Full text for dissertations and theses included in this collection dates back to 2011. For older dissertations, check the library’s catalog CatalogUSMAI or Dissertations and Theses database.

Recent Submissions

  • Developing Therapies to Overcome Immunosuppressive Myeloid Cells in the Tumor Microenvironment

    Ceradoy, Justine Anne; Davila, Eduardo, Ph.D.
    Myeloid cells in the tumor microenvironment represent significant barriers to the development of successful cancer immunotherapies. A multi-kinase inhibitor, Regorafenib (Reg), and a DNA-PK inhibitor, NU7441 (NU) were shown in a previous study to reduce expression of immunoinhibitory proteins in adaptive immune cells while increasing stimulatory MHC-I on cancer cells. In this study, we explored whether these drugs could reverse the suppressive activity of myeloid-derived suppressor cells (MDSCs) and alternatively activated macrophages. To test this idea, we used splenocytes from tumor-bearing mice and a human monocytic cell line differentiated into suppressive macrophages and assessed Arginase activity, their ability to suppress effector T cells, and mRNA expression of immunosuppressive and activating markers. We showed that Reg/NU decrease arginase activity and increase immunoactivating markers. These data demonstrate that treatment of suppressive myeloid cells with Reg/NU confers a less suppressive phenotype and leads to the generation of a more activating phenotype.
  • The Role of LRP1 in Inflammation and Vasculopathies

    Au, Dianaly; Strickland, Dudley K.; Catania, Selen M.; 0000-0002-8797-833X
    The prevalence of overweight and obesity and a growing aging population has resulted in higher incidences of type 2 diabetes (T2D) and cardiovascular disease (CVD). Although risk factors for T2D and CVD have been known for decades, the molecular mechanisms involved in the pathophysiology of these multifaceted diseases and their interrelationship remain unclear. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundantly expressed in several tissues and possesses diverse biological functions, including chylomicron remnant clearance, involvement in insulin signaling and glucose homeostasis, modulation of the inflammatory response, atheroprotection, and maintenance of vascular integrity. We hypothesized that LRP1 may serve as a molecular link between metabolic processes and CVD development and employed tissue-specific LRP1 knockout mouse models to identify potential molecular mechanisms. Studies performed on macrophage-specific LRP1-deficient mice generated on an LDL receptor knockout background (LDLR-/-; macLRP1-/-) and challenged with a Western diet revealed that LRP1 expression in macrophages promotes hepatic inflammation and the development of glucose intolerance and insulin resistance by modulating Wnt signaling. Interestingly, studies performed on smooth muscle-specific LRP1-deficient (smLRP1-/-) mice identified a novel and critical role for LRP1 in modulating vascular smooth muscle cell (VSMC) contraction by regulating calcium signaling events. These results uncovered a potential mechanism by which LRP1 protects against aneurysm development. Studies performed on VSMCs isolated from an aneurysm patient, who also contains two heterozygous missense mutations within the LRP1 gene, showed dysregulation of the TGF-β and p53 signaling pathways. These results provide further biological evidence supporting the association of LRP1 with aortic aneurysms. The role of LRP1 in vascular remodeling was also investigated by inducing remodeling in smLRP1-/- mice using the carotid artery ligation model. Our results suggest that LRP1 protects against excessive vascular remodeling by modulating angiotensin II-mediated signaling. Taken together, this work reveals the complex roles of LRP1 in various tissues and provides evidence supporting LRP1 as a critical molecule that integrates metabolic processes with inflammation and vascular disease.
  • Immunosuppressive Drug Regimens that May Help Improve Survival and Reduce the Risks of Rejection, Infection, and Malignancy after a Lung Transplant

    Wijesinha, Marniker; Hirshon, Jon Mark; 0000-0002-6609-8456
    Background: Median survival among lung transplant recipients is below 6 years, and there is minimal knowledge regarding modifiable factors that may help improve long-term survival. Identifying induction and maintenance immunosuppressive regimens associated with optimal survival can potentially improve outcomes. Methods: We classified lung transplant recipients in the United States from 2003-2016 according to their induction and prophylactic cell cycle inhibitor maintenance therapies, within a tacrolimus-based regimen. We compared the different therapies via multivariable Cox Proportional Hazards models, generating adjusted hazard ratios for death, rejection, infection, and malignancy, the latter three of which utilized semi-competing risks methods. Since prophylactic sirolimus initiation is delayed by up to 1 year post-transplant, adjustments were made to avoid immortal time bias. Multiple imputation was utilized to handle missing data. Results: Sirolimus had the best survival among cell cycle inhibitor maintenance therapies: adjusted Hazard Ratio (HR)=0.71, p=0.003, compared to mycophenolate mofetil [MMF]; chronic rejection incidence was also reduced with sirolimus (HR=0.75, p=0.005). Azathioprine also had slightly better survival than MMF (HR=0.92, p=0.05), and reduced infection incidence (HR=0.62, p<0.0001). Among induction therapies, equine ATG had the best survival: HR=0.79, p=0.003, compared to no induction, as well as reduced rejection (HR=0.75, p=0.02) and infection (HR=0.57, p=0.008) incidence. The combination of induction and maintenance therapies associated with the most favorable survival was sirolimus + tacrolimus maintenance with no induction; HR=0.48, p=0.002 compared to MMF + tacrolimus with induction, and HR=0.41, p<0.0001 compared to MMF + tacrolimus with no induction. Conclusions: Sirolimus initiated in the first year within a tacrolimus-based regimen may significantly improve long-term survival compared to MMF in lung transplant recipients. Out of all combinations of maintenance and induction therapies studied, sirolimus + tacrolimus maintenance with no induction therapy was associated with the best survival. In patients whom sirolimus cannot be utilized for any reason, azathioprine may modestly improve survival compared to MMF. Additional long-term studies in lung transplantation are needed to confirm these findings.
  • Histomorphometric Analysis of Mouse Growth Plate during Maturation and Senescence.

