Quakerly conflict: The cultural logic of conflict in the Religious Society of Friends

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Evidence for the requirement of 14-3-3eta (YWHAH) in meiotic spindle assembly during mouse oocyte maturation

Background The 14-3-3 (YWHA) proteins are central mediators in various cellular signaling pathways regulating development and growth, including cell cycle regulation. We previously reported that all seven mammalian 14-3-3 isoforms are expressed in mouse oocytes and eggs and that, 14-3-3η (YWHAH) accumulates and co-localizes in the region of meiotic spindle in mouse eggs matured in vivo. Therefore, we investigated the role of 14-3-3η in spindle formation during mouse oocyte maturation. Results Examination of oocytes matured in vitro demonstrated that 14-3-3η accumulates in both meiosis I and II spindles. To explore if 14-3-3η interacts directly with α-tubulin in meiotic spindles, we performed an in situ proximity ligation assay that can detect intracellular protein-protein interactions at the single molecule level and which allows visualization of the actual interaction sites. This assay revealed a marked interaction between 14-3-3η and α-tubulin at the metaphase II spindle. To demonstrate a functional role for 14-3-3η in oocyte maturation, mouse oocytes were microinjected with a translation-blocking morpholino oligonucleotide against 14-3-3η mRNA to reduce 14-3-3η protein synthesis during oocyte maturation. Meiotic spindles in those cells were examined by immunofluorescence staining of 14-3-3η and α-tubulin along with observation of DNA. In 76% of cells injected with the morpholino, meiotic spindles were found to be deformed or absent and there was reduced or no accumulation of 14-3-3η in the spindle region. Those cells contained clumped chromosomes, with no polar body formation. Immunofluorescence staining of 14-3-3η and α-tubulin in control eggs matured in vitro from uninjected oocytes and oocytes microinjected with the ineffective, inverted form of a morpholino against 14-3-3η, a morpholino against 14-3-3γ, or deionized water showed normal, bipolar spindles. Conclusions The results indicate that 14-3-3η is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with α-tubulin, to regulate the assembly of microtubules. These data add to our understanding of the roles of 14-3-3 proteins in mouse oocyte maturation and mammalian reproduction

Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

<p>Abstract</p> <p>Background</p> <p>The 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ). These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms.</p> <p>Results</p> <p>We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ) are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development.</p> <p>Conclusions</p> <p>We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the differential expression of these 14-3-3 isoforms in female germ cells and ovarian follicles provides the foundation for further investigating 14-3-3 isoform-specific interactions with key proteins involved in ovarian development, meiosis and oocyte maturation. This will lead to a better understanding of the individual functional roles of the 14-3-3 protein isoforms in mammalian oogenesis and female reproductive development.</p

Protein 14-3-3 eta is essential for normal meiotic spindle assembly in mouse eggs

The 14-3-3 proteins constitute a family of conserved, acidic proteins in a variety of organisms, regulating important intracellular events including cell cycle control, apoptosis, signal transduction and embryonic development. However, little is known about the functions of 14-3-3 in mammalian reproduction. There are seven mammalian isoforms of 14-3-3 (beta, gamma, epsilon, eta, zeta, tau/theta and sigma) encoded by different genes. We previously reported accumulation and co-localization of a specific isoform of 14-3-3, namely 14-3-3 eta, in the region of meiotic spindle apparatus in mouse eggs. To determine the role of 14-3-3 eta, we microinjected mouse oocytes with a translation-blocking morpholino oligonucleotide to knock down the expression of 14-3-3 eta mRNA. Overnight incubation of the morpholino-injected oocytes followed by in vitro maturation revealed absence or impairment of meiotic spindle assembly in eggs, as shown by immunocytochemical staining of 14-3-3 eta and alpha-tubulin along with observation of chromosomes. Duolink In Situ Proximity Ligation Assay indicated marked interaction of 14-3-3 eta with alpha-tubulin in the region of meiotic spindle in all eggs examined, with prominent cortical accumulation about the spindles. These results suggest that 14-3-3 eta is necessary for normal meiotic spindle formation in mouse eggs. The study will help to elucidate the functional importance of 14-3-3 proteins in regulating mammalian oocyte maturation and female reproductive development

Case Management

This chapter describes the roles and responsibilities of the MISSION-VET Case Manager and how it relates to MISSION-VET service delivery. The chapter begins with an overview of the MISSION-VET Case Manager’s responsibilities. Settings in which MISSION-VET can be delivered and implications for case management are then reviewed. The importance of teamwork with the Peer Support Specialist is stressed, and the Case Manager’s role is distinguished from that of the Peer Support Specialist. The Chapter then reviews each of the Case Manager’s primary responsibilities. Because case management is seen as the foundation of the MISSION-VET model, this chapter refers to a number of appendices that will be useful tools for the Case Manager to use as MISSION-VET is implemented

Development of a Self-report Measure of Dual Diagnosis Capability for Addiction and Mental Health Programs

