The role of the dopaminergic neurotrophin growth/differentiation factor-5 in the developing rat ventral midbrain

Growth differentiation factor-5 (GDF-5) is a member of the transforming growth factor-β superfamily, a family of proteins that play diverse roles in many aspects of cell growth, proliferation and differentiation. GDF-5 has also been shown to be a trophic factor for embryonic midbrain dopaminergic neurons in vitro (Krieglstein et al. 1995) and after transplantation to adult rats in vivo (Sullivan et al. 1998). GDF-5 has also been shown to have neuroprotective and neurorestorative effects on adult dopaminergic neurons in the substantia nigra in animal models of Parkinson’s disease (Sullivan et al. 1997, 1999; Hurley et al. 2004). This experimental evidence has lead to GDF-5 being proposed as a neurotrophic factor with potential for use in the treatment of Parkinson’s disease. However, it is not know if GDF-5 is expressed in the brain and whether it plays a role in dopaminergic neuron development. The experiments presented here aim to address these questions. To that end this thesis is divided into five separate studies each addressing a particular question associated with GDF-5 and its expression patterns and roles during the development of the rat midbrain. Expression of the GDF-5 in the developing rat ventral mesencephalon (VM) was found to begin at E12 and peak on E14, the day that dopaminergic neurons undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicated a role for GDF-5 in the development and maintenance of dopaminergic neurons. The appropriate receptors for GDF-5 (BMPR-II and BMPR-Ib) were found to be expressed at high levels in the rat VM at E14 and BMPR-II expression was demonstrated on dopaminergic neurons in the E13 mouse VM. GDF-5 resulted in a three-fold increase in the numbers of dopaminergic neurons in cultures of E14 rat VM, without affecting the numbers of neurones or total cells. GDF-5 was found to increase the proportion of neurons that were dopaminergic. The numbers of Nurr1-positive cells were not affected by GDF-5 treatment, but GDF-5 did increase the numbers of Nurr1- positive cells that expressed tyrosine hydroxylase (TH). Taken together this data indicated that GDF-5 increases the conversion of Nurr1-positive, TH-negative cells to Nurr1-positive, TH-positive cells. In GDF-5 treated cultures, total neurite length, neurite arborisation and somal area of dopaminergic were all significantly increased compared to control cultures. Thus this study showed that GDF-5 increased the numbers and morphological differentiation of VM dopaminergic neurones in vitro. In order to examine if GDF-5 could induce a dopaminergic phenotype in neural progenitor cells, neurosphere cultures prepared from embryonic rat VM were established. The effect of the gestational age of the donor VM on the proportion of cell types generated from neurospheres from E12, E13 and E14 VM was examined. Dopaminergic neurons could only be generated from neurospheres which were prepared from E12 VM. Thus in subsequent studies the effect of GDF-5 on dopaminergic induction was examined in progentior cell cultures prepared from the E12 rat VM. In primary cultures of E12 rat VM, GDF-5 increased the numbers of TH-positive cells without affecting the proliferation or survival of these cells. In cultures of expanded neural progenitor cells from the E12 rat VM, GDF-5 increased the expression of Nurr1 and TH, an action that was dependent on signalling through the BMPR-Ib receptor. Taken together, these experiments provide evidence that GDF-5 is expressed in the developing rat VM, is involved in both the induction of a dopaminergic phenotype in cells of the VM and in the subsequent morphological development of these dopaminergic neurons

Team-teaching for inclusive learning: Purposes, practices and perceptions of a team-teaching initiative in Irish post-primary schools

