Anatomy, Cell Biology & Physiology Department Theses and Dissertations
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Item Investigating the Long-Term Outcomes of Service-Learning(2022-10) Schmalz, Naomi Alexandra; Byram, Jessica N.; Hoffman, Leslie A.; Organ, Jason M.; Palmer, Megan M.; Wisco, Jonathan J.Anatomy Academy (AA) is a service-learning program in which pre- and current health professional students (Mentors) work in pairs to teach anatomy, physiology, and nutrition to children in the community. The purpose of this study was to investigate the short- and long-term Mentor outcomes in personal, social, civic, academic, and professional domains. Former Mentors were invited to complete a survey of Likert-style and free response questions evaluating the perceived impact of their AA experience on: teaching skills, personal and interpersonal development, civic engagement, and academic and professional development. Follow-up interviews with a subset of survey respondents were performed. The survey was completed by 219 Mentors and 17 survey respondents were interviewed. Over 50% of former Mentors reported moderate or major impact of AA participation on elements of personal and interpersonal development (e.g., selfesteem [57.6%], altruism [67.9%], communication skills [60.1%], and ability to work with others [72.6%]) and community service participation (54.2%) that endures in the years after the program. Mentors who worked with low-income or Special needs populations reported unique impacts in personal, interpersonal, and civic domains. A majority of former Mentors agreed that AA participation helped them learn practical skills (76.3%) and factual knowledge (65.4%) relevant to the their careers, with several current health professionals reported that they regularly employ teaching and interpersonal skills learned while Mentors in their roles as physicians, nurses, or physician’s assistants. A majority of former Mentors reported that AA validated their choice to either pursue a healthcare career or not (59.7%), increased their confidence in performing professional tasks (64.7%), and helped shape their professional identity (58.9%). These results indicate that a health education-based service-learning program offers undergraduate, graduate, and professional students interested in or actively pursuing a healthcare career benefits across personal, interpersonal, civic, and professional domains that support their academic progress and preparation for professional practice. This study contributes much-needed evidence of the long-term student outcomes of service-learning to the literature, with a particular focus on how the pedagogy can supplement the education and professional development of pre- and current health professional students.Item Disabling the Transcription Factor Nmp4 from Osteogenic Precursors Enhances the Skeleton's Response to the Osteoporosis Drug Parathyroid Hormone(2022-08) Atkinson, Emily Grace; Bidwell, Joseph P.; Robling, Alexander G.; Plotkin, Lilian I.; Wallace, Joseph; Organ, Jason M.; Evans-Molina, CarmellaActivation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis. Yet, FDA-approved osteoanabolics, e.g., parathyroid hormone (PTH) have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice, exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues, therefore, to determine whether the suppression of PTHinduced bone formation is cell autonomous, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+ targeting mature osteoblasts and (iii) Dmp1Cre+ to disable Nmp4 in transitional osteocytes. Virgin female Cre+ and Cre- mice (10wks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1–34 at 30μg/kg, or vehicle for 4wks or 7wks. The skeletal response was assessed using dual-energy X-ray absorptiometry, microcomputed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl;Prx1Cre+ mice recapitulated the global Nmp4-/- skeletal phenotype in the femur, i.e., an enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared to their Nmp4fl/fl;Prx1Cre- controls. This was not observed in the spine, where Prx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl;BglapCre+) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl;Dmp1Cre+) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- MSPCs drive the enhanced response to PTH therapy, and Nmp4 has stage-specific effects on osteoanabolism.Item CaMKK2 Signaling in Metabolism and Skeletal Disease: A New Axis with Therapeutic Potential(2022-07) Williams, Justin N.; Sankar, Uma; Evans-Molina, Carmella; Bonewald, Lynda; Burr, David; Allen, MatthewType 2 diabetes mellitus (T2DM) is a growing problem globally and is associated with increased fracture risk and delayed bone healing. Novel approaches are needed in the treatment of T2DM and the resulting diabetic osteopathy. Recent studies highlight the role of bone as an endocrine organ producing factors that communicate with distant tissues to modulate systemic glucose metabolism. Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is a potent regulator of whole-body energy metabolism, inflammation, bone remodeling and fracture healing. Genetic ablation of CaMKK2 protects from diet-induced obesity, insulin resistance and inflammation, while enhancing pancreatic β cell survival and insulin secretion. Deletion or inhibition of CaMKK2 promotes bone accrual by stimulating osteoblast-mediated bone formation and suppressing osteoclast-mediated bone resorption; however, its specific role in osteocytes, the master regulator of bone remodeling remains unknown. Here we demonstrate that conditional deletion of CaMKK2 from osteocytes enhances bone mass in 3-month-old female, but not male mice, due to suppression of osteoclasts. Conditioned media experiments and proteomics analysis revealed that female osteocytes lacking CaMKK2 suppressed osteoclast formation and function through enhanced secretion of calpastatin, a potent inhibitor of calpains, which are calciumdependent cysteine proteases that support osteoclasts. Further, to determine if CaMKK2- deficient osteocytes regulate whole-body glucose homeostasis, we placed these mice on a high-fat diet (HFD) for a period of 16 weeks. Although the diet did not significantly impact bone mass or strength, we found that conditional deletion of CaMKK2 in osteocytes enhanced bone microarchitecture in 6-month-old male and female mice. We also observed that conditional deletion of CaMKK2 from osteocytes protected male and female mice from HFD-induced obesity and insulin insensitivity. Taken together, these findings highlight CaMKK2 as a potent regulator of osteocyte-mediated modulation of bone remodeling and whole-body energy metabolism.Item Molecular Studies on Calcium Oxalate Kidney Stones: A Window into the Pathogenesis of Nephrolithiasis(2022-05) Canela, Victor Hugo; Williams, James C.; Ashkar, Tarek M.; Blum, Janice S.; Sankar, UmaNephrolithiasis will affect one-in-eleven people, and more than half of those individuals will have stone recurrence within a decade of their first episode. Despite decades of biomedical research on nephrolithiasis and extraordinary advances in molecular and cell biology, the precise mechanisms of kidney stone formation are not fully understood. Currently, there are limited treatments or preventative measures for nephrolithiasis. Therefore, it is crucial to scrutinize kidney stones from a molecular and cell biology perspective to better understand its pathogenesis and pathophysiology; and to, hereafter, contribute to effective therapeutic targets and preventative strategies. Kidney stones are composed of an admixture of crystal aggregated material and an organic matrix. 80% of all kidney stones are composed of calcium oxalate (CaOx) and half of all CaOx patients grow their stones on to Randall’s plaques (RP). RP are interstitial calcium phosphate mineral deposits in the renal papilla. Thus, we developed and optimized methodologies to directly interrogate CaOx stones. CaOx stones were demineralized, sectioned, and imaged by microscopy, utilizing micro CT for precise orientation. Laser microdissection (LMD) of specific regions of stone matrix analyzed by proteomics revealed various proteins involved in inflammation and the immune response. Analyses on jackstone calculi, having arm protrusions that extend out from the body of the stone, revealed that they are a rare subtype of CaOx stone formation. Micro CT analyses on 98 jackstones showed a radiolucent, organic-rich core in the arm protrusions. Fluorescence imaging on RP stones showed consistent differences in autofluorescence patterns between RP and CaOx overgrowth regions. Moreover, cell nuclei were discovered with preserved morphology in RP regions, along with variable expressions of vimentin and CD45. In comparing spatial transcriptomic expression of reference and CaOx kidney papillae, CaOx patients differentially expressed genes associated with pathways of immune cell activation, reactive oxygen damage and injury, extracellular remodeling, and ossification. Our findings provide novel methodologies to better understand the role of molecules and cells in CaOx stone matrix. Several of the proteins and cells identified in these studies may serve as potential biomarkers, and future therapeutic targets in preventing kidney stone disease.Item Neurodegeneration Risk Factor TREM2 R47H Mutation Causes Distinct Sex- and Age- Dependent Musculoskeletal Phenotype(2022-05) Essex, Alyson Lola; Plotkin, Lillian I.; Bonetto, Andrea; Allen, Matthew; Landreth, Gary E.Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone has been proposed as a link between brain and bone disease. Previous studies identified an AD-associated mutation (R47H) which is known to confer an increased risk for developing AD. In these studies, we used a heterozygous model of the TREM2 R47H variant (TREM2R47H/+), which does not exhibit cognitive defects, as a translational model of genetic risk factors that contribute to AD, and investigated whether alterations to TREM2 signaling could also contribute to bone and skeletal muscle loss, independently of central nervous system defects. Our study found that female TREM2R47H/+ animals experience bone loss in the femoral mid-diaphysis between 4 and 13 months of age as measured by microCT, which stalls out by 20 months of age. Female TREM2R47H/+ animals also experience significant decreases in the mechanical and material properties of the femur measured by three-point bending at 13 months of age, but not at 4 or 20 months. Interestingly, male TREM2R47H/+ animals do not demonstrate any discernable differences in bone geometry or strength until 20 months of age, where we observed slight changes in the bone volume and material properties of male TREM2R47H/+ bones. Ex vivo osteoclast differentiation assays demonstrate that only male TREM2R47H/+ osteoclasts differentiate more after 7 days with osteoclast differentiation factors compared to WT, but qPCR follow-up showed sexdependent differences in intracellular signaling. However, bone is not the only musculoskeletal tissue affected by the TREM2 R47H variant. Skeletal muscle strength measured by both in vivo plantar flexion and ex vivo contractility of the soleus is increased and body composition is altered in female TREM2R47H/+ mice compared to WT, and this is not likely due to bone-muscle crosstalk. These studies suggests that TREM2 R47H expression in the bone and skeletal muscle are likely impacting each tissue independently. These data demonstrate that AD-associated variants in TREM2 can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of the presence of central neuropathology.Item Approaches to Improve the Structure and Function of the Skeleton in Chronic Kidney Disease(2022-03) Swallow, Elizabeth Anne; Allen, Matthew R.; McNulty, Margaret A.; Moe, Sharon M.; Wallace, Joseph M.Chronic kidney disease (CKD) currently affects ~37 million Americans and causes substantially increased risk of skeletal fracture and fracture-related mortality. Current methods to treat CKD-related bone loss remain alarmingly ineffective. Skeletal fragility in CKD is predominately driven by deteriorations in cortical bone, highlighted by significant cortical porosity development. It is hypothesized that cortical porosity is largely driven by chronically high levels of parathyroid hormone (PTH), which alters the balance of bone remodeling in favor of rampant osteoclast activity and bone resorption. Restricting cortical bone deterioration and the development of cortical pores is likely essential to improve CKD patients’ bone health and reduce their fracture risk. The goal of this series of studies was to answer the following key questions: (1) to what degree do bisphosphonates, an approved pharmacological agent used in metabolic bone disease, accumulate in the skeleton of animals with CKD; (2) can smaller and more frequent doses of bisphosphonates alter skeletal accumulation and improve cortical architecture and the mechanical integrity of bone; (3) can non-bisphosphonate pharmacological interventions more specifically affect cortical bone deterioration. Utilizing epi-fluorescence and two-photon microscopy, our results show that bisphosphonates accumulate more in rats with renal impairment and fractionating bisphosphonates lowered skeletal accumulation irrespective of disease state. Further, studies in both rat and mouse models of CKD demonstrated different bisphosphonate treatments alone do not recover declines in cortical microarchitecture or mechanical properties in CKD. These findings demonstrate that a single intervention is not sufficient in managing CKD-induced bone alterations. Utilizing individual pore tracking analysis, we demonstrated cortical pores can be modulated with therapeutic interventions and can infill, despite the presence of CKD. Potent suppression of PTH led to significant pore infilling while more subtle reductions in PTH, via a calcimimetic, had less striking effects on bone. Calcimimetics mitigated cortical microarchitecture deterioration and reduced the rate of cortical pore expansion. Overall, these findings highlight the importance of PTH management for treating cortical deterioration in CKD. Although bisphosphonates can be utilized in ways that reduce skeletal accumulation, they appear to need co-therapies to reduce skeletal fragility associated with CKD.Item Three-Dimensional Visualization Technology in the Medical Curriculum: Exploring Faculty Use in Preclinical, Clinical, and Postgraduate Anatomy Education(2021-01) Helbling, Shannon Amara; Torbeck, Laura J.; Byram, Jessica