ELUCIDATING FUNCTIONAL ROLES FOR MYOGENIN IN ADULT SKELETAL MUSCLE METABOLISM, EXERCISE CAPACITY, AND REGENERATION

The four basic helix-loop-helix myogenic transcription factors, myogenin, Myf5, MRF4, and MyoD are critical for embryonic skeletal muscle development. Myogenin is necessary for the terminal differentiation of myoblasts into myofibers during embryogenesis, but little is known about the roles played by myogenin in adult skeletal muscle function and metabolism. Furthermore, while metabolism is a well-studied physiological process, how it is regulated at the transcriptional level remains poorly understood. In this study, my aim was to determine the function of myogenin in adult skeletal muscle metabolism, exercise capacity, and regeneration. To investigate this, I utilized a mouse strain harboring the Myogflox allele and a Cre recombinase transgene, enabling the efficient deletion of myogenin in the adult mouse. Myogflox/flox mice were stressed physically through involuntary treadmill running and by breeding them with a strain harboring the Duchenne’s muscular dystrophy (DMDmdx) allele. Surprisingly, Myog-deleted animals exhibited an enhanced capacity for exercise, running farther and faster than their wild-type counterparts. Increased lactate production and utilization of glucose as a fuel source indicated that Myog-deleted animals exhibited an increased glycolytic flux. Hypoglycemic Myog-deleted mice no longer possessed the ability to outrun their wild-type counterparts, implying the ability of these animals to further deplete their glucose reserves confers their enhanced exercise capacity. Moreover, Myog-deleted mice exhibited an enhanced response to long-term exercise training. The mice developed a greater proportion of type 1 oxidative muscle fibers, and displayed increased levels of succinate dehydrogenase activity, indicative of increased oxidative metabolism. Mdx:Myog-deleted mice exhibited a similar phenotype, outperforming their mdx counterparts, although lagging behind wild-type animals. The morphology of muscle tissue from mdx:Myog-deleted mice appears to mimic that of mdx animals, indicating that myogenin is dispensable for adult skeletal muscle regeneration. Through global gene expression profiling and quantitative (q)RT-PCR, I identified a unique set of putative myogenin-dependent genes involved in regulating metabolic processes. These data suggest myogenin’s functions during adulthood are distinctly different than those during embryogenesis, and myogenin acts as a high-level transcription factor regulating metabolic activity in adult skeletal muscle.

