysis of a novel msp domain – containing protein, MOSPD1

MOSPD1 belongs to a class of proteins that have a major sperm protein (MSP) domain at the N terminus and two transmembrane domains at the C terminus and are thought to act as membrane adaptor proteins. Previous work in the laboratory indicated that the closely related, mammalian-specific, Mospd 3 plays a role in the development and function of the heart as homozygous Mospd 3 gene trap neonates displayed a right ventricle defect characterised by a thinning of the right ventricle wall. The function of Mospd 1 is not known. Whilst Mospd 3 is mammalian specific Mospd 1 is conserved in all vertebrates including Danio rerio (zebrafish). The aims of this thesis were to investigate the possibility of genetic redundancy between Mospd 1 and Mospd 3 by identifying the sub-cellular localisation of MOSPD1 and MOSPD3 in both cells and tissues and to investigate the function of MOSPD1. Mouse monoclonal antibodies specific for MOSPD1 and MOSPD3 were generated and tested to ensure they did not cross react. MOSPD1 was found to be localised to the nucleus whilst MOSPD3 was located in the nucleus and cytoplasm. The sub-cellular localisation of these proteins changes during the cell cycle as they were localised to the cytoplasm during cell division, possible due to the breakdown of the nuclear membrane during cell division. To investigate the function of Mospd 1 during early development Mospd 1 gene expression was knocked down using morpholino anti-sense knockdown technology. A morpholino was generated against the splice-site between exons 2 and 3 of the zebrafish Mospd 1 gene and injected into early embryos. At doses that significantly reduced the level of Mospd 1, to below 50 %, the embryos developed normally and did not exhibit any gross morphological phenotypes when compared to both noninjected and 5 mispair control morpholino-injected embryos. A morpholino targeted to the start site of the Mospd 1 gene confirmed the lack of a gross morphological phenotype. In conjunction with the zebrafish functional tests the tools were generated to assess the role of Mospd 1 in a mammalian system. A conditional allele of Mospd 1 was generated in mouse embryonic stem (ES) cells which could be used to generate a conditional Mospd 1 mouse. The electroporation of a Cre recombinase plasmid into the conditional Mospd 1 ES cell line resulted in the generation of Mospd 1 null ES clones which could be used for functional studies both in vitro and in vivo. The Mospd 1 null ES cells were able to self-renew, expressed ES cell specific markers and were able to differentiate into cardiomyocytes. However, Mospd 1 null cells showed a reduced ability to differentiate into osteoblasts compared to wild type cells and showed changes in the expression of genes involved in Epithelial to mesenchymal transition (EMT) indicating Mospd 1 may be involved in this process

A role for mospd1 in mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1‐null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self‐renewal capacity of MOSPD1‐null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)‐like cells expressing CD73, CD90, and CD105 were generated from MOSPD1‐null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1‐null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA‐sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre‐MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation. Stem Cells 2015;33:3077–308

The UK clinical eye research strategy: refreshing research priorities for clinical eye research in the UK

To validate and update the 2013 James Lind Alliance (JLA) Sight Loss and Vision Priority Setting Partnership (PSP)'s research priorities for Ophthalmology, as part of the UK Clinical Eye Research Strategy. Twelve ophthalmology research themes were identified from the JLA report. They were allocated to five Clinical Study Groups of diverse stakeholders who reviewed the top 10 research priorities for each theme. Using an online survey (April 2021-February 2023), respondents were invited to complete one or more of nine subspecialty surveys. Respondents indicated which of the research questions they considered important and subsequently ranked them. In total, 2240 people responded to the survey (mean age, 59.3 years), from across the UK. 68.1% were female. 68.2% were patients, 22.3% healthcare professionals or vision researchers, 7.1% carers, and 2.1% were charity support workers. Highest ranked questions by subspecialty: Cataract (prevention), Cornea (improving microbial keratitis treatment), Optometric (impact of integration of ophthalmic primary and secondary care via community optometric care pathways), Refractive (factors influencing development and/or progression of refractive error), Childhood onset (improving early detection of visual disorders), Glaucoma (effective and improved treatments), Neuro-ophthalmology (improvements in prevention, diagnosis and treatment of neurodegeneration affecting vision), Retina (improving prevention, diagnosis and treatment of dry age-related macular degeneration), Uveitis (effective treatments for ocular and orbital inflammatory diseases). A decade after the initial PSP, the results refocus the most important research questions for each subspecialty, and prime targeted research proposals within Ophthalmology, a chronically underfunded specialty given the substantial burden of disability caused by eye disease. [Abstract copyright: © 2024. The Author(s).

