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PART I The basic helix-loop-helix (bHLH) family of transcription factors are involved in a variety of developmental processes. MyoR is the mouse homologue of the human transcription factor ABF-1 . MyoR is classified as a class II basic-helix-loop-helix transcription factor. In order to better understand the relationship between MyoR and muscle cell differentiation, we analyzed the temporal expression at both the mRNA and protein level. Unlike previous studies, we have utilized reverse transcriptase quantitative PCR to analyze mRNA expression. This allows quantitative analysis of MyoR mRNA levels during muscle cell differentiation. We have also analyzed MyoR expression at the protein level. Our studies suggest that the temporal expression of MyoR at the mRNA level is similar to the expression profile seen at the protein level. To ascertain differences in the MyoR DNA-binding activity during myogenesis we performed EMSA. Results suggest that changes in MyoR expression fail to account for differences in the DNAbinding complexes to an E-box site. Part II Members of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including lymphopoiesis, myogenesis, neurogenesis and sex determination. Screening a eDNA library prepared from silk-producing glands of the black widow spider, we have identified a new bHLH transcription factor named BW6. Within the bHLH region, BW6 shows considerable conservation with other HLH proteins, including Drosophila melanogaster achaete and scute, as well as three HLH proteins identified by gene prediction programs. The expression pattern of bw6 is restricted to a subset of silk producing glands, which includes the tubuliform and major ampullate glands. BW6 is capable of binding an E-box element as a heterodimer with E2A, but was unable to bind this motif as a homodimer. BW6 is also capable of inhibiting the transactivation of rE47 in mammalian cells. BW6 represents the first example of a silk-gland-restricted bHLH protein, and its expression pattern suggests that BW6 may play a role in regulating differentiation of cells in the spider that control silk gland formation or egg case silk gene expression.

The glenohumeral (GH) joint is the most mobile joint in the human body, but its mobility inherently increases the risk of instability. The humeral head sits in a shallow glenoid in the scapula like a golf ball sitting on a tee. The stability in this joint is provided by the rotator cuff muscles and tendons that actively pull the humerus back into the socket to prevent dislocation, especially during overhead motions. However, the rotator cuff is prone to tears, resulting in pain, loss of mobility, and recreational limitations. Surgical reattachment of the tendon to the bone is challenging due to the mechanical disparity between the two tissues, resulting in stress concentrations and a high risk of retear. Notably, the specialized tissue at the tendon-to-bone attachment, which facilitates stress transfer between tendon and bone in healthy joints, does not regenerate after surgical reattachment and healing, making tendon-to-bone repairs prone to re-tears. A comprehensive understanding of GH joint biomechanics is essential for developing early interventions to prevent rotator cuff injuries. Furthermore, improving tendon-to-bone fixation during rotator cuff repair is critical to improve post-surgery outcomes. In the last decade, bioinspired solutions have shown considerable promise for addressing several biomedical problems. This thesis draws bioinspiration from two animals that have evolved unique mechanical functions: (i) the bat shoulder joint, which facilitates repetitive overhead motions during flight and may offer insights into rotator cuff pathology and (ii) the curvature of python snake teeth, which enables secure grasping of prey without soft tissue tearing. In the first part of the thesis, the bat shoulder was studied for its unique characteristics relative to mice. Overhead motions in humans often lead to shoulder injuries, partly because the bony anatomy of the unstable GH joint places greater stress on the joint's surrounding soft tissues to stabilize these motions. Traditional animal models used to study shoulder pathology are quadrupeds, which lack the capacity for overhead motion. In contrast, bats consistently engage in overhead motion during flight, subjecting their shoulders to substantial loading throughout their relatively long lifespan. Remarkably, the biomechanical demands placed on a bat's shoulder are estimated to exceed those of a competitive swimmer’s by 45-fold, despite sharing similar coracoacromial arch anatomy with humans. We were inspired to study functional adaptations in the shoulders of bats that enable this overhead motion. We performed comparative