ΔNp63 REGULATES A COMPLEX NETWORK OF TARGET GENES IN LIMB AND EPIDERMAL DEVELOPMENT

The skin is composed of two major compartments, the dermis and epidermis. The epidermis forms a barrier to protect the body. The stratified epithelium has self-renewing capacity throughout life, and continuous turnover is mediated by stem cells in the basal layer. p63 is structurally and functionally related to p53. In spite of their structural similarities, p63 is critical for the development and maintenance of stratified epithelial tissues, unlike p53. p63 is highly expressed in the epidermis and previously has been shown to play a critical role in the development and maintenance of the epidermis. The study of p63 has been complicated due to the existence of multiple isoforms: those with a transactivation domain (TAp63) and those lacking this domain (ΔNp63). Mice lacking p63 cannot form skin, have craniofacial and skeletal defects and die within hours after birth. These defects are due to the ability of p63 to regulate multiple processes in skin development including epithelial stem cell proliferation, differentiation, and adherence programs. To determine the roles of these isoforms in skin development and maintenance, isoform specific p63 conditional knock out mice were generated by our lab. TAp63-/- mice age prematurely, develop blisters, and display wound-healing defects that result from hyperproliferation of dermal stem cells. That results in premature depletion of these cells, which are necessary for wound repair, that indicates TAp63 plays a role in dermal/epidermal maintenance. To study the role of ΔNp63, I generated a ΔNp63-/- mouse and analyzed the skin by performing immunofluorescence for markers of epithelial differentiation. The ΔNp63-/- mice developed a thin, disorganized epithelium but differentiation markers were expressed. Interestingly, the epidermis from ΔNp63-/- mice co-expressed K14 and K10 in the same cell suggesting defects in epidermal differentiation and stratification. This phenotype is reminiscent of the DGCR8fl/fl;K14Cre and Dicerfl/fl;K14Cre mice skin. Importantly, DGCR8-/- embryonic stem cells (ESCs) display a hyperproliferation defect by failure to silence pluripotency genes. Furthermore, I have observed that epidermal cells lacking ΔNp63 display a phenotype reminiscent of embryonic stem cells instead of keratinocytes. Thus, I hypothesize that genes involved in maintaining pluripotency, like Oct4, may be upregulated in the absence of ΔNp63. To test this, q-RT PCR was performed for Oct4 mRNA with wild type and ΔNp63-/- 18.5dpc embryo skin. I found that the level of Oct4 was dramatically increased in the absence of ΔNp63-/-. Based on these results, I hypothesized that ΔNp63 induces differentiation by silencing pluripotency regulators, Oct4, Sox2 and Nanog directly through the regulation of DGCR8. I found that DGCR8 restoration resulted in repression of Oct4, Sox2 and Nanog in ΔNp63-/- epidermal cells and rescue differentiation defects. Loss of ΔNp63 resulted in pluripotency that caused defect in proper differentiation and stem cell like phenotype. This led me to culture the ΔNp63-/- epidermal cells in neuronal cell culture media in order to address whether restoration of DGCR8 can transform epidermal cells to neuronal cells. I found that DGCR8 restoration resulted in a change in cell fate. I also found that miR470 and miR145 play a role in the induction of pluripotency by repressing Oct4, Sox2 and Nanog. This indicates that ΔNp63 induces terminal differentiation through the regulation of DGCR8.

The door of the tomb is open: Participatory action research to assess the impact of strengthened community capacity on maternal health care in rural Mali

This participatory action-research project addressed the hypothesis that strengthened community and women's capacity for self-development will lead to action to address maternal health problems and the prevention of maternal morbidity and mortality in Mali. Research objectives were: (1) to undertake a comparative cross-sectional study of the association of community capacity with improved maternal health in rural areas of Sanando, Mali, where capacity building interventions have taken place in some villages but not in others. (2) to describe women's maternal health status, access to and use of maternal health services given their residence in program or comparison communities.^ The participatory action research project was an integrated qualitative and quantitative study using participatory rural appraisal exercises, semi-structured group interviews and a cross-sectional survey.^ Factors related to community capacity for self-development were identified: community harmony; an understanding of the benefits of self-development; dynamic leadership; and a structure to implement collective activities.^ A distinct difference between the program and comparison villages was the commitment to train and support traditional birth attendants (TBAs). The TBAs in the program villages work in the context of the wider, integrated self-development program and, 10 years after their initial training, the TBAs continue to practice.^ Many women experience labor and childbirth alone or are attended by an untrained relative in both program and comparison villages. Nevertheless a significant change is apparent, with more women in program villages than in comparison villages being assisted by the TBAs. The delivery practices of the TBAs reveal the positive impact of their training in the "three cleans" (clean hands of the assistant, clean delivery surface and clean cord-cutting). The findings of this study indicate a significant level of unmet need for child spacing methods in all villages.^ The training and support of TBAs in the program villages yielded significant improvements in their delivery practices, and resulting outcomes for women and infants. However, potential exists for further community action. Capacities for self-development have not yet been directed toward an action plan encompassing other Safe Motherhood interventions, including access to family planning services and emergency obstetric care services. ^

The role of LMX1B and PITX2 in anterior segment development and adult ocular function