    Wilson, Kimberly; Enomoto-Iwamoto, Motomi
    The growth plate (GP) is cartilage tissue at both ends of long bones and plays an essential role for long bone formation and growth. GP changes in overall height, width, zone proportion, cell density and size during skeletal growth and becomes arrested upon skeletal maturation. There is limited information on GP microscopic changes. To establish GP histomorphometric parameters changes throughout skeletal maturation, the tibia of C57Bl/6J mice were evaluated from neonatal to young adult stages. GP histology in the proximal tibia was analyzed regarding total and zone height, width and area. The results showed unique GP changes in width, height and area during growth. The parameters also showed strong correlation with growth rate of the tibia. We studied GP proliferation activity and also found its correlation with tibia growth rate. The results of this study provide groundwork that will aid skeletal research on understanding of GP changes during bone growth.
  • Microglia Regulation of Sexually Dimorphic Amygdala Development

    VanRyzin, Jonathan; McCarthy, Margaret M., 1958-; 0000-0003-2990-6765
    Sex differences in the brain are established early in development and generate lasting changes in brain and behavior, a process known as sexual differentiation. Sexual differentiation of the amygdala produces a highly conserved sex difference in juvenile rough and tumble play behavior; however, the mechanisms underlying this sex difference are unknown. Here, we report that microglia, resident immune cells of the brain, actively shape the sexual differentiation of the amygdala. We found that microglia are more phagocytic in the amygdala of males from postnatal day 0 and 4, during which they also have a higher endocannabinoid (eCB) tone. Administering a masculinizing dose of testosterone to increase the eCB tone in females, or cannabinoid receptor agonists to female pups increased the number of phagocytic microglia and correspondingly decreased the number of newborn cells. Given these data, we hypothesized that microglia control the number of postnatally-born cells in the developing rat amygdala by phagocytosing newborn cells in an endocannabinoid-dependent manner. We found that these phagocytic microglia engulf newly proliferated cells, which are enriched for complement proteins. To directly implicate microglia phagocytosis, we used a function-blocking antibody against the complement receptor 3 (CR3) to prevent phagocytosis. Anti-CR3 antibody treatment increased the number of BrdU+ cells only in males, demonstrating that newborn cells can survive if phagocytosis is prevented. Moreover, administration of cannabinoid receptor antagonists to male pups occluded the effects of phagocytic blockade, suggesting that newborn cell phagocytosis was dependent on the developing ECB tone. Finally, to understand how these early life events manifest changes in the composition of the amygdala, we used a fate mapping approach to phenotype postnatally-born cells at the juvenile age. Our analysis found that the majority of newborn cells differentiated into astrocytes, which were overall higher in density in the posterodorsal region of the medial amygdala, an amygdalar nucleus essential to the integration of social stimuli. Together, these data indicate that sex differences in the local environment of the developing amygdala instruct microglia to actively phagocytose newborn cells as a means to sculpt later life architecture of the amygdala and produce sex differences in social play.
  • Effects of Alcohol Exposure during Developmental Phases on Brain Functional Connectivity

    Tang, Shiyu; Medina, Alexandre E.; Gullipalli, Rao P.
    Fetal alcohol spectrum disorders (FASD) is one of the most common causes of mental disability in the world. There is growing evidence that developmental alcohol exposure can cause reduced cognitive flexibility and alter multisensory processing, suggesting impairments in fronto-striatal and multisensory integrative cortical areas. The central hypothesis is that developmental alcohol exposure leads to impaired connectivity within these areas, and will result in reorganization of the large-scale brain network. We tested this hypothesis with two animal models that are known to exhibit abnormities in cognitive flexibility and sensory processing: (1) a rat FASD model with alcohol exposure from gestational day 6 to 20, similar to human gestation from the first to the late second trimester; (2) a ferret FASD model with alcohol exposure from postnatal day 10 to 30, similar to the third trimester of gestation in humans. These two models mimic alcohol exposure during early and late fetal stage humans. Our study revealed reduced resting-state functional connectivity within fronto-striatal circuit and a visual-tactile circuit in young adult animals with alcohol exposure during early fetal stage. Although alcohol exposure during late fetal stage did not alter the resting-state functional connectivity within fronto-striatal circuit, an increased functional connectivity within the visual-tactile circuit was detected in juvenile animals. Graph theoretical analysis was used to assess the alteration in brain network properties after alcohol exposure during early or late fetal stage. A reduction of large-scale brain network small-worldness was observed following alcohol exposure during early fetal stage. No significant group difference was observed following alcohol exposure during late fetal stage. In conclusion, the results supported the central hypothesis that developmental alcohol exposure alters resting-state functional connectivity within fronto-striatal circuit and visual-tactile circuit and may eventually alter the organization of large-scale brain networks. The timing of alcohol exposure plays an important role in the outcomes and should be taken into consideration in future research, the usage of diagnostic biomarkers and the application of intervention approaches.
  • Regulation of Glucokinase in Pancreatic Beta Cells