anatomy studies of the shoulders of bats and mice, similarly-sized quadrupeds. By quantifying the constraints imposed by the bony anatomy, we identified adaptations of the shoulder, including the rotator cuff tendons, that allow bats to sustain overhead motion in a high stress, repeated loading environment, without injury. In the second part of the thesis, python teeth were used as inspiration to develop a repair device optimized to grasp the rotator cuff without tearing. Rotator cuff repair surgeries fail frequently, with 20-94% of the 600,000 repairs performed annually in the United States resulting in retearing of the rotator cuff. The most common cause of failure is sutures tearing through tendons at grasping points. To address this issue, we examined the specialized teeth of snakes of the Pythonoidea superfamily, which effectively grasp soft tissues without tearing. To apply this non-damaging and effective gripping approach to the surgical repair of tendons, we developed and optimized a python-tooth inspired array as an adjunct to current rotator cuff suture repair, and found that it nearly doubled repair strength. Integrated simulations, 3D printing, and ex vivo experiments revealed a relationship between tooth shape and grasping mechanics, and enabled optimization of a tooth array device to enhance rotator cuff repair to distribute stresses and increase tendon-bone contact. The efficacy of the approach was demonstrated via human cadaver tests, suggesting an alternative to traditional suturing paradigms that may reduce tendon re-tearing. Collectively, these studies contribute to a better understanding of the biomechanics of the GH joint and offer novel, bioinspired approaches for rotator cuff repair. The functional adaptations of bats provide insight into developing new approaches to treat GH joint instability, and a clinically relevant python-tooth inspired device can ultimately reduce the high rates of re-rupture currently observed in rotator cuff repair.

This thesis documents three main projects performed during my PhD. Chapter 3 describes a published project in which detailed behavioral analysis based on machine learning approaches for pose-estimation were used to characterize a novel sensorimotor transformation in which mice use whisker information to rapidly modify their gait in order to rapidly avoid an obstacle in their path (Warren et al., 2021). I contributed to designing experiments, data collection and analysis related to this project spanning roughly from Aug. 2018 to Aug. 2019. Appendix 1 describes a follow-up study in which I performed multi-site silicon probe recordings and anatomical reconstruction of recording sites across the deep cerebellar nuclei in head-fixed mice performing the same obstacle avoidance behavior mentioned above. Data collection for this project spanned roughly from May 2019 to Jan. 2021. This data was initially analyzed in collaboration with Richard Warren and is currently being analyzed in collaboration with Ramin Kajeh in Dr. Larry Abbott’s group. Finally, Chapter 2 reports on the major independent work undertaken as part of my thesis, spanning from Sept. 2021 to present. As such, the Introduction relates solely to Chapter 2. The goal of this ongoing project is to extend the Sawtell laboratory studies of the mechanisms for sensory prediction and cancellation in the cerebellum-like circuitry of the electrosensory lobe (ELL) of electric fish to a cerebellum-like circuit in mammals, the dorsal cochlear nucleus (DCN) in the auditory brainstem. In particular, my work provides initial insights into the function of the cartwheel cell (CWC), a previously enigmatic cell type that occupies a similar place in the circuitry of the dorsal cochlear nucleus as the Purkinje cell of the cerebellum and the medium ganglion (MG) cell of the ELL. We have demonstrated that CWCs convey tonotopically-specific signals that are well-suited for canceling self-generated auditory responses in fusiform cells (FCs), the principal output cells in the DCN. Additionally, our findings reveal that the two characteristic types of spikes observed in CWCs—the axonal simple spikes (comparable to simple spikes in Purkinje cells and narrow spikes in MG cells) and dendritic complex spikes (similar to complex spikes in Purkinje cells and broad spikes in MG cells)—are distinctly modulated by both self-generated behavior and external acoustic stimuli, suggesting that these two types of spikes serve separate functional roles in the processing of the cancellation signal, as well as auditory information, within the DCN circuitry. This finding is consistent with the reported distinct functions of narrow and broad spikes in MG cells within the circuitry of the ELL, suggesting an evolutionarily conserved role of Purkinje-like cells in cerebellum-like circuits.