N.; Deane, Andrew S.; Nelson Laird, Thomas F.Background: The advancement of three-dimensional visualization technology provides exciting new opportunities in medical education, including new methods for teaching complex anatomical relationships and promising tools for the training of postgraduate physicians. Information on how faculty use three-dimensional visualization technology for anatomy education is essential for informed discussions surrounding their effectiveness as a teaching tool and use in the medical curriculum, yet the current literature lacks necessary contextual details on how faculty integrate these technologies into actual medical curricula. Methods: Fifteen medical educators from North American medical schools and teaching hospitals completed semi-structured interviews and discussed how they use three-dimensional visualization technology for teaching in preclinical courses, clinical clerkships, and postgraduate programs. Transcripts were analyzed using the constant comparative method and resulting themes were used to inform the creation of a questionnaire. Results: The resulting themes of analysis were organized according to a curricular framework that describes how faculty use these technologies as an instructional resource and how this use is related to the purposes, content, sequence, instructional processes and evaluation of medical curricula. The results demonstrate how three-dimensional visualization technology is being is implemented in a variety of ways in the curriculum and revealed numerous similarities of use across the levels of medical education. Analyses revealed minimal use of three-dimensional visualization technology for assessment and indicated faculty face significant challenges in designing such assessment. Conclusions: Results suggest continuing assessment of the effectiveness of these technologies as a teaching tool needs to encompass broader aspects of use, such as those described in this study. Additionally, results showing similarities of use across levels suggest that educators and administrators should consider how threedimensional visualization technology can be thoughtfully integrated to address the changing needs of learners as they progress through medical education. Findings also suggest that administrators who want to support the integration of three-dimensional visualization technology into the curriculum need to provide adequate support and training to help faculty overcome time limitations and difficulties designing assessment methods.Item A Foucauldian Archaeology of Modern Medical Discourse(2020-09) Azim, Homaira M.; Scheurich, James J.; Brokaw, James J.; O'Loughlin, Valerie D.; Byram, Jessica N.Medical education researchers have long been interested in understanding medical professional identity formation and its implications for the healthcare system. Various theories have been proposed to explain identity formation. Among them, Foucault’s discourse theory maintains that it is the discourse of medicine that constitutes medical professional identities. This study deployed a Foucauldian archaeological methodology to analyze the structure of modern medical discourse and establish links between discourse and professional identity formation in medical students. A total of forty-six medical students at Indiana University School of Medicine participated in either individual or focus group interviews. Direct observation of the clinical and educational settings was also performed, which resulted in additional textual data in the form of fieldnotes. Archaeological analysis of discourse was undertaken in three levels of the statements, the discursive elements, and the discursive rules and relations. Results entailed a detailed depiction of the structure of medical discourse including discursive objects and modes of enunciation, discursive concepts, and theoretical strategies related to each object. Discursive objects are things that are talked about in modern medical discourse. This study identified four discursive objects as disease and treatment, the doctor, the human body, and the sick person. Modes of enunciation are the different ways in which people talk about objects of medicine, whereas concepts consist of the notions people draw from when talking about objects of medicine. Theoretical strategies indicate certain positions that people take in relation to the objects of medicine. Rules of formation and conditions of existence for each discursive element were also established. Since Identities are entrenched through language and interaction, developing a systematic understanding of the structure of medical discourse will shed new light on medical professional identity formation. Results of this study also have profound implications for teaching professionalism and medical humanities in medical curricula. Furthermore, as a