Anatomy, Cell Biology & Physiology Department Theses and Dissertations

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    What's My Role Again? Cultivating Interprofessionalism, Role Knowlege, and Role Clarity Through Case-Based Learning
    (2024-03) Herriott, Hannah Laine; McNulty, Margaret A.; Byram, Jessica N.; Agosto, Elizabeth R.; Deane, Andrew S.; Rebman, Rebecca; Scheurich, Jim J.
    As healthcare has shifted away from physician-centered practice, in favor of a patient-centered model, the importance of interprofessional team-based practice was recognized. Early exposure, practice working through clinical cases in teams, and learning each profession’s roles are essential determinants of successful interprofessional collaboration (IPC). Although interprofessional role comprehension is widely accepted as one of four core competencies of interprofessional education (IPE) and lack of role comprehension is associated with medical errors; literature measuring said construct is lacking. Role knowledge and clarity are two crucial skills that encompass identifying the roles and limitations of various professions’ scopes of practice and discerning which professional is best equipped to undertake a task in a particular situation. The present study investigated a novel IPE intervention employing role centered, small group casebased learning (CBL) sessions, integrated throughout an anatomy course for firstsemester occupational therapy (OTD), physical therapy (DPT), and physician assistant (MPAS) students. Additionally, fourth-year medical (MD) students participated in the IPE intervention by serving as near-peer facilitators for each of the small groups. A sequential explanatory, mixed methods design was employed to examine participants’: 1) acquisition of role knowledge, 2) demonstration of role clarity, 3) views of IPC after engaging in CBL sessions, and 4) perceptions of the intervention itself. The present study addressed the previously mentioned gap in the literature by exploring a CBL intervention’s influence on role comprehension (a previously unquantified aspect of IPE), in addition to their IPE-related perceptions. The aforementioned CBL intervention effectively improved role knowledge and clarity when implemented in-person; however, no significant changes were demonstrated in the virtual cohort. While many positive perceptions of the intervention and IPE experience were found, some misconceptions about professions and inhibitory power dynamics were also identified. Despite the latter findings, the CBL intervention examined in this study can serve as an effective model for cultivating IPE through enhanced role knowledge and clarity among health professional students.
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    Identification of a Biofilm-Inducible Wound Macrophage Subset Characterized by Unique MARCO Expression: Significance in Chronic Wound Infection
    (2023-08) Das Ghatak, Piya; Roy, Sashwati; Gordillo, Gayle M.; Khanna, Savita; Xuan, Yi
    Staphylococcus aureus (SA) biofilm infection, a common occurrence in human chronic wounds, exhibits sustained inflammation resulting in wound chronicity. Current studies claim that SA biofilm transforms host macrophages (m) towards an alternatively activated phenotype that suppresses the inflammatory response. Given a key role of m in wound healing, we investigate the phenotype and function of wound m (wm) in SA biofilm infection. To address the biofilm component of SA pathogenicity, two isogenic mutant strains of wild-type SA (USA300) were used that possess varying degrees of biofilm-forming ability. The strains, USA300::sarA (ΔsarA) and USA300::rexB (ΔrexB), have hypo- and hyper biofilm-forming ability, respectively. Mechanistic studies were performed on peripheral blood monocytes subjected to conditioned media (CM) derived from ΔsarA and ΔrexB biofilm cultures. scRNAseq and bulk RNAseq studies revealed presence of a unique m subtype that displays a distinct gene expression signature when exposed to CM derived from ΔrexB mutant as compared to CM from ΔsarA mutant. We identified macrophage receptors with collagenous structure (MARCO) as one of the highly abundant uniquely expressed biofilm-responsive genes in wm. MARCO, a member of the scavenger receptor, belongs to a larger group of pattern recognition receptors primarily involved in immunosurveillance and plays important roles in host defense and apoptotic cell clearance activities. scRNAseq and immunohistochemistry from human chronic biofilm-infected wounds demonstrated abundance of MARCO expression on wm. Significant upregulation of MARCO was observed in ΔrexB CM treated m in a dose and time-dependent manner as compared to ΔsarA CM treated m. Knockdown of biofilm-induced MARCO in m resulted in dysregulated m functions and inflammatory responses, suggesting its critical significance in biofilm infection. Immunohistochemistry studies in SA biofilm-infected porcine wounds validated the in vitro findings. In summary, this study identifies a SA biofilm inducible m receptor that has a significant role in wound chronicity.
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    The Effects of FNDC5/Irisin on Osteocyte Functions
    (2023-07) Shimonty, Anika; Plotkin, Lilian I.; Bonewald, Lynda F.; Brault, Jeff; Robling, Alexander G.; White, Kenneth E.