Background. Schools unequivocally privilege solo-teaching. This research seeks to enhance our understanding of team-teaching by examining how two teachers, working in the same classroom at the same time, might or might not contribute to the promotion of inclusive learning. There are well-established policy statements that encourage change and moves towards the use of team-teaching to promote greater inclusion of students with special educational needs in mainstream schools and mainstream classrooms. What is not so well established is the practice of team-teaching in post-primary settings, with little research conducted to date on how it can be initiated and sustained, and a dearth of knowledge on how it impacts upon the students and teachers involved. Research questions and aims. In light of the paucity and inconclusive nature of the research on team-teaching to date (Hattie, 2009), the orientating question in this study asks ‘To what extent, can the introduction of a formal team-teaching initiative enhance the quality of inclusive student learning and teachers’ learning at post-primary level?’ The framing of this question emerges from ongoing political, legal and educational efforts to promote inclusive education. The study has three main aims. The first aim of this study is to gather and represent the voices and experiences of those most closely involved in the introduction of team-teaching; students, teachers, principals and administrators. The second aim is to generate a theory-informed understanding of such collaborative practices and how they may best be implemented in the future. The third aim is to advance our understandings regarding the day-to-day, and moment-to-moment interactions, between teachers and students which enable or inhibit inclusive learning. Sample. In total, 20 team-teaching dyads were formed across seven project schools. The study participants were from two of the seven project schools, Ash and Oak. It involved eight teachers and 53 students, whose age ranged from 12-16 years old, with 4 teachers forming two dyads per school. In Oak there was a class of first years (n=11) with one dyad and a class of transition year students (n=24) with the other dyad. In Ash one class group (n=18) had two dyads. The subjects in which the dyads engaged were English and Mathematics. Method. This research adopted an interpretive paradigm. The duration of the fieldwork was from April 2007 to June 2008. Research methodologies included semi-structured interviews (n=44), classroom observation (n=20), attendance at monthly teacher meetings (n=6), questionnaires and other data gathering practices which included school documentation, assessment findings and joint examination of student work samples (n=4). Results. Team-teaching involves changing normative practices, and involves placing both demands and opportunities before those who occupy classrooms (teachers and students) and before those who determine who should occupy these classrooms (principals and district administrators). This research shows how team-teaching has the potential to promote inclusive learning, and when implemented appropriately, can impact positively upon the learning experiences of both teachers and students. The results are outlined in two chapters. In chapter four, Social Capital Theory is used in framing the data, the change process of bonding, bridging and linking, and in capturing what the collaborative action of team-teaching means, asks and offers teachers; within classes, between classes, between schools and within the wider educational community. In chapter five, Positioning Theory deductively assists in revealing the moment-to-moment, dynamic and inclusive learning opportunities, that are made available to students through team-teaching. In this chapter a number of vignettes are chosen to illustrate such learning opportunities. These two theories help to reveal the counter-narrative that team-teaching offers, regarding how both teachers and students teach and learn. This counter-narrative can extend beyond the field of special education and include alternatives to the manner in which professional development is understood, implemented, and sustained in schools and classrooms. Team-teaching repositions teachers and students to engage with one another in an atmosphere that capitalises upon and builds relational trust and shared cognition. However, as this research study has found, it is wise that the purposes, processes and perceptions of team-teaching are clear to all so that team-teaching can be undertaken by those who are increasingly consciously competent and not merely accidentally adequate. Conclusions. The findings are discussed in the context of the promotion of effective inclusive practices in mainstream settings. I believe that such promotion requires more nuanced understandings of what is being asked of, and offered to, teachers and students. Team-teaching has, and I argue will increasingly have, its place in the repertoire of responses that support effective inclusive learning. To capture and extend such practice requires theoretical frameworks that facilitate iterative journeys between research, policy and practice. Research to date on team-teaching has been too focused on outcomes over short timeframes and not focused enough on the process that is team-teaching. As a consequence team-teaching has been under-used, under-valued, under-theorised and generally not very well understood. Moving from classroom to staff room and district board room, theoretical frameworks used in this research help to travel with, and understand, the initiation, engagement and early consequences of team-teaching within and across the educational landscape. Therefore, conclusions from this study have implications for the triad of research, practice and policy development where efforts to change normative practices can be matched by understandings associated with what it means to try something new/anew, and what it means to say it made a positive difference.

