For biomass derived molecules to serve as precursors for biofuel and other related energy sources, more stable and efficient catalysts are needed. Drawing inspiration from enzymes, our group has recently shown that a bifunctional acid–base metal–organic framework (MOF) with co-localized acid and base sites outperforms a MOF with randomly dispersed acid and base sites as a catalyst for the aldol condensation of biomass-derived carbonyls. These active acid–base sites are composed of a primary amine and carboxylic acid. However, to further improve catalytic activity a templated framework with secondary amine and carboxylic acid active sites can be developed. Relative to primary amines, secondary amines should favor the formation of the key enamine intermediate and increase catalytic rates. Framework synthesis and characterization show success of incorporation of the secondary amine, and preliminary catalysis results indicate how successful this secondary amine has been. Overall, this work expands on the previous introduction of metal-organic framework catalysts as an alternative to common industrial catalysts in the biomass upcycling process by exploring the utility of a new templated secondary amine acid–base MOF. 

Rising atmospheric carbon dioxide levels have driven research into efficient gas separation materials. Polymers of intrinsic microporosity (PIMs) is one promising solution due to their rigid, porous structures and processability, allowing them to be turned into thin films for membrane-based gas separations. My research focuses on enhancing the carbon dioxide selectivity of helicene-based PIMs through post-synthetic modification of these polymers. I have synthesized a small molecule model of the PIM to screen for amine substitution conditions and ensure the viability of post-synthetic modification on the larger helicene-based PIM. Characterization techniques, multinuclear NMR and mass spectrometry, have verified the synthesis and amination of my model system. By incorporating nucleophilic amines into PIMs, these polymers can feature enhanced binding to electrophilic carbon dioxide, thereby increasing the interactions with carbon dioxide over other mixed gases, leading to separation. In my future studies, I will extend these modifications to the helicene-base PIM, fabricate films and evaluate their properties. Surface area measurements using N₂ gas sorption methods and CO₂ absorption isotherms will quantify gas-binding affinity and separation performance.

Dynamic marine environments require long-term spatiotemporal datasets to successfully monitor and understand patterns in ecosystem composition on a decadal scale. High-resolution photography is often used to compensate for the field logistical constraints associated with marine sites, such as personnel availability or weather conditions, and works well to quickly capture data in the field. However, these photos require extensive manual analysis after the fact. As a research mentee, I asked the question: “Can image annotation with machine learning models provide enough clear data to inform community ecology studies of sessile organisms in subtidal habitats?” Using images collected through transect/quadrat sampling by the Sebens Lab’s Salish Sea Long-term Monitoring Project (University of Washington Biology and Friday Harbor Laboratories), a variation of an open-source model (CoralNet) was trained in the identification of relevant sessile flora and fauna. I then used CoralNet to continue the work of the lab by generating sessile assemblage metrics for a single site in the Friday Harbor Laboratories Biological Preserve. This site included images from transects at two different depths, with sampling from 2014 and 2024. After being uploaded, CoralNet identified 200 random points per image to the lowest possible taxon. I then reviewed all annotations for accuracy and corrected them whenever necessary. This technique greatly reduced the time spent per identification, without sacrificing accuracy. Next, I calculated species richness and Simpson’s Diversity for each quadrat and transect, comparing between depth and year. My analysis showed significant increases in both metrics from 2014 to 2024 and no significant differences between depths, demonstrating a successful report on the dynamics of a subtidal marine community. Application of this method to the entire existing dataset (7 sites, 63 transects per year), and others, provides opportunities to streamline analysis of sessile community composition.