    Irisin is a myokine generated when Fibronectin type III Domain Containing protein 5 (FNDC5) is proteolytically cleaved during exercise. While irisin has been shown to be beneficial in the functions of the brain, heart, and adipose tissue, its effect on bone cells remains contradictory. Osteocytes are the most abundant and longest-living bone cells, with different transcriptomes based on sex and age. One of the major functions of osteocytes is osteocytic osteolysis, the removal of their perilacunar matrix. A previous study showed that irisin deletion protects bone from ovariectomy-induced loss due to less osteoclastic resorption and less osteocytic osteolysis. Therefore, we hypothesized that FNDC5/irisin modulates the osteocyte function of osteocytic osteolysis in a sex-dependent manner. Under normal conditions, there was no difference in bone parameters between wildtype and FNDC5-null adult female mice starting from 5 to 20 months of age. However, 5-month-old null male mice had higher bone mass, but weaker bone compared to wildtype males, which persisted up to 20 months. Both 5-month-old female and male null mice had significantly lower TRAP-positive osteocytes, suggesting a role of irisin in priming the osteocytes for bone resorption. Osteocytes from female wildtype mice show higher lacunar area and upregulated resorption genes compared to wildtype males. However, null females and null males do not have significant differences in the lacunar area or resorption genes. Under calcium-deficient conditions, both 5 and 18-month-old female null mice lost less bone compared to their wildtype counterparts. In contrast, both 5 and 18-month-old null male mice lost more bone than age-matched wildtype males. Additionally, the percentage of bone loss was greater in the aged null male mice compared to 5-month-old null males. In summary, in female osteocytes, irisin works to release calcium from bone during lactation, ensuring offspring survival; however, with aging and hypocalcemia, irisin exerts a negative effect on bone mass. In contrast, irisin works to maintain bone mass and strength by modulating male osteocyte function. This study is the first to demonstrate a sex and age-specific irisin effect on the osteocyte function of osteocytic osteolysis, which has implications for the development of osteoporosis treatment.
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    Identification of Extracellular Wnt Inhibitors for Novel Synergistic Anabolic Action on the Skeleton
    (2023-07) Choi, Roy Byung-Jun; Robling, Alexander; Bidwell, Joseph; Thompson, William; Pavalko, Fredrick; White, Kenneth
    The Wnt pathway has been an obvious target for designing skeletal therapies, mainly based on the high-bone-mass phenotypes in patients with activating mutations in the Wnt co-receptors Lrp5 and Lrp6, or with inactivating mutations in the Lrp5/6 inhibitor Sost. An attractive property of the Wnt pathway is that it stimulates anabolic action in bone cells. The powerful anabolic potential of manipulating Wnt signaling in bone has been demonstrated by the recent FDA approval of sclerostin antibody (Scl-mAb) EvenityTM (Romosozumab) for the treatment of osteoporosis. However, an increased risk of cardiovascular events was reported, triggering the FDA to issue a ‘black box warning’ requirement for romosozumab. One potential solution to lower the risk of adverse events is to reduce the medication dose. Reducing the dose of Scl-mAb, while maintaining the anabolic potential of the drug, will likely provide a safer and more cost-effective strategy to harness Wnt for fracture prevention. Here, we found that dual inhibition of sclerostin and Dkk1 produced extremely potent synergistic bone anabolic effects in the cancellous compartment, using both genetic and pharmacological models. Further, much lower total doses of dual antibody treatment, given at optimized proportions, generated equivalent trabecular bone anabolic effects as Scl-mAb alone. On the contrary, we looked for other candidates that might potentiate the cortical effects of sclerostin inhibition. We found that either Sostdc1 or Notum deletion results in high bone mass, specifically in the cortical compartment, with little to no effect in the cancellous compartment. Inhibition/deletion of Sostdc1 or Notum alone had no detectable effects (Sostdc1) or mild (Notum) cortical effects, but suppression of either target while co-supressing/deleting Sost improved bone mass disproportionately. In summary, these findings highlight the potential therapeutic role that combinational inhibition of different Wnt inhibitors generates, resulting in synergistic bone anabolic action in different/select skeletal compartments.
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    Metacognition in Anatomical Sciences Education
    (2023-06) Cale, Andrew Stephen; McNulty, Margaret A.; Byram, Jessica N.; Hoffman, Leslie A.; Longtin, Krista; Palmer, Megan; Williams, James C.
    Metacognition, the ability to self-regulate one’s learning and performance, is well-known to provide numerous academic and professional benefits for students, educators, and clinicians. However, few studies have studied metacognition specifically in the context of anatomical sciences education. Therefore, the overarching purpose of this dissertation was to explore the metacognition of students and educators who are learning and teaching the anatomical sciences. This dissertation investigated the metacognition of allied health students (physical therapy, physician assistant, and occupational therapy; n=109), first-year medical students (n=1802), and anatomy educators (faculty, associate instructors, and teaching assistants; n=13) in anatomy courses through three multiple-methods studies. Quantitative data were collected using assessment data and either the Metacognition Awareness Inventory (MAI), Practice-Based Learning and Improvement (PBLI) assignments, or Teacher Metacognition Inventory (TMI). These data were then analyzed using the appropriate descriptive and inferential statistics. Qualitative data were also collected through reflective writing activities (e.g., online discussion boards or reflective journals) and analyzed using thematic or framework analysis. Overall, both students and educators improved their metacognition across a semester of either learning or teaching anatomy, with certain subgroups demonstrating greater metacognitive ability or growth than others. Higher performing allied health and medical students were both more accurate at predicting their exam performances compared to their lower performing peers. Faculty also demonstrated the greatest teaching-specific metacognition, though teaching assistants exhibited the greatest growth in their teaching-specific metacognition. These improvements were primarily in their reflective ability and awareness of personal strengths and weaknesses as teachers. Several notable themes relating to metacognition were also identified such as student willingness to monitor learning diminishing over time due to competing academic or professional commitments. Additionally, novice educators were more inwardly-focused on personal traits and content mastery, whereas experienced educators were more outwardly-focused on interpersonal factors (e.g., student rapport and inclusive language). These insights into the metacognition of both students and educators can inform how to best support and improve teaching and learning in the anatomical sciences. Given the significance of metacognition, it may be beneficial to incorporate educational activities that can support the metacognition of both students and educators, simultaneously.
