Recent advances in ultra-high-resolution frequency comb spectroscopy have enabled the observation of previously unresolved spectroscopic details in small molecular systems. However, current theoretical frameworks are insufficient to fully describe the complex interactions between internal and overall rotational angular momenta, and higher frequency vibrational modes, particularly in molecules with multiple internal rotors. This work focuses on elucidating the coupled torsional, rotational, and vibrational kinematics of dimethyl sulfide (DMS), an asymmetric top with two internal methyl rotors which generate a rich and highly structured spectrum. We develop a general theoretical approach that incorporates torsional angular momenta into the overall molecular framework by systematically coupling the individual degrees of freedom, which are initially described in their well-known primitive bases, into a fully symmetrized torsion-rotation-vibration Hamiltonian. Through this systematic approach, interactions between the overall rotational and internal angular momenta of the methyl groups are explicitly addressed, capturing the effects of intrinsic Coriolis couplings and the tunneling splittings of the rotors. The resulting eigenstates and energy spectrum are analyzed to predict spectroscopic transitions, which are then compared with experimental findings, allowing the assignment of observed peaks to specific ground and excited quantum states. This rigorous treatment provides insights into nontrivial state mixing and previously unresolved splittings observed in high-resolution spectra. The methods developed in this work offer a pathway toward more accurate analysis of complex molecular systems and clusters, with broader applicability to high-resolution spectroscopy in atmospheric, astrochemical, and low-temperature environments.

BK channels are potassium channels activated concomitantly by membrane depolarization and the elevation of intracellular calcium. We have previously shown that BK channels form clusters at the plasma membrane in heterologous cells and primary neurons, but the mechanism for their clustering is unknown. Our research seeks to discover important components that generate and maintain BK channel clusters. We hypothesize that membrane lipidic composition can be essential in BK clustering. Given the known role of PIP2 in increasing the activity of BK channels, we evaluated the role of PIP2 in their spatial organization. We expressed BK channels in a human cell line and assessed the organization in clusters using super-resolution microscopy and proximity ligation assay. We also measured ion channel mobility using fluorescence recovery after photobleaching. We expressed PIP5K and Ins5P to increase and decrease PIP2 levels, respectively. Preliminary experiments found that the expression of PIP5Kγ did not affect the mobility of single or cluster BK channels, but decreased density of channels at the plasma membrane.

Skin serves two key functions: hardened cells at the surface of the skin form a superficial layer to protect against the environment, while the inner layers of the skin are packed with diverse sensory machinery which allow us to perceive and navigate the world. Incredibly, the basal most layer of the epidermis houses stem cells which allow the skin to constantly renew itself, fortifying its protective function and maintaining somatosensation by replenishing all these diverse cell types. Perhaps unsurprisingly, these multipotent and highly active skin stem cells are emerging as an effective way to treat genetic skin conditions, promote wound healing, and rejuvenate ageing skin. To understand how skin stem cells contribute to these different functions, investigators are studying the many niches within the skin which may house diverse skin stem cells. Zebrafish are an excellent model to dissect this topic due to their translucent skin and the many genetic tools available. However, the anatomy and molecular characteristics of zebrafish skin is poorly described. Recently, we performed single cell RNA-sequencing of zebrafish skin and identified seven presumptive skin stem cell subpopulations. Informed by this data, I performed whole-mount hybridization chain reaction, a form of in-situ hybridization, to investigate molecular and spatial heterogeneity in zebrafish skin stem cells. My results have identified three novel skin stem cell subpopulations which occupy distinct spatial domains along the anterior-posterior axis. I found that the appearance of each subpopulation and the establishment of their spatial domain is dynamic throughout skin development. Finally, we have constructed a tool to interrogate their behavioral and functional differences. Moving forward, I aim to determine each subpopulation’s role in skin development, homeostasis, and regeneration, as well as whether they serve as specific progenitors for certain cell types.