    Seckinger, Kendra; Rizzo, Mark A.
    Glucose homeostasis is a tightly coordinated process that ensures an adequate source of cellular energy. Blood glucose concentrations are coupled to changes in activity of glucokinase (GCK) in β-cells of the pancreas. GCK acts as the glucose sensor and is responsible for the phosphorylation of glucose that leads to insulin secretion from the pancreas to constrain glucose concentrations within physiologic levels. However, the underlying mechanistic details explaining how GCK maintains this tight control over glucose metabolism are unclear. First we elucidated the role of intracellular Ca2+, particularly Ca2+ mobilized from the endoplasmic reticulum, in GCK activation. We then investigated the cellular regulation of GCK through post-translational S-nitrosylation. Finally, we assessed changes in GCK activity that occur during the development of diabetes. To quantitatively assess this GCK activity in vitro, we used Förster resonance energy transfer (FRET) spectroscopy, fluorescence microscopy and GCK biosensors expressed in cultured pancreatic β-cells and primary mouse islets. This work describes our homotransfer FRET-GCK biosensor and improved data analysis methodology to understand the cellular regulation of GCK activity. To create the homotransfer biosensor, we attached two mVenus fluorescent proteins to GCK and transfected this single-color biosensor into cultured β-cells. We used the inherent polarization of light in combination with FRET principles to precisely measure GCK activation in living cells under various conditions.
  • Repurposing Oxaliplatin for the Treatment of Glioblastoma

    Roberts, Nathan; Woodworth, Graeme
    Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults, accounting for approximately 40% of primary brain tumors. Even with the most aggressive therapy, the mean survival for patients with GBM is still less than 18 months, highlighting the critical need for new therapeutic strategies for this deadly cancer. Among the strategies under consideration is a repurposing of platinum-based chemotherapeutics. Traditionally considered DNA damaging cytotoxic agents, recent findings suggest that platinum-based chemotherapeutics, especially oxaliplatin (OXA), can induce multi-faceted anti-tumor effects, including modulation of cytokines, transcription factors, and tumor immunosuppressive mechanisms, even at lower concentrations that are not directly cytotoxic. Data suggests that a major alternative effect of OXA is the inhibition of signal transducer and activator of transcription 3 (STAT3), a transcription factor at the core of GBM pathobiology. STAT3 signaling is constitutively active in many gliomas and dictates diverse aspects of glioma biology including angiogenesis, invasion, chemotherapeutic resistance, and immunosuppression. STAT3 also controls and co-opts the primary gliomainfiltrating immune cell, the macrophage, which composes up to 40% of the tumor mass. OXA treatment may overcome the pleiotropic glioma-supporting functions of STAT3. It is likely that OXA therapeutic formulations designed to maximize the multi-faceted effects of OXA, including STAT3 inhibition, will have potent anti-GBM effects, including reprogramming of the tumor microenvironment. Although high-dose platinum-based chemotherapeutics have been investigated for CNS tumors, systemic and direct neuronal toxicity at high doses has thus far limited their use. However, new therapeutic delivery strategies including polymeric nanoparticle formulations capable of improving drug delivery to tumor cells, providing a sustained release of chemotherapeutic at the target site, and significantly reducing toxicity are facilitating the adaptation of these compounds in the CNS. We sought to investigate the multi-faceted anti-tumor effects of low-dose OXA in glioma cells and macrophages, with a particular focus on STAT3 modulation. We hypothesized that OXA will reduce STAT3 activity in glioma cells as well as macrophages and that OXA nanoparticle formulations will sustain STAT3 inhibition in vivo, thereby enabling the use of OXA as a biomaterial inhibitor of STAT3 for the treatment of glioma.
  • Aging effects on Kinematics, Kinetics, and Neuromuscular Control of Landing Movements in Response to Sudden Loss of Ground Support Surface while Standing