Relative adrenal insufficiency in mice deficient in 5α-reductase 1

Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as ‘relative adrenal insufficiency’. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5β-reductase, resulting in compensatory downregulation of the hypothalamic–pituitary–adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause ‘relative adrenal insufficiency’ in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease

Characterisation and functional analysis of a novel MSP domain-containing protein, MOSPD1

MOSPD1 belongs to a class of proteins that have a major sperm protein (MSP) domain at the N terminus and two transmembrane domains at the C terminus and are thought to act as membrane adaptor proteins. Previous work in the laboratory indicated that the closely related, mammalian-specific, Mospd 3 plays a role in the development and function of the heart as homozygous Mospd 3 gene trap neonates displayed a right ventricle defect characterised by a thinning of the right ventricle wall. The function of Mospd 1 is not known. Whilst Mospd 3 is mammalian specific Mospd 1 is conserved in all vertebrates including Danio rerio (zebrafish). The aims of this thesis were to investigate the possibility of genetic redundancy between Mospd 1 and Mospd 3 by identifying the sub-cellular localisation of MOSPD1 and MOSPD3 in both cells and tissues and to investigate the function of MOSPD1. Mouse monoclonal antibodies specific for MOSPD1 and MOSPD3 were generated and tested to ensure they did not cross react. MOSPD1 was found to be localised to the nucleus whilst MOSPD3 was located in the nucleus and cytoplasm. The sub-cellular localisation of these proteins changes during the cell cycle as they were localised to the cytoplasm during cell division, possible due to the breakdown of the nuclear membrane during cell division. To investigate the function of Mospd 1 during early development Mospd 1 gene expression was knocked down using morpholino anti-sense knockdown technology. A morpholino was generated against the splice-site between exons 2 and 3 of the zebrafish Mospd 1 gene and injected into early embryos. At doses that significantly reduced the level of Mospd 1, to below 50 %, the embryos developed normally and did not exhibit any gross morphological phenotypes when compared to both noninjected and 5 mispair control morpholino-injected embryos. A morpholino targeted to the start site of the Mospd 1 gene confirmed the lack of a gross morphological phenotype. In conjunction with the zebrafish functional tests the tools were generated to assess the role of Mospd 1 in a mammalian system. A conditional allele of Mospd 1 was generated in mouse embryonic stem (ES) cells which could be used to generate a conditional Mospd 1 mouse. The electroporation of a Cre recombinase plasmid into the conditional Mospd 1 ES cell line resulted in the generation of Mospd 1 null ES clones which could be used for functional studies both in vitro and in vivo. The Mospd 1 null ES cells were able to self-renew, expressed ES cell specific markers and were able to differentiate into cardiomyocytes. However, Mospd 1 null cells showed a reduced ability to differentiate into osteoblasts compared to wild type cells and showed changes in the expression of genes involved in Epithelial to mesenchymal transition (EMT) indicating Mospd 1 may be involved in this process.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

U.S. POLICY TOWARD IRAN AFTER THE NUCLEAR DEAL

Created as part of the 2016 Jackson School for International Studies SIS 495: Task Force.While the landmark Joint Comprehensive Plan of Action will be the cornerstone of U.S.-Iran relations for years to come, a new set of policies for engagement between the U.S. and Iran is both possible and necessary if the U.S. wishes to help bring Iran into the international community as a more pragmatic actor. To prevail in this, Washington will need a new, more balanced approach that recognizes Tehran’s saber-rattling for what it is, and a groundbreaking cooperative agenda that balances the strategic interests of its allies in the Middle East with the U.S.’ overarching objective of stability in the region – both of which Iran is integral to. This is not to say that the U.S. should at any point overlook Tehran’s history of fomenting violence and discord in the region for the sake of compromise; such antagonistic policies must be systematically opposed wherever Tehran seeks to implement them. But at this critical juncture where tensions between Iran and the Arab states are at a fever pitch, Iran will be more likely to sit at the table if the U.S. can convince its allies that their security is best served through diplomatic measures. Moreover, if Iran can be shown that its more aggressive tendencies do more harm than good to its national interests, it may yet shift its attention inward to more cooperative and prosperous endeavors worthy of the regional power it aspires to be. Reintegrating Iran into the international community will be by no means easy, and hopes for swift reform must be tempered even as a new generation of young Iranians begins to exert a more moderate influence on domestic politics. But if the following U.S. policies can be implemented to deter Iranian actions that destabilize the region, demonstrate commitment to regional allies, and incentivize acceptable behavior from Iran with opportunities for economic and diplomatic integration, ideology may give way to reveal instances of progress toward a more beneficial state of relations between the U.S. and Iran

The UK clinical eye research strategy: refreshing research priorities for clinical eye research in the UK