Hypothalamic pro-opiomelanocortin (POMC) neurons are canonically recognized as key anorexigenic neurons in the melanocortin circuit with a role in satiety and energy homeostasis. However, optogenetic stimulation does not decrease feeding behavior during ad-lib fed animals in a physiologically relevant manner. This suggests that there are possible nuances in their activity dynamics such as timing (when a neuron is active in relation to a specific behavior), direction (inhibition or excitation), or specificity (if only certain subgroups of POMC neurons are active). POMC neurons in the hypothalamus are a molecularly diverse population, which suggests that they would display diverse neuronal activity responses during various ingestive behaviors. Currently, single-cell recordings of hypothalamic POMC neurons has never been investigated in behaving animals. Using one-photon microendoscopic calcium imaging, we characterized the neuronal activity dynamics of individual hypothalamic neurons during a broad range of feeding behaviors prior to, during, and after ingestion, during different metabolic states in mice. We show that hypothalamic POMC neurons are highly engaged during food-seeking, consumption of different nutrients, and post-ingestive responses related to circulating molecules relaying metabolic information. Individual hypothalamic POMC neurons show diverging responses in terms of valence, duration, magnitude, and timing to different feeding behaviors that are responsive to intercurrent metabolic status. Our results suggest that hypothalamic POMC neurons may integrate moment-to-moment metabolic status with feeding and food-seeking actions at short- and long-term scales to implement behaviors and autonomic responses to coordinate complex components of energy homeostasis.

Calcium (Ca²⁺) influx through Caᵥ1.2 channels mediates cardiac excitation-contraction coupling, tunes cardiac action potential duration and excitability, and regulates cardiomyocytes’ (CM) gene expression. Mechanisms regulating the sub-cellular localization, trafficking, and dynamics of surface Caᵥ1.2 in ventricular CMs are poorly understood though these are critical determinants of cardiac function. To gain new insights into Caᵥ1.2 organization, dynamics, and regulation at the CM surface we generated transgenic mice expressing an αMHC controlled cardiac-specific, dihydropyridine (DHP)- resistant α₁_ᴄ construct, tagged at the N-terminus with FLAG and HA epitopes, at the C- terminus with YFP, a 13-residue bungarotoxin binding site (BBS) inserted into in the third extracellular loop of domain II, and mutations that prevent cleavage of the C-terminus. We found robust inducible expression of DHP-resistant FLAG-HA-BBS-α₁_ᴄ-YFP in the heart that targeted to dyadic junctions, generated nisoldipine-resistant Ca²⁺ currents, supported cardiac excitation-contraction coupling, and was normally up-regulated by β-adrenergic activation with isoproterenol. Incubating transgenic CMs with AlexaFluor₆₄₇-conjugated α- bungarotoxin (BTX₆₄₇) enabled selective labeling of surface BBS-tagged Caᵥ1.2 channels. We used total internal fluorescence (TIRF) microscopy to investigate the spatiotemporal organization and dynamics of surface Caᵥ1.2 channels. Similar to endogenous Caᵥ1.2, transgenic α1C-YFP forms clusters with exponentially distributed sizes at the cell surface. A flow cytometry-based optical pulse-chase assay revealed surface Caᵥ1.2 channels in adult cardiomyocytes fully turn over within two hours. Application of angiotensin II (Ang II) decreased transgenic Caᵥ1.2 surface density and this effect was blocked by the selective Ang II receptor type I (AT1R) blocker losartan. Application of losartan by itself increased Caᵥ1.2 surface density, suggesting the potential presence of constitutively active Ang II receptors in adult CMs. Our results provide new insights into spatiotemporal organization, dynamics, and regulation of Caᵥ1.2 channels in adult CMs and introduce an approach that can be widely applied to elucidate spatiotemporal dynamics of cardiac ion channels and membrane proteins.