    Sanders, Ozell Phillip; Rogers, Mark William
    Background. With advancing age, the capacity to maintain balance after perturbations deteriorates due to a number of age-related sensorimotor deficits, and likely increases the risk for falls. The unexpected nature of falls triggers startle-like whole body postural responses. Startle responses are characterized by exaggerated whole body postural responses with increased muscle co-activity causing co-contraction during the first trial response (FTR) and normally diminish with repeated exposure due to behavioral habituation. Previous work suggested that age-related abnormalities of exaggerated startle responses and habituation might influence protective balance and startle FTRs to sudden loss of the ground support surface. Furthermore, while startle-like effects in younger adults are modifiable during self-activated (SLF) drop perturbations due to motor prediction, the extent to which this capacity is retained with aging is unknown. Aim. This dissertation 1) compared changes in protective balance and startle responses to unexpected and expected drop perturbations in relation to age during a) FTRs and b) subsequent trials; and 2) determined the modulatory effects of repeated self-triggered drop perturbations on reducing FTR magnitude evoked by externally triggered drop perturbations. Methods. Participants stood atop a moveable platform and received blocks of twelve consecutive trials of externally triggered (EXT) and self-triggered (SLF) drop perturbations. Following the last SLF trial, participants received an additional EXT trial spaced 20 minutes apart to assess retention (EXT RTN) of any modulation effects. Electromyographic (EMG) activity was recorded bilaterally over the sternocleidomastoid (SCM), middle deltoid (DLT), biceps brachii (BIC), vastus lateralis (VL), biceps femoris (BF), medial gastrocnemius (MG), and tibialis anterior (TA). Whole-body kinematics were recorded with motion analysis. Stability in the antero-posterior direction was quantified using the margin of stability (MoS). To quantify landing strategies, the mechanical work performed during drop landings was measured. Results. Incidence of early onset of bilateral SCM activation within 120ms after drop onset was present during the first trial response (FTR) for all participants. Co-contraction indices (CoI) during FTRs between VL and BF as well as TA and MG were significantly greater in older adults compared to young (VL/BF by 26%, p<.05 and TA/MG by 37%, p<0.05). Reduced shoulder abduction between FTR and last trial responses (LTR) was present across both groups and indicative of habituation. Significant age-related differences in landing strategy were present between groups as older adults had greater trunk flexion (p<0.05) and less knee flexion (p<0.05) which resulted in greater peak vGRFs and decreased MoS compared to young adults. Motor prediction via self-triggered drop perturbations (SLF) reduced peak SCM response and VL/BF and TA/MG CoIs (p <0.05) across both groups. Older adults significantly reduced peak vGRFs during SLF FTR compared to EXT FTR (2.40 ± 0.07 vs. 2.74 ± 0.07, p<0.05). Similarly, young adults significantly reduced peak vGRF during SLF FTR compared to EXT FTR (1.43 ± 0.08 vs. 1.83 ± 0.07, p<0.001). Lastly, in both groups, more eccentric work was performed during SLF trials compared to EXT (p <.05). Conclusion. Drop perturbations of standing balance evoke startle-like reactions in young and older adults. Age-associated abnormalities of delayed, exaggerated, and poorly habituated startle/postural FTRs linked with less balance stability and increased joint stiffening with increased impact forces were present among older adults. However, protective balance and startle responses were modulated with motor prediction resulting in reduced knee and ankle co-contraction, reduced ground impact forces and improved balance stability. The observed difference coincided with a reduced SCM response amplitude indicative of a reduced startle influence.
  • Glutathione S-Transferase π Gene Amplification in Oropharyngeal Cancer

    Sadegh, Nahal Mir; Zou, Ying, M.D., Ph.D.; Mitchell, Braxton D.; 0000-0002-4025-1678
    Head and neck cancers make up about 3% of all new cancer cases in the United States. In addition to the traditional risk factors such as tobacco and alcohol use, recent studies have shown that Human Papillomavirus (HPV) is another risk factor for this type of cancer. Since Traditional prognostic markers such as tumor extent and size are not adequate for risk stratification, clinically relevant and practical biomarkers are needed, especially in the context of non-HPV related cancers, in order to define high-risk patients who might benefit from more aggressive treatment strategies. This project explores the suitability of GSTP1 gene amplification as a biomarker for risk stratification in a subset of head and neck cancers, oropharyngeal cancer (OPC), by evaluating the suitability of a previously established algorithm for defining GSTP1 gene amplification and using that information to evaluate GSTP1 gene amplification status as a prognostic indicator for OPC patients.
  • Pediatric Cell-Mediated Immune System and Response to Ty21a Typhoid Vaccination Compared to Adults

    Rudolph, Mark Edward; Sztein, Marcelo B.; 0000-0002-0942-0349
    Typhoid fever is a life-threatening disease caused by the human-restricted pathogen Salmonella enterica serovar Typhi (S. Typhi). The oral live-attenuated Ty21a typhoid vaccine protects against this severe disease by eliciting robust, multifunctional cell mediated immunity (CMI), shown to be associated with protection in wild-type S. Typhi challenge studies. Ty21a induces S. Typhi-responsive CD8+ and CD4+ T cells but little is known about the response to this vaccine in children. To address this important gap in knowledge, we have used mass cytometry to analyze pediatric and adult pre- and post-Ty21a vaccination T cells with both an HLA-E restricted and an autologous S. Typhi-antigen presentation model. Here, using conventional supervised analytical tools, we show adult T cells are more multifunctional at baseline than those obtained from children. Moreover, pediatric and adult T cells respond similarly to Ty21a vaccination, but adult responders remain more multifunctional. The use of the unsupervised dimensionality reduction tools allowed us to confirm these findings, as well as to identify increases and decreases in well-defined specific CD4+ and CD8+ T cell populations that were not possible to uncover using the conventional gating strategies. These findings evidenced age-associated maturation of multifunctional S. Typhi-responsive T cell populations, including those which have previously shown to be associated with protection from, and/or delayed onset of, typhoid disease. Further, in depth analysis of control stimulation conditions also found age-associated multifunctional T cell heterogeneity. These findings are likely to play an important role in improving pediatric vaccination strategies against S. Typhi and other enteric pathogens.
  • A Role for Taurine in Food Sensitivities in Fish