Cell shape opens a powerful window into the genetic and mechanical processes that drive cell behavior and, ultimately, tissue morphogenesis during development. By identifying cell shape, we can track specific cells and their responses to different gene expressions - creating a clearer mapping of which cells are affected by various manipulations. In this project we combine computational tools with quantitative microscopy to measure nucleus shape, and use these readouts to identify different cell types in the pectoral fins of zebrafish embryos. High resolution images of pectoral fin nuclei were taken using confocal microscopy - a technique commonly used when capturing tissue and cell data. Following nucleus identification and segmentation during data pre-processing, the FlowShape analysis package was utilized to extract quantitative "shape vectors" that encode the morphology of each nucleus. We plan to leverage the spherical harmonic weights produced within FlowShape to cluster and identify key shape-types that emerge from the collected nuclei. These shape readouts will serve as the basis for future analyses aimed at classifying different nucleus morphologies within the pectoral fin. Ultimately we hope to use nucleus morphology to predict the expression of key marker genes. This approach provides a powerful method for bridging the gap between the rich gene expression information provided by single-cell RNA-seq atlases, and the dynamical and morphological information produced by in vivo microscopy.

Female mammals possess two X chromosomes in every cell, but one is silenced by condensing into a barr body, making its genetic information largely inaccessible. While X inactivation is stable in somatic cells, it is reversible in germ cells, raising the intriguing question of what proteins maintain this silenced state. My project aims to identify the protein composition of both active and inactive X chromosomes in mice. To achieve this, I will use in situ hybridization to target proximal labeling with biotin of X chromosome-associated proteins. This is accomplished by targeting a biotinylation enzyme, such as HRP, to the X chromosomal region, where it will selectively biotinylate neighboring proteins. After affinity purification, these proteins can be identified using mass spectrometry-based quantitative proteomics. To direct the enzyme to the correct location, a two-probe system is employed. The primary oligonucleotide probe complements a specific X chromosome region which also contains landing sites for a secondary probe. Hybridization of the secondary probe which is tagged with HRP enables precise labeling of chromosome-associated proteins. This approach enables in situ biotinylation, preserving proteins in their native context for accurate identification. Since the two X chromosomes are homologous, distinguishing between the active and inactive X requires careful probe design. By utilizing Single Nucleotide Polymorphisms (SNPs) that exist in the X chromosomes, the maternal and paternal X chromosomes can be differentially targeted by primary probes, allowing for homolog specific protein labeling and analysis of their distinct regulatory environments. 

Somatosensory neurons innervate the skin, where their peripheral axons detect signals like touch and pain. The neurons relay stimuli to the brain via peripheral axons in the skin and spinal cord axons in the spinal cord. Given their superficial location, somatosensory axons are susceptible to damage. Axon damage can cause tingling, increased pain, or sensory inhibition, and reinnervation in mammals is often slow or incomplete. I use injury models in zebrafish to study the mechanisms of successful axon regeneration in an adult vertebrate with optically accessible skin. I aim to reveal conserved regeneration patterns of somatosensory neurons. Furthermore, I seek to understand the extent of reinnervation success and observe the prevalence of hyperinnervation post-injury. Using in vivo confocal microscopy and adult zebrafish skin models, I created a methodology to capture somatosensory reinnervation over a three-week span following a scale pluck injury. Zebrafish scales separate epidermal and dermal layers of skin, and scale removal induces regeneration of epidermal skin and surrounding dermal tissue. I use transgenic zebrafish with fluorescent labels for dorsal root ganglion DRG neurons and osteoblast cells Tg(p2rx3a:mCherry);Tg(sp7:EGFP). DRG neurons are the primary somatosensory neuron in adult zebrafish, and osteoblasts allow me to view the scale alongside axon reinnervation. For image acquisition, I designed a 3d-printed chamber for zebrafish mounting and intubation within our confocal microscope. For analysis, I developed Image J macros which use threshold analysis to quantify changes in axon density of specific regions of regenerating axons. Dermal axons tend to regenerate first while superficial axons in the epidermis regenerate secondarily in conjunction with the novel scale. To examine skin layer differences, I separate epidermal and dermal layers to compare the reinnervation trends between superficial and dermal axons. With this data, I can gain insight in the regeneration potential of somatosensory neurons.