Proteome turnover, the process by which proteins are continuously synthesized and degraded, is a crucial biological process for gene expression regulation, cell state maintenance, cellular homeostasis, and response to stimuli. This dissertation outlines novel methods to quantify proteome turnover in both mouse-derived organoid disease models and in vivo using stable isotope labeling combined with mass spectrometry-based methods. In Chapter 1, we review recent LC-MS/MS techniques for measuring proteome turnover, highlighting their applications and limitations. Chapter 2 focuses on a systematic analysis of proteome turnover in an organoid model of pancreatic ductal adenocarcinoma (PDA) using dynamic Stable Isotope Labeling of Organoids (dSILO). This study reveals faster proteome turnover in metastatic organoids compared to primary tumors and identifies several differentially regulated protein complexes in metastatic tumors, particularly from the mitochondrial respiratory chain. In Chapter 3, we present the exploration of various methods for quantifying in vivo proteome turnover in mice. We employed an isotopic pulse-labeling strategy, dynamic Stable Isotopic Labeling of Mammals (dSILAM), then compared four combinations of mass spectrometry-based data acquisition and half-life modeling methods. We uncovered moderate differences in coverage, reproducibility, and half-life estimations between datasets, although further optimization is required for robust conclusions. Chapter 4 discusses potential limitations and future directions for all of the work described herein. Overall, our findings contribute to the optimization of proteomic workflows for studying protein turnover, which can be applied to enhance our understanding of cellular physiology and the molecular mechanisms underlying disease.

Chiu, Wang Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 95-110). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Contents --- p.v / Acknowledgements --- p.viii / Abbreviations --- p.ix / List of tables --- p.xii / List of figures --- p.xii / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Cell cycle and chromosome replication --- p.1 / Chapter 1.2 --- Telomere-associated proteins --- p.4 / Chapter 1.3 --- Telomere repair protein --- p.6 / Chapter 1.4 --- Function of telomere --- p.9 / Chapter 1.5 --- Telomerase --- p.10 / Chapter 1.6 --- Factors affecting telomere length --- p.14 / Chapter 1.7 --- Stress and telomere length --- p.14 / Chapter 1.8 --- Definition of Stress --- p.16 / Chapter 1.9 --- Central nervous system components involved in stress response --- p.17 / Chapter 1.10 --- Glucocorticoid --- p.17 / Chapter 1.11 --- Physiological effects under the activation of stress system --- p.19 / Chapter 1.12 --- Effects of chronic hyperactivation of the stress system --- p.20 / Chapter 1.13 --- Hypothesis --- p.23 / Chapter 2. --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.2 --- Experiment animals --- p.25 / Chapter 2.3 --- Methods --- p.26 / Chapter 2.3.1 --- Treatment schedule --- p.26 / Chapter 2.3.2 --- Organ extraction and serum preparation --- p.27 / Chapter 2.4 --- Serum corticosteroid assay --- p.27 / Chapter 2.5 --- Telomere length assay --- p.30 / Chapter 2.5.1 --- Genomic DNA extraction --- p.30 / Chapter 2.5.2 --- Genomic DNA digestion --- p.31 / Chapter 2.5.3 --- Southern blotting procedure --- p.32 / Chapter 2.6 --- Telomeric Repeat Amplification Protocol (TRAP) assay --- p.37 / Chapter 2.6.1 --- Extract preparation and protein concentration quanititation --- p.38 / Chapter 2.6.2 --- Real-time PCR reaction --- p.39 / Chapter 2.6.3 --- Melt Curve --- p.41 / Chapter 2.7 --- Detection of mouse telomerase reverse transcriptase component (mTERT) mRNA expression by reverse transcriptase- polymerase chain reaction (RT-PCR) --- p.42 / Chapter 2.7.1 --- Total RNA extraction --- p.42 / Chapter 2.7.2 --- RT-PCR --- p.44 / Chapter 2.7.3 --- Agarose gel electrophoresis of RT-PCR products --- p.45 / Chapter 2.8 --- Detection of mouse TERT (mTERT) by Western blotting --- p.46 / Chapter 2.8.1 --- Nuclear protein extraction --- p.46 / Chapter 2.8.2 --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.47 / Chapter 2.9 --- Statistics --- p.49 / Chapter 3. --- Results --- p.51 / Chapter 3.1 --- Body and spleen weights of study animals --- p.51 / Chapter 3.2 --- Serum corticosterone level --- p.54 / Chapter 3.3 --- Telomere lengths of spleen --- p.54 / Chapter 3.4 --- Telomerase activity of spleen tissue --- p.58 / Chapter 3.5 --- Correlation between serum corticosterone level and telomere length --- p.65 / Chapter 3.6 --- Correlation between serum corticosterone level and telomerase activity --- p.66 / Chapter 3.7 --- Correlation between telomere length and telomerase activity --- p.67 / Chapter 3.8 --- Detection of telomerase reverse transcriptase component (mTERT) mRNA expression by RT-PCR --- p.69 / Chapter 3.9 --- Detection of mTERT by Western blotting --- p.72 / Chapter 3.10 --- Correlation between serum corticosterone level and mTERT mRNA /protein expression --- p.75 / Chapter 4. --- Discussion --- p.77 / Chapter 4.1 --- Serum corticosterone level in mice --- p.78 / Chapter 4.2 --- Mice body weight and spleen weight --- p.80 / Chapter 4.3 --- Telomere lengths in the spleen tissue --- p.83 / Chapter 4.4 --- Telomerase activity in spleens --- p.84 / Chapter 4.5 --- Correlation between serum corticosterone level and telomere length --- p.87 / Chapter 4.6 --- Correlation between serum corticosterone level and telomerase activity --- p.89 / Chapter 4.7 --- Mouse telomerase reverse transcriptase component (mTERT) mRNA expression --- p.89 / Chapter 4.8 --- Expression of mTERT protein --- p.90 / Chapter 4.9 --- Conclusion --- p.92 / Chapter 5. --- References --- p.95 / Chapter 6. --- Appendix --- p.111

Indiana University-Purdue University Indianapolis (IUPUI) / Sensitization to the locomotor stimulant effects of alcohol (ethanol) is thought to be a heritable risk factor for the development of alcoholism that reflects progressive increases in the positive motivational effects of this substance. However, very little is known about the genetic influences involved in this phenomenon or the extent to which ethanol’s positive motivational effects are altered in parallel to its development. The first goal of this work was to determine the heritability of ethanol-induced locomotor sensitization in mice using short-term behavioral selection. Genetically heterogeneous C57BL/6J (B6) x DBA/2J (D2) F2 mice were generated from B6D2F1 progenitors, phenotyped for the expression of locomotor sensitization, and bred for high (HLS) and low (LLS) expression of this behavior. A secondary goal was to characterize possible line differences in ethanol’s positive motivational effects using a conditioned place preference assay. There were large and significant differences in locomotor sensitization between HLS and LLS lines by the fourth generation. Twenty-two percent of the observed line difference(s) were attributable to genes (h2=.22). However, there were no significant differences in conditioned place preference between lines despite significant line differences in ethanol-stimulated locomotion following repeated exposures. The results of this work have several implications. First, that changes in ethanol sensitivity following repeated exposures are in part genetically regulated highlights the relevance of studies aimed at determining how genes regulate susceptibility to ethanol-induced behavioral and neural adaptations. Additionally, the lack of line differences in ethanol-induced CPP, and the observation that CPP and ethanol sensitization are dissociable, suggests that 1) different genes regulate these two behaviors and 2) the utility of locomotor sensitization as a model of alterations in ethanol’s positive motivational effects is, at best, still unclear. Together these studies provide evidence that genes are capable of regulating alterations in ethanol-induced locomotor behavior but provide little support for ethanol-induced locomotor sensitization as a model for increases in ethanol’s positive subjective effects in mice.