    Larkin, Mary Elizabeth; Place, Allen R.; 0000-0002-9421-0943
    As the aquaculture industry expands to feed an ever-growing world population, it seeks to develop more low-cost, environmentally sustainable feed ingredients. Fish meal replacement by plant sources of protein, including those derived from soybeans, wheat, and peas may have unintended effects. Certain fish species have reacted poorly to particular ingredients, including components of soy and pea protein. We present here evidence for potential negative health effects, including possible inflammation, elicited by wheat gluten incorporation into the feed of Cobia (Rachycentron canadum). European sea bass (Dicentrarchus labrax), on the contrary, seem to well tolerate this ingredient. We sought to evaluate the ability of taurine, a known immunomodulator, to alleviate inflammation in cases of adverse effects caused by particular plant ingredients in feed. We saw evidence for this in the cobia study, and though our study did not induce any inflammation in sea bass, we detected dramatically increased levels of taurine levels in the plasma of fish consuming a diet containing 4% wheat gluten. In another study, supplemental taurine in European sea bass shifted spectral sensitivity to a longer wavelength, though there were no apparent anatomical differences in the retina between the un-supplemented and supplemented groups. We observed changes to the microbiome induced by dietary wheat gluten, and in a separate study, taurine. Wheat gluten addition to the diet greatly increased the number of predominant orders represented in the intestinal microbiota. Taurine caused less of a shift, but interestingly, the predominant orders were very uniform throughout the sections of the intestines of the taurine-fed fish. In two different studies, we attempted to characterize a dietary taurine-dependent zebrafish that was incapable of endogenous taurine synthesis. However, both of our potential strains turned out to be producing a wild-type CSAD (cysteine sulfinic acid decarboxylase) protein even in the presence of early termination codons in the csad gene. We also observed the expression of two smaller sizes of CSAD, ~53 and ~55 kDa, in addition to the previously described ~59 kDa protein. The two smaller sizes appear to be produced early in development and are not detectable by 3.5 weeks post-fertilization.
  • Beyond the Dinoflagellate Transcriptome: Validation of Protein Production via Biochemical Analysis and Mass Spectrometry

    Haq, Saddef; Place, Allen R.; 0000-0002-5274-6299
    Dinoflagellates are members of the Alveolata (meaning “with cavities”), a monophyletic group of single cell protists which includes apicomplexans and ciliates that exhibit a diverse mode of nutrition, ranging from predation to photo autotrophy to intracellular parasitism. Dinoflagellates are both primary producers and consumers in the food web, sometimes at the same time, and best known for their dominant role in causing harmful algal blooms. In keeping with their unusual nuclear chromatin architecture (condensed throughout the cell cycle) the degree to which dinoflagellates use transcriptional responses to alter mRNA expression levels appears limited. In addition, it appears dinoflagellates have extensive gene duplications for most biological processes. For these reasons, the goal for this dissertation was to measure protein levels directly for important cellular phenotypes. We began with characterizing a proton selective channel (Hv1) that was hypothesized to be involved in bioluminescence in Lingulodinium polyedrum with goals of elucidating function, location, and production of this protein. Next, we focused on acetyl CoA carboxylases (ACC) which are responsible for the rate limiting step in fatty acid (FAS) and polyketide synthases (PKS). We find the presence of these proteins in thirteen dinoflagellates surveyed and show there is distribution in the plastid and cytosol. In Amphidinium carterae we verify protein production with high performance liquid chromatography-coupled to tandem mass spectrometry (LC-MS/MS), otherwise known as shotgun proteomics as well as compare transcript and protein levels across a diel cycle. Lastly, we focused on a multi-modular polyketide protein in A. carterae with implications in toxin and fatty acid production due to the domain arrangement which is indicative of a noniterative trans-AT PKS process. We show successful inhibition of 14C acetate incorporation into toxins and fatty acids with the addition of the ketosynthase specific (KS) fatty acid inhibitor cerulenin. Partial protein production is verified by western blotting and LC-MS/MS analysis of KS peptides reveals a strong covalent bond with cerulenin addition. Taken together the work discussed in this thesis has resulted in a better understanding of three different cellular processes in dinoflagellates using a shotgun proteomic approach. Future work continuing this trend of protein characterization in dinoflagellates will help elucidate many uncharacterized pathways in dinoflagellate biology.
  • Oral Anticoagulation Medication Usage in Older Adults with Atrial Fibrillation Residing in Long-term Care Facilities