The angiopoietin-Tie2 signaling pathway is central to regulating vascular stability, remodeling, and permeability. Angiopoietin-1 (Ang1) promotes pAKT activation and vascular stability and regeneration, whereas Ang2 antagonizes these effects, leading to leaky vasculature. Although Tie2’s association with α5β1 integrin has been implicated in mediating these divergent outcomes, the requirement of direct F-domain ligand binding for integrin recruitment remains unclear. Here, we report the development and mechanistic evaluation of a de novo designed Tie2 mini binder (Tmb) that selectively targets the Tie2 receptor without engaging α5β1 integrin. Using an AI-based protein design pipeline, we designed Tmb with high affinity (KD ≈ 0.65 nM) for Tie2, as confirmed by CryoEM analysis, which demonstrated that Tmb accurately recapitulates its designed structure. When conjugated to multivalent scaffolds, Tmb effectively clusters Tie2 receptors, recapitulating the signaling profile of native Ang1. Notably, high valency Tmb constructs (e.g., H8T) robustly activated pAKT and induced nuclear FOXO1 exclusion, mirroring the pro-survival and vascular stabilizing effects of Ang1, despite lacking the capacity to bind α5β1 integrin directly. Detailed cellular assays revealed that Tie2 clustering leads to the formation of two distinct complexes: a Tie2–α5β1 integrin complex that facilitates focal adhesion assembly and cell migration via pCAS recruitment, and a Tie2–tight junction complex (comprising ZO1, claudin-5, and occludin) that underpins vascular barrier integrity. Importantly, competitive binding studies demonstrated that integrin recruitment to the Tie2 complex does not require direct F-domain engagement. In human iPSC-derived diabetic blood vessel organoids, treatment with Tmb-based Tie2 agonists ameliorated diabetic vasculopathy phenotypes by reducing pathogenic collagen IV deposition, restoring tight junction organization, and lowering nuclear FOXO1 levels. These findings provide novel insights into the mechanistic interplay between Tie2, integrin, and junctional proteins, and underscore the therapeutic potential of synthetic Tie2 agonists in vascular repair and diabetic vasculopathy.

Over a hundred years after the Spanish Flu, the influenza A virus (IAV) remains a leading cause of respiratory infections and mortality worldwide. The proliferation of IAV causes many of the symptoms associated with IAV clinical disease. However, the severity of acute lung injury (ALI) from IAV is primarily driven by the host's immune response to infection. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated protein kinase (ERK) signaling cascade is a highly conserved pathway that is activated during lung injury and inflammation in both rodents and humans. Two MEK isoforms, MEK1 (Map2k1) and MEK2 (Map2k2), activate downstream effectors, ERK1 and ERK2, and control critical cellular processes, including the intensity and duration of inflammatory signaling. Our prior research revealed that MEK2-deficient mice exhibited improved weight recovery and overall fitness during IAV infection, suggesting that MEK2 is a host response exacerbating ALI during IAV infection. During IAV infection, excessive pro-inflammatory cytokine production drives immune cell recruitment into the lungs, leading to collateral tissue damage that impairs organ function and exacerbates disease. We hypothesize that MEK2 enhances immune cell recruitment to the lungs, enhancing inflammation, which may occur through exuberant chemokine signaling. To investigate this, we infected MEK2-deficient and wild-type mice with mouse-adapted IAV (H1N1, PR8) and collected cells from bronchoalveolar lavage (BAL) and lung homogenates. Using flow cytometry, we found reduced immune cell recruitment, including decreased numbers of monocytes, dendritic cells, monocyte-macrophages, CD4+ T-cells, CD8+ T-cells, and B-cells. Next, we assessed levels of key chemokines known to attract monocytes and lymphocytes by measuring their gene expression in the lungs and protein levels in the BAL. Investigating MEK2’s impact on chemokine signaling will elucidate the mechanism by which MEK2 perpetuates lung inflammation and injury during IAV infection and will guide the development of future host-directed therapies for IAV-induced lung damage.

The Kinnar are a South Asian genderqueer community, who possess the power of shraap or ashirwaad - the power to curse or bless. The use their powers to perform blessing (badhai), sex, and begging work. While they are regarded as deities and a "third gender," they have historically been othered in South Asia's dominant colonial, cultural, and religious archives. These archives paradoxically narrate the Kinnar as less-than-human and more-than-human, trapping them in the ontological category of the "non-human." In my multi-genre, multi-modal book, Of Ghosts & Gods, I seek to understand the development of Kinnar identity and de-humanization. I ask three main, interwoven questions: (1) How do Kinnar peoples narrativize themselves? (2) How have their identities and lives been (de)constructed under various empires (Mughal, British, contemporary Hindu fundamentalist)? (3) How have they managed to survive - despite and with - a paradoxical identity under empire? I amalgamate several Kinnar testimonies, Hindu epics, Burke Museum archives, and personal experiences to get at this inquiry. I bring these sources together through ethnographic and critically fabulative methodologies, in an effort to amplify and ally with Kinnar voices. Through this book, I want to help visibilize the Kinnar peoples in ways they may wish to be made visible. It is important to do so, as imperial projects invested in eliminating the Kinnar relied on gross misrepresentation of the community to justify their dehumanization. The urgency of this work increases when we recognize that the Hindu-fundamentalist administration of 2025 India continues this work, limiting Kinnar livelihoods through its unquestioned religious assumptions. I am not a member of the Hijra, Kinnar, Khwajasarai or other South Asian genderqueer communities. To re-write archival violence therefore, Of Ghosts & Gods strives to place Kinnar voices before my own. 