research methodology used for the first time in medical education, archaeology not only opens new territories to be explored by future research, it also provides an entirely new way to look at them.Item The Role of Wnt Signaling in Bone Mechanotransduction(2019-11) Bullock, Whitney Ann; Robling, Alexander; Bidwell, Joseph; Plotkin, Lilian; Sankar, Uma; White, KennethThe aging US population is experiencing a growing incidence of osteoporosis, characterized by increased fracture risk and low bone mass. In skeletal tissue, canonical Wnt signaling is a critical regulator of bone mass, and dysregulation of the Wnt pathway has been implicated in numerous skeletal displasias. Some components of the Wnt signaling pathway have a clear role in bone homeostasis, particularly in the response of bone to altered mechanical environment. Other pathway components are more poorly defined. One important intracellular signal transduction node in the Wnt cascade is β- catenin, which modulates gene expression and cell-cell junctions, among other functions. During periods of disuse, β-catenin is degraded, leading to inhibition of Wnt targets. Here, I characterize the role of β-catenin in bone during a disuse challenge, using a genetic mouse model expressing an inducible constitively-active mutant form of β- catenin in the osteocyte population. I hypothesize that prevention of β-catenin degradation during disuse will prevent the bone wasting effects of mechanodeprivation. As a second goal, I focus on upstream (membrane-bound) modulation of Wnt. Here, I investigate the low-density lipoprotein receptor-related receptor 4 (Lrp4), in the regulation of bone mass and mechanotransduction. I generated an Lrp4 knockin mouse model harboring a missense mutation found among human patients with abnormally high bone mass. I hypothesize that the mutation compromises sclerostin action on bone cells. Understanding how each of these components of the Wnt signaling pathway interact, may lead to novel therapeutic targets for treatment of bone diseases.Item Neural Repair by Enhancing Endogenous Hippocampal Neurogenesis Following Traumatic Brain Injury(2019-10) Wang, Xiaoting; Xu, Xiao-Ming; Chen, Jinhui; Jones, Kathryn J.; Meyer, Jason; Pollok, Karen E.Traumatic brain injury (TBI) is a critical public health issue in the United States, affecting about 2.8 million people annually. Extensive cell death and neural degeneration directly and diffusively caused by the initial mechanical insult results in a wide range of neurological complications post-trauma. Learning and memory dysfunction is one of the most common complains. Hippocampal neuronal loss, together with other mechanisms, largely contributes to learning and memory impairment as well as other cognitive dysfunctions post-trauma. To date, no FDA-approved drug is available to target cell death or improve learning and memory following TBI. It is of great interest to develop alternative approaches targeting neural repair instead. Neural stem/progenitor cells (NSCs) in the adult hippocampus undergo life-long neurogenesis supporting learning and memory functions, thus hold great promise for post-traumatic neuronal replacement. The previous studies demonstrated that TBI transiently increase NSC proliferation. However, it is debated on whether TBI affects neurogenesis. The mechanism of TBI-enhanced NSC proliferation remains elusive. In the current studies, I have investigated post-traumatic neurogenesis after different injury severities, evaluated integration of post-injury born neurons, illustrated a molecular mechanism mediating TBI-enhanced NSC proliferation, proposed a de novo state of NSCs, and tested effects of a pharmacological approach on spatial learning and memory function recovery. My results demonstrated that post-traumatic neurogenesis is affected by injury severities, partially explained the pre-existing inconsistency among works from different groups. Post-injury born neurons integrate in neural network and receive local and distal inputs. TBI promotes functional recruitment of post-injury born neurons into neural circuits. Mechanistically, mechanistic target of rapamycin (mTOR) pathway is required primarily for TBI-enhanced NSC proliferation; NSCs feature a de novo alert state, in which NSCs are reversibly released from quiescence and primed for proliferation. Furthermore, my data demonstrated a beneficial role of ketamine in improving post-traumatic spatial learning possibly by activating mTOR signal in NSCs and/or promoting neuronal activity of post-injury born neurons. Together, my data support the feasibility of neurogenesis mediated neuronal replacement, provide a target for enhancing post-traumatic NSC proliferation and subsequent neurogenesis, and prove a potential pharmacological approach benefiting post-traumatic functional recovery in learning and memory.