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    Interactions Between Aging and Chronic Kidney Disease on the Skeleton
    (2023-05) Tippen, Samantha P.; Allen, Matthew R.; White, Kenneth E.; Moe, Sharon M.; Wallace, Joseph M.
    In the US, 15% of adults have chronic kidney disease (CKD). While CKD occurs across all ages, the prevalence is highest in the aged, with ~40% of individuals over age 65 having some form of CKD. CKD and aging are each independently associated with higher fracture risk, and thus overlaying CKD in the aging population presents an additive fracture risk. Cortical porosity is a central tenet underlying skeletal fragility and occurs in CKD and aging. Previous research on cortical porosity has focused on preventing pore formation, while research on pore reversal (infilling) is lacking. Pore infilling is dependent on proper osteoblast function, and previous research has shown that infilling is possible in young mice. However, it is unclear whether infilling is possible in aging mice due to aging-associated osteoblast dysfunction. Therefore, we proposed that aging animals with CKD may require both suppression of CKD-induced elevations in parathyroid hormone (PTH) and anabolic therapy to infill cortical pores. Romosozumab, a humanized monoclonal sclerostin antibody, uses PTH-independent mechanisms to increase osteoblast activity, making it an attractive therapeutic for CKD. CKD was induced by feeding aging (78-week) male mice 0.2% adenine for six weeks followed by two weeks of maintenance on control diet for a total study duration of eight weeks of CKD; mice were then treated with calcium water, romosozumab, or the combination and their effectiveness in improving skeletal quantity and quality was evaluated. Romosozumab treatment was associated with higher trabecular bone volume, lower cortical porosity, and higher mechanical properties compared to control animals. Combination treatment also resulted in benefits to trabecular bone volume and mechanical properties. These results demonstrate that both romosozumab alone and when combined with PTH suppression can be effective at improving bone microarchitecture and mechanical properties in aged individuals with CKD who are at high risk of fracture.
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    Experiences of Residency Program Directors in Their Roles: Exploring Well-Being Through Burnout and Engagement
    (2022-11) Robertson, Kyle A.; Byram, Jessica N.; Hayes, Cleveland; Agosto, Elizabeth R.; McNulty, Margaret A.; Organ, Jason M.
    Recent literature on well-being of physicians in general, and residency program directors (PD) specifically, has demonstrated those meeting the criteria of burnout reaching almost 50% in physicians, and 20-30% in PDs. However, few studies have explored engagement, or the positive or meaningful aspects, in physicians and no studies have explored engagement in the PD and Assistant PD community. Therefore, this study employed a qualitative approach to explore the experiences of PDs and APDs as they encountered burnout, engagement, and every combination in between through their multifaceted, roles, responsibilities, and tasks embedded in their institutional context and personal lives. Phase 1 participants (n=3) included two PDs and one APD from Indiana University School of Medicine (IUSM). Participants in Phase 1 took part in three semi-structured interviews at 6-month intervals, and direct observations in their clinical, administrative, and education roles. Phase 2 participants (n=5) were PDs from IUSM who completed a single semi-structured interview based on preliminary results and exploration of Phase 1 participants’ experiences. Interviews and field notes from observations were analyzed using inductive thematic analysis, followed by a deductive application of Job Demands-Resources (JD-R) theory. Document analysis was incorporated to add context, understanding, and a rich description of the participants’ experiences. This study found multiple sub-themes situated within four major themes: It Takes a Village, Integration of the “Hats” They Wear, Motivation and the Meaning of Their Career, and Coping. Exploring the sub-themes to JD-R theory allowed contextualization of how job demands, job resources, personal resources, absence of resources, job crafting, recovery, self-undermining, and strain, interact to add context, nuance, and broader conceptualization of how PD and APD experienced their multifaceted roles. This study provides a rich description of the experiences of PDs and APDs embedded in their social context of roles, tasks, and responsibilities. These results indicated that understanding how the individual experiences their job demands as they interact with their experiences of job and personal resources, and how the individual proactively engages with their environment through job crafting and recovery enables for a nuanced appreciation of engagement and burnout.
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    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.
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    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, Carmella
    Activation 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.
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    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, Matthew
    Type 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.