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.

In the developing brain of a fruit fly (Drosophila melanogaster), neural stem cells, called neuroblasts, divide to produce new cells that will become neurons. These divisions follow strict biological rules, but because many factors influence how and when neuroblasts divide, predicting their behavior is challenging. While lab experiments provide crucial insights, they are often limited in how many conditions can be tested at once (genetic, physical, or otherwise). To address these limitations, we developed an agent-based computer model that simulates neuroblast divisions and their interactions with neighboring cells. Our model allows exploration of different conditions to predict how neuroblasts behave in complex environments. This work focuses on three key hypotheses about neuroblast behavior: (1) post stem cell division, the larger cells are more likely to remain as stem cells, (2) the cell positioned on top during division will keep its stem cell identity, and (3) clustering of differentiated neural cells on the membrane of a neuroblast suppresses their division. To investigate these hypotheses, we examine emergent behaviors in our model through size-based, location-based, and clustering-based differentiation rules. By adjusting parameters such as cell placement, division timing, and proximity to other neuroblasts, we analyze how these factors influence neuroblast fate. We validate model predictions against experimental data by comparing division patterns observed in simulations to those seen in Drosophila brains through live imaging. By combining computational modeling with experimental data, this work provides a framework for understanding the factors responsible for neural development. Our findings will refine existing models of neural stem cell behavior and help guide future experiments, making it easier to uncover the fundamental rules of brain development.

This project analyzes the Blade Runner films in order to rethink the cyborg, a theory articulated by Donna Haraway as a metaphor that transgresses binaries which uphold systems of oppression, such as the distinctions between male/female, organic/inorganic, and human/nonhuman. For Haraway, the cyborg is a paragon of agency and liberation; however, Blade Runner imagines a world in which cyborgs – in this case human-like androids called replicants – entrench capitalist ideals in addition to racist and anthropocentric hierarchies. In the films, the replicants are coded as ambiguous racial Others who flexibly inhabit the symbolic position of Black, Asian, and white persons. Yet they are still placed above non-white humans within the racial hierarchy of the films because of their contributions to the capitalist and colonial projects of the future. In other words, they take on the role of a “model minority” desired for their production of capital, yet despised for being quintessentially non-human. The replicant-cyborg in Blade Runner reflects the societal desire for a class of laborers that will submit to capitalist interests, demonstrating (contra Haraway) its failure to disrupt established systems of power. However, while this paper interrogates our faith in the potential of the cyborg, it would be remiss to disavow this figure completely. In light of the cyborg’s associations with capitalist ideals, how might we reconsider it and our relationship with new technologies as they emerge? How can we conceptualize a future in which entanglements with technology are liberating rather than oppressive? These are ongoing questions for this project, which I explore in returning to Blade Runner.

Downregulation of the mTOR complex has been shown to increase lifespan and delay development of multiple organisms, including Drosophila melanogaster. Rapamycin, an inhibitor of this complex, is undergoing FDA-approved clinical trials as a promising anti-aging drug. However the impact of genetic variation on rapamycin's response is unknown. Our study of 140+ genetically diverse Drosophila strains revealed significant variation in pupation time after rapamycin exposure, however, the underlying mechanisms of this variation remain poorly understood. Surprisingly, this sensitivity does not correlate with genetic variation in or around the mTOR gene. We therefore hypothesize that differences in phosphorylation of downstream mTOR targets may explain this variation. Currently, we are using multiple approaches to investigate how activation of downstream targets differs between highly resistant and sensitive strains. We aim to characterize the phosphoproteome of first instar Drosophila larvae from highly sensitive and resistant strains. First instar larvae were treated with rapamycin for 12 hours, followed by mass spectrometry analysis to identify phosphorylation changes in mTOR pathway targets. To validate that 12 hours of treatment induces a rapamycin response, we monitored the growth of a parallel group of larvae until 72 hours and measured their size. Sensitive DGRP strains, 348 and 517, showed a twofold reduction in length when treated with 20uM rapamycin compared to control (p-value <0.0001), while the resistant strain, 441, showed no significant decrease. Comparing the phosphoproteome of multiple resistant and sensitive lines will uncover molecular factors associated with resistance or sensitivity. Additionally, whole-larvae RNA-seq will assess the expression profile of these factors, revealing whether gene expression of tor pathway-related genes contributes to sensitivity. Understanding the mechanisms behind rapamycin resistance or sensitivity is critical for its clinical application. This project highlights the value of accounting for genetic variation in drug development, guiding future approaches for developing new drugs.