    Gill, Christine; Baumgarten, Mona
    Statement of the problem: Oral anticoagulants (AC) reduce the risk of ischemic stroke (IS) in older adults with atrial fibrillation (AF) but increase the risk of major hemorrhage. Treatment with ACs requires prescribers and patients to weigh benefits against risks. Many older adults with AF residing in long-term care (LTC) facilities may not be using ACs, even in the absence of absolute contraindications. This study (1) examined the prevalence of AC use, (2) assessed which factors were associated with AC use, and (3) estimated the net effect of ACs weighing the benefit (IS prevention) against the risk (intracranial hemorrhage [ICH]) in older adults with AF residing in LTC facilities. Methods: An observational cohort study was performed using a 5% random sample of older adults with AF residing in LTC facilities for at least 101 days from 2007 to 2013 using a Medicare administrative claims database linked to the Minimum Dataset assessments. Results: Of the 21,877 Medicare beneficiaries meeting the study eligibility criteria, over half (54.6%) were 85 years or older, most were female (75.9%) and white (88.1%). The prevalence of AC use was 36.2% (95% confidence interval [CI]: 35.6%-36.8%). History of stroke or transient ischemic attack and history of thromboembolism were associated with an increased likelihood of AC use, while history of internal bleed was associated with a decreased likelihood of AC use. The net effect of AC use was 1.07 per 100 person-years, 95% CI: 0.31-3.01; this is the difference between, on the one hand, the difference in the estimated rate of IS while not using ACs and using ACs and, on the other hand, the difference between the estimated rate of ICH while using ACs and not using ACs . Conclusions: The majority of older adults with AF residing in LTC facilities are not being managed with ACs. While this study provides evidence suggestive of a net benefit of AC use in older adults with AF residing in LTC facilities, health status and the burden of medication monitoring are among the other factors that patients, their caregivers and providers should consider when making the decision about initiating ACs.
  • The regulation and role of dendritic mitochondrial fission during long-term potentiation

    Divakaruni, Sai Sachin; Blanpied, Thomas A.; 0000-0003-3478-3229
    Neurons continuously modify their synaptic strength to encode memories and to adapt to experience and the environment. Long-term potentiation (LTP) is a critical cellular mechanism of this adaptation and is the prevailing form of synaptic plasticity. Baseline synaptic function is bioenergetically demanding, and this demand is elevated during episodes of synaptic plasticity. Therefore, mitochondrial functions, such as ATP synthesis and calcium handling, are likely essential for plasticity. Furthermore, mitochondrial functions themselves are regulated by mitochondrial dynamics including fission, fusion, and motility. Although axonal mitochondria have been extensively studied, LTP induction predominantly occurs postsynaptically, where the roles of mitochondria are less well understood. Additionally, mitochondrial fission has recently garnered interest because it is necessary for development and is required for normal mitochondrial function, and because perturbed fission is associated with many neurological and psychiatric diseases. However, whether or how fission in dendrites supports ongoing synaptic transmission and plasticity is still unclear. Furthermore, although the molecular mechanisms underlying mitochondrial fission have been well described in other cell types, little is known about how mitochondrial fission is accomplished in neurons, particularly in dendrites, or how neuronal activity might modulate these mechanisms. Here I tested the hypothesis that dendritic mitochondrial fission is triggered during LTP induction, and is necessary for LTP expression. Mitochondria in dendrites at rest are stationary and rarely undergo fission. However, I found that chemical induction of LTP (cLTP) by NMDAR activation in cultured rat hippocampal neurons prompted a rapid burst of dendritic mitochondrial fission. Mitochondrial fission canonically requires actin nucleation and membrane constriction by the GTPase dynamin-related protein 1 (Drp1). Consistent with this, inhibition of actin polymerization or expression of a dominant negative (DN) mutant or knockdown of Drp1 each suppressed the cLTP fission burst. Furthermore, the GTPase Dynamin 2 (Dyn2) was recently implicated in fission in cell lines, and I found similarly that expressing DN Dyn2 abolished the fission burst. Drp1 function is also known to be regulated by phosphorylation, with CaMKII as a possible activator based on studies of non-neuronal cells. In line with this, I found that fission was triggered by cytosolic calcium elevation via glutamate photolysis at dendritic spines, and also that the fission burst was prevented by acutely inhibiting CaMKII activation or by prohibiting Drp1 phosphorylation. I then tested whether mitochondrial fission is required for LTP expression. Knocking down Drp1 or expressing DN Drp1 suppressed dendritic spine growth and synaptic AMPA receptor trafficking following LTP induction. Furthermore, NMDAR-dependent LTP induction by high-frequency stimulation (HFS) of Schaffer collaterals in acute hippocampal slices decreased dendritic mitochondrial length in area CA1. Remarkably, postsynaptic expression of DN Drp1 prevented HFS LTP at Schaffer collateral-CA1 synapses in slices, with no effect on basal transmission or intrinsic electrophysiological properties of neurons. Furthermore, I found that cLTP stimulation produced transient elevations of dendritic mitochondrial calcium (i.e. mCaTs), and that expression of DN Drp1 suppressed the frequency, amplitude, and duration of evoked mCaTs. These data illustrate a novel pathway whereby synaptic activity controls mitochondrial fission, and show that dynamic control of fission is required for LTP induction perhaps by modulating mitochondrial calcium handling. Impaired synaptic function is implicated in myriad neuropsychiatric diseases, many of which are also associated with mitochondrial dysfunction. Therefore, our findings raise the important question of whether neuronal mitochondrial dysfunction contributes to cognitive impairment in these diseases by perturbing dendritic and/or synaptic plasticity.
  • Effects of Idebenone on the Mitochondrial Respiration of Neurons, Astrocytes, and Microglia