The purpose of this study is to develop and test the psychometrics of a self-report version of a measure of the capacity of addiction and mental health programs to deliver dual-diagnosis treatment, that is, to provide treatment for both addiction problems and mental health problems. Traditionally these services are provided by very different service providers that did not until recently interact very well, if at all. The increasing recognition that patients who suffer from both kinds of problems – who are dually diagnosed – would benefit from integrated delivery of addiction and mental health services has led to efforts to encourage provision of such integrated services in programs that have tended to focus primarily on the delivery of either addiction or mental health services to the exclusion of the other. In order to assess how well the integration of these services is progressing, various measures have been developed, one of which is the original Dual Diagnosis Capability in Addiction Treatment (DDCAT) Index. The DDCAT, as it now stands, however, is a very time-intensive tool. It requires a rater to visit a site and spend one half to a full day there interviewing administrators, therapists, and patients, reviewing medical records, and attending meetings. The purpose of this study is to test a self-report version of the DDCAT that will be administered to administrators and therapists to see how well it performs compared to the more time- intensive procedures of the original DDCAT

YWHA (14-3-3) Protein Isoforms and Their Interactions with CDC25B Phosphatase in Mouse Oogenesis and Oocyte Maturation

Background Immature mammalian oocytes are held arrested at prophase I of meiosis by an inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1). Release from this meiotic arrest and germinal vesicle breakdown is dependent on dephosphorylation of CDK1 by the protein, cell cycle division 25B (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the cytoplasm of immature oocytes and is thus maintained in an inactive form. The importance of YWHA in meiosis demands additional studies. Results Messenger RNA for multiple isoforms of the YWHA protein family was detected in mouse oocytes and eggs. All seven mammalian YWHA isoforms previously reported to be expressed in mouse oocytes, were found to interact with CDC25B as evidenced by in situ proximity ligation assays. Interaction of YWHAH with CDC25B was indicated by Förster Resonance Energy Transfer (FRET) microscopy. Intracytoplasmic microinjection of oocytes with R18, a known, synthetic, non-isoform-specific, YWHA-blocking peptide promoted germinal vesicle breakdown. This suggests that inhibiting the interactions between YWHA proteins and their binding partners releases the oocyte from meiotic arrest. Microinjection of isoform-specific, translation-blocking morpholino oligonucleotides to knockdown or downregulate YWHA protein synthesis in oocytes suggested a role for a specific YWHA isoform in maintaining the meiotic arrest. More definitively however, and in contrast to the knockdown experiments, oocyte-specific and global deletion of two isoforms of YWHA, YWHAH (14-3-3 eta) or YWHAE (14-3-3 epsilon) indicated that the complete absence of either or both isoforms does not alter oocyte development and release from the meiotic prophase I arrest. Conclusions Multiple isoforms of the YWHA protein are expressed in mouse oocytes and eggs and interact with the cell cycle protein CDC25B, but YWHAH and YWHAE isoforms are not essential for normal mouse oocyte maturation, fertilization and early embryonic development

Assess Intermediate Force Capabilities (IFC) and concept of operations for application during the Competition Phase in an environment of GPC

NPS NRP Executive SummaryIntermediate force capabilities represent a strategic risk mitigation investment that are designed to provide warfighters tools to compete below the level of major armed conflict without losing credibility in the information. This research examines the effects of a set of intermediate force capabilities to assess the strategic impact on a near peer adversary during the competition phase in a 'gray zone' scenario. The effort will seek to gain insights and identify challenges to the employment of IFCs, through several venues, including leveraging defense analysis and operations research department faculty and students and utilizing the NPS warfighting continuum of Joint Campaign Analysis and Wargaming Applications courses.HQMC Plans, Policies & Operations (PP&O)This research is supported by funding from the Naval Postgraduate School, Naval Research Program (PE 0605853N/2098). https://nps.edu/nrpChief of Naval Operations (CNO)Approved for public release. Distribution is unlimited.

Assess Intermediate Force Capabilities (IFC) and concept of operations for application during the Competition Phase in an environment of GPC

NPS NRP Project PosterIntermediate force capabilities represent a strategic risk mitigation investment that are designed to provide warfighters tools to compete below the level of major armed conflict without losing credibility in the information. This research examines the effects of a set of intermediate force capabilities to assess the strategic impact on a near peer adversary during the competition phase in a 'gray zone' scenario. The effort will seek to gain insights and identify challenges to the employment of IFCs, through several venues, including leveraging defense analysis and operations research department faculty and students and utilizing the NPS warfighting continuum of Joint Campaign Analysis and Wargaming Applications courses.HQMC Plans, Policies & Operations (PP&O)This research is supported by funding from the Naval Postgraduate School, Naval Research Program (PE 0605853N/2098). https://nps.edu/nrpChief of Naval Operations (CNO)Approved for public release. Distribution is unlimited.