The role of the IL-1 type 1 receptor in the life, death and differentiation of adult hippocampal neural precursor cells

Neurogenesis occurs in two distinct regions of the adult brain; the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the subventricular zone (SVZ) lining the lateral ventricles. It is now well-known that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors e.g. local signalling molecules, exercise, environmental enrichment and learning. Moreover, levels of adult hippocampal neurogenesis decrease with age, at least in rodents, and alterations in hippocampal neurogenesis have been reported in animal models and human studies of neuropsychiatric and neurodegenerative conditions. Neuroinflammation is a common pathological feature of these conditions and is also a potent modulator of adult hippocampal neurogenesis. Recently, the orphan nuclear receptor TLX has been identified as an important regulator of adult hippocampal neurogenesis as its expression is necessary to maintain the neural precursor cell (NPC) pool in the adult DG. Likewise, exposure of animals to voluntary exercise has been consistently demonstrated to promote adult hippocampal neurogenesis. Lentivirus (LV)- mediated gene transfer is a useful tool to elucidate gene function and to explore potential therapeutic candidates across an array of conditions as it facilitates sustained gene expression in both dividing and post-mitotic cell populations. Both intrinsic and extrinsic factors are important regulators of adult hippocampal neurogenesis. Examining how these factors are affected by an inflammatory stimulus, and the subsequent effects on adult hippocampal neurogenesis provides important information for the development of novel treatment strategies for neuropsychiatric and neurodegenerative conditions in which adult hippocampal neurogenesis is impaired. The aims of the series of experiments presented in this thesis were to examine the effect of the pro-inflammatory cytokine interleukin-1β (IL-1β) on adult hippocampal NPCs both in vitro and in vivo. In vitro, we have shown that IL-1β reduces proliferation of adult hippocampal NPCs in a dose and time-dependent manner. In addition, we have demonstrated that TLX expression is reduced by IL-1β. Blockade of IL-1β signalling prevented both the IL-1β-induced reduction in cell proliferation and TLX expression. In vivo, we examined the effect of short term and long term exposure to LV-IL-1β in sedentary mice and in mice exposed to voluntary running. We demonstrated that impaired hippocampal neurogenesis is only evident after long term exposure to IL-1β. In mice exposed to voluntary running, hippocampal neurogenesis is significantly increased following short-term but not long-term exposure to running. Moreover, short-term running effectively prevents any IL-1β-induced effects on hippocampal neurogenesis; however, no such effects are seen following long-term exposure to running.

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Differentiation and fine structure of larval epaulets in the echinoid Paracentrotus lividus

info:eu-repo/semantics/published

Differentiation of the genital apparatus in juvenile echinoids (Paracentrotus lividus)

info:eu-repo/semantics/published

Teaching yoga to seniors: essential considerations to enhance safety and reduce risk in a uniquely vulnerable age group.

BACKGROUND: Seniors age 65 and older represent the fastest-growing sector of the population and, like many Americans, are increasingly drawn to yoga. This presents both an extraordinary opportunity and a serious challenge for yoga instructors who must be both a resource and guardians of safety for this uniquely vulnerable group. A typical class of seniors is likely to represent the most diverse mix of abilities of any age group. While some may be exceedingly healthy, most fit the profile of the average older adult in America, 80% of whom have at least one chronic health condition and 50% of whom have at least two. OBJECTIVES: This article discusses the Therapeutic Yoga for Seniors program, offered since 2007 at Duke Integrative Medicine to fill a critical need to help yoga instructors work safely and effectively with the increasing number of older adults coming to yoga classes, and explores three areas that pose the greatest risk of compromise to older adult students: sedentary lifestyle, cardiovascular disease, and osteoporosis. To provide a skillful framework for teaching yoga to seniors, we have developed specific Principles of Practice that integrate the knowledge gained from Western medicine with yogic teachings.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

WNT3 is a biomarker capable of predicting the definitive endoderm differentiation potential of hESCs.

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However, the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here, we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition, manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally, analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively, our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs.

Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro.

