Mitochondria are well known for their function in providing energy supply to the cell. Aside from these "bioenergetic" functions, mitochondria perform many other essential processes. To accomplish these functions, mitochondria must import many proteins from the cytosol. This import process can sometimes become dysfunctional and induce a severe stress on the cell via the mistargeting of mitochondrial proteins to the cytosol. Our lab termed this specific type of cell stress as mitochondrial precursor overaccumulation stress (mPOS). Our work has focused on demonstrating that mPOS is able to occur in various models of disease, both in vivo and in vitro. In this thesis we demonstrate that mPOS can occur in the heart of mice and induce significant signaling and functional changes over the lifespan (chapter 2). Additionally, in a related work, we found that mitochondrial protein import clogging can induce mPOS in the central nervous system (CNS) and potentiate pathology in a mouse model of Parkinson's disease (appendix I). Most importantly, all the changes occurring in both animal models do not necessarily co-occur with bioenergetic deficiencies. The implication of this is that mPOS may be a bioenergetic independent mechanism liking mitochondrial dysfunction with tissue dysfunction. Speaking more generally, mPOS may occur in many clinically relevant conditions such as heart failure, normative ageing, muscle loss, and neurodegenerative diseases. This work and future work therefore aims to establish the basic mechanisms by which mPOS may occur within cells and how cells can in turn respond to this stress.

Data pertaining to risk factor analysis in coronavirus disease 2019 (COVID-19) is confounded by the lack of data from an ethnically diverse population. In addition, there is a lack of data for young adults. This study was conducted to assess risk factors predicting COVID-19 severity and mortality in hospitalized young adults. A retrospective observational study was conducted at two centers from China and India on COVID-19 patients aged 20-50 years. Regression analysis to predict adverse outcomes was performed using parameters including age, sex, country of origin, hospitalization duration, comorbidities, lymphocyte count, and National Early Warning Score 2 (NEWS2) score at admission. A total of 420 patients (172 East Asians and 248 South Asians) were included. The predictive model for intensive care unit (ICU) admission with variables NEWS2 Category II and higher, diabetes mellitus, liver dysfunction, and low lymphocyte counts had an area under the curve (AUC) value of 0.930 with a sensitivity of 0.931 and a specificity of 0.784. The predictive model for mortality with NEWS2 Category III, cancer, and decreasing lymphocyte count had an AUC value of 0.883 with a sensitivity of 0.903 and a specificity of 0.701. A combined predictive model with bronchial asthma and low lymphocyte count, in contrast, had an AUC value of 0.768 with a sensitivity of 0.828 and a specificity of 0.719 for NEWS2 score (5 or above) at presentation. NEWS2 supplemented with comorbidity profile and lymphocyte count could help identify hospitalized young adults at risk of adverse COVID-19 outcomes.

Background: High levels of childhood trauma (CT) have been observed in adults with mental health problems. Herein, we investigated whether self-esteem (SE) and emotion regulation strategies (cognitive reappraisal (CR) and expressive suppression (ES)) affect the association between CT and mental health in adulthood, including depression and anxiety symptoms. Methods: We performed a cross-sectional study of 6057 individuals (39.99% women, median age = 34 y), recruited across China via the internet, who completed the Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7), Childhood Trauma Questionnaire (CTQ), Self-esteem Scale (SES), and Emotion Regulation Questionnaire (ERQ). Multivariate linear regression analysis and bias-corrected percentile bootstrap methodologies were used to assess the mediating effect of SE, and hierarchical regression analysis and subgroup approach were performed to examine the moderating effects of emotion regulation strategies. Results: After controlling for age and sex, we found that (1) SE mediated the associations between CT and depression symptoms in adulthood (indirect effect = 0.05, 95% confidence interval [CI]: 0.04-0.05, 36.2% mediated), and CT and anxiety symptoms in adulthood (indirect effect = 0.03, 95% CI: 0.03-0.04, 32.0% mediated); (2) CR moderated the association between CT and SE; and (3) ES moderated the association between of CT and mental health in adulthood via SE, and such that both the CT-SE and SE-mental health pathways were stronger when ES is high rather than low, resulting the indirect effect was stronger for high ES than for low ES. Conclusions: These findings suggested that SE plays a partially mediating role in the association between CT and mental health in adulthood. Furthermore, ES aggravated the negative effect of CT on mental health in adulthood via SE. Interventions such as emotional expression training may help reduce the detrimental effects of CT on mental health. Trial registration: The study was registered on http://www.chictr.org.cn/index.aspx and the registration number was ChiCTR2200059155.