Directly converting fibroblasts (that make up scar tissue) into skeletal or heart muscle without a pluripotent intermediate (direct skeletal muscle or cardiac reprogramming) is one of the most promising methods for regenerating lost muscle tissue, but its low efficiency in human cells remains a significant obstacle toward clinical application. In collaboration with the Institute of Protein Design, UW, we have designed several synthetic minibinders against receptor kinases which are highly specific to their cognate receptor. Utilizing these minibinders we have created a new class of designed protein, called heterofusions, that fuse two unrelated minibinders together to force the two cognate receptor kinases together in an unnatural pairing, which could elicit novel signaling responses not achievable using natural ligands. However, which heterofusions elicit novel signaling is unknown. We aim to use direct skeletal muscle and cardiac reprogramming systems, which would benefit from this novel signaling, to screen which heterofusions elicit novel signaling to increase efficiency. To do this I developed an inducible direct cardiac reprogramming system and we also used a previously established inducible direct skeletal muscle reprogramming system to be backgrounds for screening heterofusions, with efficiency determined by imaging cardiac and skeletal muscle development makers. We found a few heterofusions, including that which brings together TrkA and BMPRII (TAB2), increased the efficiency of skeletal muscle reprogramming. I found in signaling experiments using Chinese hamster ovary cells modified to express human TrkA and BMPRII that TAB2 upregulates pERK and pCREB. Interestingly, pCREB is not part of native TrkA or BMPRII signaling, meaning novel signaling is occuring. Additionally, I have shown pCREB inhibition with a small molecule impairs direct skeletal reprogramming and TAB2’s ability to increase efficiency, showing pCREB is TAB2’s mechanism of increasing efficiency. These results show heterofusions novel signaling abilities and its applications in revolutionizing regenerative therapies.

Upon nerve injury and neurodegeneration, neuron regeneration is crucial to maintain proper function. However, this natural process happens infrequently and slowly. Neuron regeneration is known to be mediated by the activity of nerve growth factor (NGF) in neurons, which binds to two receptors: tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR). Previous studies have shown that engaging the receptor p75NTR activates a signaling pathway that also triggers a pain response, thus it would be ideal to have a ligand that only activates TrkA for neuron regeneration without initiating the pain response. In collaboration with the Institute for Protein Design (IPD), this study investigated an AI-designed TrkA agonist that specifically binds to and activates only the TrkA receptor. We used fibroblasts transdifferentiated into neurons as a model to study the efficiency of this TrkA agonist. Western blotting was used to study the phosphorylation of the proteins downstream of TrkA in the signaling pathway, such as pPLCγ, pAkt, and pErk, and the activity of transient receptor potential vanilloid 1 (TRPV1), a calcium channel that indicates the sensitivity of a neuron. Immunofluorescence staining was used to examine the expression of calcitonin gene-related peptide (CGRP), a neuropeptide involved in pain perception. We found that the designed TrkA agonist generates a similar level of activation of downstream proteins as NGF while successfully preventing the expression of pain response markers. Directly injecting NGF as a treatment for neurodegenerative diseases is generally not considered viable as it often induces significant pain, therefore this TrkA agonist has the potential for therapeutic use.

Natural growth factors like fibroblast growth factor (FGF) are essential for maintaining pluripotency in induced pluripotent stem cells (iPSCs). However, current limitations of native growth factors include signal instability, off-target pathway activation, and dependence of xenogenic components for production. To address these issues, we developed a synthetic protein, C6-79C, which consists of six scaffolded subunits of a de novo designed FGFR1/2c binder, mb7. While mb7 functions as an FGF pathway inhibitor, the hexameric C6-79C acts as a receptor tyrosine kinase (RTK) agonist, providing more isoform-specific and prolonged signaling compared to native FGF. We formulated SynGrow, replacing FGF with C6-79C in minimal E8 media, and compared its performance against commercial media. Our study focused on three objectives: (1) comparing the expression of pluripotency markers (Oct4, NANOG, SOX2, and TRA1-60) in cells grown in SynGrow versus commercial media, and (2) evaluating morphology and viability under different media change regimens (daily, every other day, or no change). iPSCs grown in SynGrow exhibited superior morphology compared to those in mTeSR (commercial media). Pluripotency markers (Oct4, NANOG, and SOX2) were expressed at similar levels in both media, with SynGrow also showing higher expression of TRA1-60 across passages, confirmed by flow cytometry. Future evaluations will assess germ layer marker expression following directed differentiation. Our findings demonstrate that synthetic protein-based media formulations, like SynGrow, can effectively replace native growth factor-based media. This approach offers stable, prolonged, and xeno-free alternatives for stem cell culture, with broad implications for improving reproducibility and safety in regenerative medicine and cell-based therapies. 