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

Teaching neuraxial anesthesia techniques for obstetric care in a Ghanaian referral hospital: achievements and obstacles.

Anesthesia providers in low-income countries may infrequently provide regional anesthesia techniques for obstetrics due to insufficient training and supplies, limited manpower, and a lack of perceived need. In 2007, Kybele, Inc. began a 5-year collaboration in Ghana to improve obstetric anesthesia services. A program was designed to teach spinal anesthesia for cesarean delivery and spinal labor analgesia at Ridge Regional Hospital, Accra, the second largest obstetric unit in Ghana. The use of spinal anesthesia for cesarean delivery increased significantly from 6% in 2006 to 89% in 2009. By 2012, >90% of cesarean deliveries were conducted with spinal anesthesia, despite a doubling of the number performed. A trial of spinal labor analgesia was assessed in a small cohort of parturients with minimal complications; however, protocol deviations were observed. Although subsequent efforts to provide spinal analgesia in the labor ward were hampered by anesthesia provider shortages, spinal anesthesia for cesarean delivery proved to be practical and sustainable.

TEACHING AMERICAN CIVIL WAR MUSIC IDSTORY WITH MODERN EDITIONS OF PERIOD MUSIC FOR FULL CONCERT BAND

This dissertation explores a method of teaching the history of Civil War music and musicians through modern full-band editions of original brass band music. In the study of music history the period of the Civil War is rarely discussed, or at best, mentioned only if a student takes a specific course on the history of bands and happens to look deeply into the background of some of the early band pioneers such as Patrick Gilmore, who served in the Union Army as a bandmaster. The history of the musicians, bands, and music performed during the Civil War deserves study to provide a way for students and audiences to learn this history. This project includes lesson plans that can be used with the arrangements of the period music as well as select published music that is also representative of the period. Included with the historical information are four arrangements of original brass band music now scored for full concert band. Each arrangement includes a section scored for brass only with optional brass band parts. Historical information is provided on the Civil War period bands and how each side used them, on the composers of the music, and also on the individual compositions. The historical information can be used to supplement the lesson plans to teach the history, as well as for program notes for audiences. The research involved locating information on both Union and Confederate bands available in books, other dissertations, articles, and interviews with Civil War music historians. The original brass band music is scored for full band. This method will allow teachers and conductors to highlight this period of wind band history and to share it with both students and audiences. Included with this project are photos and video footage taken during a visit with the 1st Brigade Band of Watertown, Wisconsin, an historical organization dedicated to recreating the music and performances of an actual Civil War era band.

Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation.

Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate.

TEACHING HIGH SCHOOL STUDENTS THE BEST CHORAL REPERTOIRE FROM THE GREAT COMPOSERS: MASTERWORKS AVAILABLE FOR IMMEDIATE, FREE ACCESS FROM THE CHORAL PUBLIC DOMAIN LIBRARY

Studying the choral works of the great composers of the past is always a worthy endeavor. For those aspiring to create an excellent high school choral program, it is critical to a student's musical foundation and heritage. Choral educators who teach high school are often bombarded with the most recently published new choral works, when they have a trove of excellent pieces right at their fingertips through websites like the Choral Public Domain Library (CPDL), all available at no cost. This project will explore the pedagogical reasons why this canon of public domain choral music should be taught at the high school level. A thorough guide to CPDL and an anthology of 200 works available on CPDL will provide the conductor with resources for programming this music. Though choral music in the public domain is free to all, publishers still publish this music and adhere copyright claims. This can create mistrust of legitimate editions on CPDL; why are they available at no cost when publishers are claiming copyright on similar editions? These issues will be thoroughly discussed in this project. For any given work on CPDL, there may be multiple editions available on the site. Choosing the right edition requires knowledge about basic editorial principles, especially for works written during the Renaissance period. A detailed discussion of these principles will provide the conductor with the tools needed to choose the best edition for his or her ensemble.

Rapid immunological diagnosis of active tuberculosis, and differentiation from asymptomatic infection

info:eu-repo/semantics/nonPublished