Purpose: The aim of this study was to investigate the relationship between cardiac autonomic dysfunction, cognitive impairment, and survival in patients with amyotrophic lateral sclerosis (ALS). Methods: The heart activity of 65 patients with ALS (28 with normal cognition [ALS-CN]; 37 with impaired cognition [ALS-CI]) and 38 healthy controls (HCs) was measured by 24-h Holter monitoring. Heart rate (HR) measures and heart rate variability (HRV) parameters were compared between the three study groups and, additionally, correlated with five Edinburgh Cognitive and Behavioral ALS Screen (ECAS) domains in the ALS subgroups. Age, gender, and educational level were adjusted. Factors associated with cognitive status were assessed using logistic regression. Survival predictors in patients with ALS were analyzed using the Kaplan-Meier estimator and Cox regression. Results: Compared to the HCs, patients with ALS-CI exhibited lower RRI (R-R-interval; P = 0.017), SDNN (standard deviation of all normal RR intervals; P = 0.013), SDNN Index (P = 0.044), and VLF power (very low-frequency power; P = 0.012). Total power was reduced in the ALS-CI group compared to the HCs (P = 0.036) and ALS-CN group (P = 0.048). In patients with ALS-CN, language negatively correlated with mean HR (P = 0.001) and positively with the RRI (P = 0.003), SDNN (P = 0.001), SDANN (standard deviation of the average NN intervals; P = 0.005), total power (P = 0.006), VLF power (P = 0.011), and low-frequency power (P = 0.026). Visuospatial function correlated positively with the SDNN Index (P = 0.041). In patients with ALS-CI, executive function (P = 0.015) and ECAS total score (P = 0.009) negatively correlated with the RMSSD (square root of mean sum-of-squares of differences between adjacent NN intervals), while visuospatial function correlated positively with normalized LF value (LFnu; P = 0.049). No associations were observed between the other cognitive domains and any of the 14 HRV/HR measures in patients with either ALS-CI or ALS-CN. SDNN ≤ 100 ms was linked to cognitive impairment (P = 0.039) and also showed a borderline association (P = 0.066) with poorer survival, while cognitive impairment (P = 0.010) was significantly linked to worse outcomes. Conclusions: Patients with ALS with cognitive impairment demonstrated reduced cardiac autonomic modulations and altered cognitive autonomic associations. Cognitive impairment was linked to reduced survival, with baseline SDNN ≤ 100 ms identified as a potential marker.

The vacuolar H+-ATPase (V-ATPase, V1Vo) is a dedicated proton pump that is highly conserved amongst eukaryotes, and is necessary for pH homeostasis within subcellular compartments. The V-ATPase consists of two subcomplexes: the soluble V1 responsible for hydrolyzing ATP, and the membrane integral Vo responsible for proton translocation across membranes. V1 and Vo are each comprised of multiple subunits, A3B3CDE3FG3 and ac8c'c"def Voa1 respectively. Many basic cellular functions depend on the differential pH gradient across cellular membranes to operate properly, making regulation of V-ATPases through "reversible disassembly" immensely important. Global loss of V-ATPase activity is lethal to all mammalian cell types, while aberrant activity and incorrectly localized V-ATPase results in various disease states. Current therapeutics struggle to target specific V-ATPase populations, and as a potential solution to this problem we generated 94 nanobody clones against the yeast nanodisc reconstituted Vo (VoND). Nanobodies (Nbs) are the small 15 kDa VHH domain isolated from heavy-chain only antibodies that are known for their high specificity. In this dissertation we describe the characterization of three α-yeast VoND Nbs, N27, N125, and N2149. Using an ATPase assay, we found that N27, but not N125 or N21149, fully inhibited the activity of assembled V-ATPase. Contrastingly, N2149, but not N27 or N125, was found to inhibit the assembly of the two subcomplexes. BLI was used to identify the binding affinity of each Nb, with affinities being observed in the nM-pM range. High-resolution structures obtained from cryoEM revealed the subunit specificity of each Nb, with N27 and N125 found to bind the c-ring in different stoichiometries, and N2149 found to bind the d subunit. Furthermore, we determined that N125 has cross affinity for the human enzyme. Overall, this study provides evidence that novel nanobody mediated inhibition of assembly or activity of V-ATPases is an effective technique with broader implications of nanobody development into therapeutics.