    Jaber Taha, Sausan Mousa; Polster, Brian M.; Fiskum, Gary
    Neuroinflammation and mitochondrial bioenergetic dysfunction are present in most neurodegenerative diseases. Microglia, the resident immune cells of the brain, release cytotoxic factors such as nitric oxide (NO), superoxide, and cytokines following proinflammatory activation. NO mediates mitochondrial respiratory inhibition in microglia and neighboring cells by competing with oxygen at complex IV of the electron transport chain. Additional modes of inhibition by NO include post-translational modifications to upstream complexes I and II. Idebenone is a clinically safe prodrug that, in its reduced form idebenol, can act in place of ubiquinol and donate electrons directly to complex III. This dissertation tested the overarching hypothesis that idebenone can support mitochondrial respiration in cells with sufficient quinone-reduction capacity to convert idebenone to idebenol, despite complex I and II impairment by NO. In astrocytes but not neurons, idebenone and two related quinones could rescue maximal oxygen consumption rate (OCR) when a complex I inhibitor was present. This difference between astrocytes and neurons was due to a disparity in cellular quinone-reduction capacity mediated by the expression of NADPH:quinone oxidoreductase 1 (NQO1). Astrocytes were sensitive to respiratory impairment by an NO donor or co-cultured proinflammatory microglia at a physiologically-relevant oxygen level and idebenone was able to partially reverse this impairment. Interestingly, microglia upregulated their quinone-reduction capacity following proinflammatory stimulation and idebenone was also able to partially reverse respiratory impairment in microglia following activation. Surprisingly, in contrast to astrocytes, NQO1 was not responsible for idebenone reduction in activated microglia. Biochemical isolation of the responsible enzyme identified inducible nitric oxide synthase (iNOS) among the few candidates common to three distinct fractionation approaches. Assays performed with recombinant iNOS revealed a novel idebenone reduction activity with exciting implications for future studies. This dissertation’s findings suggest that insufficient quinone-reduction capacity in diseased target cells may be a mechanistic reason for the failure of idebenone in clinical trials. These results support new strategic approaches for the use of idebenone and similar drugs to overcome mitochondrial bioenergetic dysfunction.
  • The Effects of Mindfulness Based Stress Reduction on Brain Gray Matter Volume and Psychosocial Co-Morbidities in Episodic Migraine Patients

    Burrowes, Shana; Seminowicz, David A.; 0000-0001-6857-6207
    Background: Studies show that migraine patients have altered brain structure. The morbidity associated with migraine is due to both the headaches and comorbid psychosocial factors, such as anxiety and depression. Current pharmacological therapies either prevent headaches or treat the ongoing pain, but fail to address the comprehensive migraine experience. Objectives: The comprehensive effects of Mindfulness Based Stress Reduction (MBSR) in episodic migraine patients enrolled in the MRI Outcomes of Mindfulness Meditation for Migraine Clinical Trial were assessed. We examined longitudinal changes in brain grey matter volume (GMV) of patients and healthy controls (HC), changes in psychosocial well-being and the manner in which these psychosocial factors were associated with treatment response (50% reduction in headache frequency post-intervention). Methods: Patients were randomized to receive MBSR or stress management for headache (SMH). Patients were scanned at three time points approximately 3 months apart (baseline, mid-intervention and post-intervention), completing psychosocial questionnaires and headache diaries. HC were also enrolled and completed the same MRI sessions and questionnaires. Results: From baseline to six months HC had reductions in six regions: bilateral superior frontal gyrus (SFG), anterior cingulate cortex, right middle frontal gyrus and anterior insula. Compared to HC, patients had increased GMV from baseline to six months in the right SFG and in a cluster located in the left parietal cortex. At baseline patients without prior clinical care for migraine had reduced GMV in the right dorsal medial prefrontal cortex compared to those with prior care. There was no difference between MBSR and SMH in psychosocial and quality of life measures (stress, anxiety, depression or sleep) from baseline to six months. Sleep quality improved significantly in both patient groups at 6 months. Baseline stress, depression and anxiety scores were not associated with treatment response. Mediation analysis showed that small significant changes in psychosocial scores over time were responsible for 6-8% of the proportion of the treatment response. Conclusion: Migraine patients had increased GMV compared to controls. These changes are likely linked to treatment effects. While there were no significant longitudinal psychosocial changes, small improvements were important in mediating the effect of the treatment on headache frequency.
  • Cortical enhancement of posture and movement planning, initiation, and execution during standing voluntary reach following stroke