Several congenital syndromes associated with anterior segment (AS) anomalies can lead to impaired vision and glaucoma, such as nail-patella syndrome (NPS), caused by mutations in the LIM homeodomain transcription factor LMX1B and Axenfeld-Rieger's syndrome (ARS), caused by mutations in the bicoid-related homeodomain transcription factor PITX2. Targeted mutations in lmx1b and pitx2 and RNA in situ analysis reveal that both genes are required for AS development and are co-expressed within the periocular mesenchyme, suggesting they participate in a shared genetic pathway. Lmx1b homozygous mutants display iris and corneal stroma hypoplasia, and defects in ciliary body formation. In contrast, pitx2 homozygous mutants exhibit a more severe phenotype: the AS chamber, corneal endothelium, and extraocular muscles (EOM) fail to develop. The absence of EOM in pitx2 mutants suggests pitx2 acts upstream of lmx1b, or that other lmx1b family members, such as lmx1a, can compensate for lmx1b function. Lmxla/lmx1b double homozygous mutants have a reduced capacity to generate EOM, implying that lmx1 gene products have a redundant function in EOM development and that lmx1 family members may act downstream of pitx2. However, analysis of pitx2 expression in the AS tissues of lmx1b mutants and reciprocal studies of lmx1b expression in pitx2 mutants indicate that these genes do not function in a simple linear pathway. Instead, lmx1b and pitx2 may regulate a shared set of downstream targets or both genes may work in parallel transcribing unique targets required for a common biological process. Ultrastructural analysis of lmx1b and pitx2 mutant corneas indicates that collagen fibrillogenesis is perturbed, revealing a common role for both genes in the deposition of extracellular matrix. Furthermore, lmx1b/pitx2 double heterozygotes develop corneal opacities not observed in single heterozygotes demonstrating that lmx1b and pitx2 genetically interact. Data suggests that defects in the basement membrane of the corneal endothelium underlie the opacities observed in double heterozygotes. Additionally, double heterozygotes develop anterior synechias that occlude the trabecular meshwork, potentially blocking aqueous humor drainage. These data suggest that lmx1b and pitx2 are responsible for ECM deposition in multiple cell types and imply that such defects may contribute to the glaucomas observed in NPS and ARS patients. ^

A summative evaluation of a HIV/AIDS Early Childhood Care, Education and Development Teacher Training Workshop in Mongu, Zambia

Objective. To conduct a summative evaluation of an Early Childhood Care, Education and Development (ECCED) Teacher Training Workshop in Mongu, Zambia by assessing changes in knowledge, attitudes and intent to use the information. ^ Study design. A matched cohort survey design was used with additional qualitative data collected by structured observation of workshop sessions, daily facilitator and participant debriefs and participant interviews. ^ Results. Matching pre and post tests were completed by 27 individuals in addition to daily debriefs, structured workshop observation and participant interviews with 22% of the group. The participant population was predominantly female individuals aged 15-44 years old that had completed high school and additional post-secondary training, been teaching children aged 0 – 8 years for 2-5 years in the Western Province and received other HIV/AIDS and ECCED education. Pre-tests indicated a strong understanding of ECCED principles and misconceptions regarding HIV transmission, prevention and the disease's impact on early childhood development. The workshop was found to significantly increase the participants' knowledge of topics covered by the curriculum (paired t-test, N=27, p = 0.004, 95% CI 1.8, 8.6). Participants began with a more limited understanding of HIV/AIDS than ECCED, but the mean gain was much greater at 7.4 +/- 12.3 points. Significantly more participants believed at post-test that HIV/AIDS education should increase for future educators. The 77.8% of participants that increased their knowledge scores at post-test expressed significantly less fear of having a child with HIV/AIDS in the classroom (Independent Samples t-test, N= 27, p = 0.011). Overall participant fear decreased 15.5%. 92.6% and 88.9% of participants planned at post-test to respectively use and share the taught information in their daily professional lives and reported on innovative strategies to communicate with the community. ^ Conclusions. Teacher training workshops can significantly increase HIV/AIDS awareness and promote positive attitudes in educators working with children affected by HIV/AIDS. Using participant suggested teaching techniques such as poems and songs and translating the materials to the local language could assist future facilitators to both culturally and professionally relate to the workshop audience as well as increase participant capacity to share the information with the local community. ^