To validate and update the 2013 James Lind Alliance (JLA) Sight Loss and Vision Priority Setting Partnership (PSP)'s research priorities for Ophthalmology, as part of the UK Clinical Eye Research Strategy. Twelve ophthalmology research themes were identified from the JLA report. They were allocated to five Clinical Study Groups of diverse stakeholders who reviewed the top 10 research priorities for each theme. Using an online survey (April 2021-February 2023), respondents were invited to complete one or more of nine subspecialty surveys. Respondents indicated which of the research questions they considered important and subsequently ranked them. In total, 2240 people responded to the survey (mean age, 59.3 years), from across the UK. 68.1% were female. 68.2% were patients, 22.3% healthcare professionals or vision researchers, 7.1% carers, and 2.1% were charity support workers. Highest ranked questions by subspecialty: Cataract (prevention), Cornea (improving microbial keratitis treatment), Optometric (impact of integration of ophthalmic primary and secondary care via community optometric care pathways), Refractive (factors influencing development and/or progression of refractive error), Childhood onset (improving early detection of visual disorders), Glaucoma (effective and improved treatments), Neuro-ophthalmology (improvements in prevention, diagnosis and treatment of neurodegeneration affecting vision), Retina (improving prevention, diagnosis and treatment of dry age-related macular degeneration), Uveitis (effective treatments for ocular and orbital inflammatory diseases). A decade after the initial PSP, the results refocus the most important research questions for each subspecialty, and prime targeted research proposals within Ophthalmology, a chronically underfunded specialty given the substantial burden of disability caused by eye disease

The UK clinical eye research strategy: refreshing research priorities for clinical eye research in the UK.

ObjectivesTo validate and update the 2013 James Lind Alliance (JLA) Sight Loss and Vision Priority Setting Partnership (PSP)'s research priorities for Ophthalmology, as part of the UK Clinical Eye Research Strategy.MethodsTwelve ophthalmology research themes were identified from the JLA report. They were allocated to five Clinical Study Groups of diverse stakeholders who reviewed the top 10 research priorities for each theme. Using an online survey (April 2021-February 2023), respondents were invited to complete one or more of nine subspecialty surveys. Respondents indicated which of the research questions they considered important and subsequently ranked them.ResultsIn total, 2240 people responded to the survey (mean age, 59.3 years), from across the UK. 68.1% were female. 68.2% were patients, 22.3% healthcare professionals or vision researchers, 7.1% carers, and 2.1% were charity support workers. Highest ranked questions by subspecialty: Cataract (prevention), Cornea (improving microbial keratitis treatment), Optometric (impact of integration of ophthalmic primary and secondary care via community optometric care pathways), Refractive (factors influencing development and/or progression of refractive error), Childhood onset (improving early detection of visual disorders), Glaucoma (effective and improved treatments), Neuro-ophthalmology (improvements in prevention, diagnosis and treatment of neurodegeneration affecting vision), Retina (improving prevention, diagnosis and treatment of dry age-related macular degeneration), Uveitis (effective treatments for ocular and orbital inflammatory diseases).ConclusionsA decade after the initial PSP, the results refocus the most important research questions for each subspecialty, and prime targeted research proposals within Ophthalmology, a chronically underfunded specialty given the substantial burden of disability caused by eye disease

The UK clinical eye research strategy: refreshing research priorities for clinical eye research in the UK

Objectives To validate and update the 2013 James Lind Alliance (JLA) Sight Loss and Vision Priority Setting Partnership (PSP)’s research priorities for Ophthalmology, as part of the UK Clinical Eye Research Strategy. Methods Twelve ophthalmology research themes were identified from the JLA report. They were allocated to five Clinical Study Groups of diverse stakeholders who reviewed the top 10 research priorities for each theme. Using an online survey (April 2021-February 2023), respondents were invited to complete one or more of nine subspecialty surveys. Respondents indicated which of the research questions they considered important and subsequently ranked them. Results In total, 2240 people responded to the survey (mean age, 59.3 years), from across the UK. 68.1% were female. 68.2% were patients, 22.3% healthcare professionals or vision researchers, 7.1% carers, and 2.1% were charity support workers. Highest ranked questions by subspecialty: Cataract (prevention), Cornea (improving microbial keratitis treatment), Optometric (impact of integration of ophthalmic primary and secondary care via community optometric care pathways), Refractive (factors influencing development and/or progression of refractive error), Childhood onset (improving early detection of visual disorders), Glaucoma (effective and improved treatments), Neuro-ophthalmology (improvements in prevention, diagnosis and treatment of neurodegeneration affecting vision), Retina (improving prevention, diagnosis and treatment of dry age-related macular degeneration), Uveitis (effective treatments for ocular and orbital inflammatory diseases). Conclusions A decade after the initial PSP, the results refocus the most important research questions for each subspecialty, and prime targeted research proposals within Ophthalmology, a chronically underfunded specialty given the substantial burden of disability caused by eye disease