    Yang, Chieh-ling; McCombe-Waller, Sandra (2018)
    Stroke is the leading cause of disability that frequently includes impairments of postural control and upper extremity function. The ability to maintain balance while performing reaching during everyday activities requires appropriate sequencing of anticipatory postural adjustments (APAs) and the goal-directed reaching movement. Although studies have shown that these abilities are frequently compromised following stroke, the interaction of posture and upper extremity movement coupling (APA-reach sequence) regarding movement planning, preparation, and execution is not well understood. We investigated posture and movement planning, preparation, and execution in individuals with chronic hemiparesis and healthy controls. Movement planning and preparation of APA-reach sequence were examined by StartReact (SR) responses elicited by a loud acoustic stimulus (LAS). After an instructed delayed period, subjects performed a standing reaching task in response to a "go" light cue. An LAS was randomly delivered at - 500, - 200, and 0ms relative to the "go" cue. Kinetic, kinematic, and electromyography data were recorded to characterize APA-reach movement response. Subjects with stroke demonstrated a marked reduction in the occurrence of the SR responses and impaired APA-reach performance during not only the paretic but also the nonparetic arm reaching compared to healthy controls. This indicates that the deficits in posture and movement planning, preparation, and execution are not specific to the lesioned side only but system-wide to both the lesioned and contralesional sides. Moreover, compensation strategy characterized by greater involvement of trunk and pelvic rotation was utilized during the paretic arm reaching compared to the nonparetic arm reaching and healthy controls. Lastly, we tested how transcranial direct current stimulation (tDCS) applied over the region of premotor areas and primary motor cortex (M1) affect movement planning and preparation of a standing reaching task in individuals with stroke and healthy subjects. The main finding was that stroke-related deficits in movement planning and preparation as shown by an abnormal absence of SR responses during the paretic arm reaching could be improved by application of anodal tDCS over the region of lesioned M1 and the enhancement effects are depending on the timing of the LAS.
  • The Claustrum: From Top to Bottom

    White, Michael; Mathur, Brian N. (Brian Neil); 0000-0002-7202-7515 (2018)
    Despite much speculation, the function of the claustrum, a thin telencephalic brain nucleus, remains largely unknown. The claustrum is reciprocally connected with seemingly the entire cortical mantle, which motivates hypotheses of claustrum function that include multi-sensory integration and top-down/bottom-up attention. In order to discriminate these hypotheses, we examined the claustrum at multiple levels of analysis in rodents. We find that the claustrum is well-connected with executive areas of cortex, such as anterior cingulate cortex (ACC), and highly responsive to stimulation of ACC inputs in acute brain slices. In contrast, claustrum exhibits less connectivity with and responsivity to inputs from hierarchically lower cortices. Using in vivo circuit monitoring and manipulation, we find that ACC inputs to claustrum and claustrum projection neurons mediate top-down cognitive control relative to other basic brain functions, such as motor control and stimulus-response action strategy. Consistent with a role in cognitive control, claustrum neurons projecting to visual cortices and parietal association cortex faithfully propagate ACC input, and claustrum afferents provide strong excitatory drive across cortical layers. To understand how claustrum processes top-down input, we identified claustrum neuron subtypes using membrane properties and examined claustrum microcircuit responsivity to ACC input. We find that one of two claustrum projection neuron subtypes preferentially burst fires in response to ACC input, that the two subtypes differentially target cortex, and that recruitment of inhibitory microcircuits constrains claustrum output. These findings support a model wherein claustrum mediates synchronization of cortices for cognitive control.
  • Identification and Characterization of Factors Associated with Biofilm Formation in Acinetobacter baumannii Surveillance Isolates

    Wallace, Lalena; Rasko, David A.; 0000-0002-7105-3607 (2018)
    Acinetobacter baumannii is a Gram-negative nosocomial pathogen. It is responsible for a variety of infections and is among the five most common pathogens in U.S. hospitals associated with ventilator-associated pneumonia. It has been estimated that A. baumannii is responsible for approximately 45,000 hospital infections in the U.S. per year. The two main characteristics that make this organism a formidable threat in health care settings is its ability to acquire multidrug resistance and to persist on surfaces. A better understanding of the genetic factors responsible for these virulence traits is needed. Genomic comparisons of 203 A. baumannii strains, collected as part of a surveillance study at the University of Maryland Medical Center, were performed using Large-Scale BLAST Score Ratio (LS-BSR) analysis. For these genomic comparisons, the genomes were grouped according to the date and source of the strain, as well as the carbapenem resistance status. This analysis resulted in the identification of genes unique to specific phylogenomic group, site of isolation, or resistance phenotypes. In addition, this work included identification of genes exclusive to other Acinetobacter species, which may be useful in the future to positively identify A. baumannii as this is currently a difficult clinical task. Phenotypic characterization of the A. baumannii strains resulted in the identification of a phylogenetic cluster of strains that exhibited robust biofilm formation. Genomic analysis of these strains revealed several genes unique to this group and potentially associated with biofilm formation. Mutagenesis of three of these genes was performed and the ability to form robust biofilms was determined. In particular, mutagenesis of a putative pilus assembly gene resulted in significantly decreased biofilm formation, suggesting that this gene plays a key role in the robust biofilm phenotype. In addition, a transcriptional regulator was identified that may play a role in the regulation of genes associated with biofilm formation. Overall, the work presented herein has broadened our understanding of the vast diversity among surveillance isolates of A. baumannii in a single healthcare setting, and has demonstrated the utility of genotypic/phenotypic correlations to identify novel virulence factors.

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