Mitochondrial dysfunction is strongly associated with aging-related degenerative diseases including muscle atrophy. However, whether bioenergetic defects are the sole drivers of mitochondria-induced muscle atrophy remains unknown. The Chen lab discovered that various forms of mitochondrial damage can disrupt protein import, leading to the toxic accumulation of unimported mitochondrial precursors in the cytosol. This causes a stress termed mitochondrial Precursor Over-accumulation Stress (mPOS). A mouse model of mPOS was developed in which the mitochondrial inner membrane protein, ANT1, was overexpressed to saturate the protein import machinery. Ant1Tg/+ mice were found to have a severe muscle wasting phenotype. The overarching goal of this dissertation is to investigate the mechanism by which mPOS drives muscle wasting and its implications for normative muscle aging. The findings presented in this thesis led to three conclusions. First, we identified a novel mitochondria-to-lysosomal proteostatic axis through which mPOS induces lysosomal damage. Lysosomal damage subsequently causes the release proteolytic enzymes, which leads to excessive protein degradation and subsequent progressive muscle atrophy. Importantly, we found that this pathway operates independently of mitochondrial respiratory complex activity and reactive oxygen species (ROS) production. Second, we demonstrated the presence of mPOS in physiologically aged muscle. Sarcopenic muscle exhibited phenotypes similar to those found in Ant1Tg/+ mice, evidenced by overlapping transcriptional and proteomic profiles, and lysosomal damage. These findings indicate that mitochondria-induced changes to autophagic activity may play a central role in the pathogenesis of sarcopenia. However, considering the overall protein content of muscle is elevated during aging, we propose that reduced protein quality, rather than absolute protein content, drives sarcopenia. We therefore termed this phenomenon Muscle Atrophy Independent of Protein Content (MAIPC). Finally, we explored additional cellular factors that affect proteostasis and muscle mass maintenance. We found that the GCN2 kinase, a well-established activator of the Integrated Stress Response (ISR), plays a role in protecting myofibers from mPOS-induced stress and muscle wasting in Ant1Tg/+ muscle. Interestingly, we found that this effect is ISR-independent. The data presented in this dissertation provide valuable insights into the mechanistic role of mitochondrial dysfunction in both normative aging and chronic muscle wasting conditions. Our findings conclude that mitochondria-induced muscle atrophy is induced by mechanisms that extend beyond bioenergetic defects. By characterizing these alternative pathways, this work opens new avenues for therapeutic strategies targeting mitochondrial stress in chronic muscle wasting conditions.

The present study replicates and extends previous research on the use of video modeling (VM) with voiceover instruction to train staff to implement discrete-trial instruction (DTI). After staff trainees reached the mastery criterion when teaching an adult confederate with VM, they taught a child with a developmental disability using DTI. The results showed that the staff trainees' accurate implementation of DTI remained high, and both child participants acquired new skills. These findings provide additional support that VM may be an effective method to train staff members to conduct DTI.

Direct teaching procedures are often an important part of early intensive behavioral intervention for consumers with autism spectrum disorder. In the present study, a video model with voiceover (VMVO) instruction plus feedback was evaluated to train three staff trainees to implement a most-to-least direct (MTL) teaching procedure. Probes for generalization were conducted with untrained direct teaching procedures (i.e., least-to-most, prompt delay) and with an actual consumer. The results indicated that VMVO plus feedback was effective in training the staff trainees to implement the MTL procedure. Although additional feedback was required for the staff trainees to show mastery of the untrained direct teaching procedures (i.e., least-to-most and prompt delay) and with an actual consumer, moderate to high levels of generalization were observed.

The primary method of data analysis in applied behavior analysis is visual analysis. However, few investigations to date have taught the skills necessary for accurate visual analysis. The purpose of the present study was to evaluate computer-based training on the visual analysis skills of adults with no prior experience. Visual analysis was taught with interactive computer-based training that included written instructions and opportunities for practice with textual feedback. Generalization of participant skills from simulated to handwritten and authentic data graphs was programmed for and assessed during the study. A multiple-baseline design was used across visual analysis properties (i.e., variability, level, and trend), with continuous overall intervention effect generalization probes, replicated across 4 participants to evaluate computer-based training for accurate visual analysis of A-B graphs. The results showed that all participants accurately visually analyzed A-B graphs following the computer-based training for variability, level, trend, and overall intervention effect. These visual analysis skills generalized to handwritten and authentic data graphs and maintained approximately 1 day, 1 week, 2 weeks, and 1 month following mastery of each property for all participants. Implications of the results suggest that computer-based training improved accurate visual analysis skills for adults with no prior experience.

Providing a choice of reinforcers is a commonly used strategy with children with autism spectrum disorder; however, less is known about the differential effectiveness and efficiency of providing choices before or after responding during acquisition tasks. Therefore, we evaluated reinforcer choice using untaught targets prior to and following responding. Results showed faster acquisition of targets in the consequence condition for 2 of 3 participants. These data provide preliminary support that providing choice prior to responding may not result in the most efficient acquisition for some individuals.