Salivary glands are organs in the mouth which produce and secrete saliva, a multifunctional fluid crucial for processes including oral cavity lubrication, digestion, and antimicrobial functions. Diabetes mellitus has been associated with salivary gland dysfunction and harmful oral consequences including severe tooth decay and disrupted wound healing, yet it is not currently known what cell populations are affected in salivary glands and how this disease affects cell organization, function, and metabolic response. One model for diseases in human tissues are organoids, three-dimensional multicellular systems derived from stem cells which self-organize to mimic the structure and function of tissues in vivo when given the right cues. Dr. Devon Ehnes in the Ruohola-Baker Lab recently created a protocol to develop salivary gland organoids from induced pluripotent stem cells (iPSCs), and through additional culture in a high-glucose media along with inflammatory cytokines, this organoid has been used to study how diabetes affects salivary glands. Preliminary analysis has suggested acinar and ductal cell dysfunction and mitochondrial stress as causes of salivary gland dysfunction, but further work is necessary to understand how this diabetic environment leads to changes in cell function and mitochondrial activity. Here, I use a human iPSC-derived organoid model to assess how diabetic conditions affect the expression and localization of the acinar marker AMY1A, the ductal marker KRT19, the cell stress marker FOXO1, and the mitochondrial marker ATPB to determine the mechanisms for salivary gland dysfunction in diabetes.

Human tooth development is a complex and tightly regulated process that involves multiple signaling pathways and specialized proteins coordinating enamel formation. Enamel, the hardest tissue in the human body, is secreted by ameloblasts, which follow a distinct developmental process. Disruptions in these processes can lead to enamel-related disorders, such as amelogenesis imperfecta, a genetic condition characterized by defective enamel formation. A key factor in this disorder is WDR72, a gene that encodes the tryptophan-aspartate repeat domain 72 (WDR72) protein, which is critical for intracellular trafficking during enamel maturation. Although WDR72 has been studied in animal models, its precise localization and function in human fetal tooth buds remain incompletely understood. To address this question, I cryosectioned human fetal tooth samples at 19 and 22 gestational weeks and performed immunochemistry staining to visualize WDR72 alongside key enamel proteins. I performed cryosectioning to prepare thin tissue sections of each tooth bud sample, followed by immunohistochemical staining with antibodies specific to WDR72. I then imaged selected sections under a fluorescence microscope. Preliminary results suggest distinct WDR72 distribution in regions corresponding to secretory ameloblasts. These findings offer insights into the localization of WDR72 during tooth formation and lay the groundwork for future studies on the mechanisms of tooth regeneration. 

This project critically examines online narratives about human and more-than-human cloning, with a focus on the spread of misinformation, radicalization, conspiracies, and their dangerous impact. At first glance, discussions about human vs. more-than-human cloning differ significantly. Human cloning is commonly considered morally objectionable, with supporters often forming part of controversial communities. In contrast, more-than-human cloning frequently sparks curiosity and, in some contexts, is encouraged. It is viewed not as an "act against God" but as a testament to human intelligence and dominance. This difference in responses raises many questions: Why are responses so dissimilar? How does online discourse drive these reactions? And can these distinctions--these different understandings of personhood and "life"--reinforce or perpetuate ideologies that cause harm? To answer these questions, I examine academic explorations of cloning and compare them with ones found all across the digital sphere-from social sites such as Reddit, X (formerly known as Twitter), and 4chan, to YouTube comment sections. Using a digital, “websplorer” approach, I analyze different perspectives on cloning, ranging from the "manosphere"-- interconnected misogynist online communities, scientism, and how they relate to the more-than-human. After a critical interrogation of these perspectives, I invite the user to consider an alternative, perhaps more ethical, approach to discussing cloning, one that does not reinforce heteronormativity, human exceptionalism, or pro-eugenic views. This alternative approach includes an exploration and critique of the Western concept of “personhood” and its limitations regarding cloned life, human and more-than-human.

Mitochondrial fusion is essential for cellular function, metabolism, apoptosis, and stress responses. Mitochondrial outer membrane fusion is mediated by two mitofusin paralogs, Mfn1 andMfn2, which are large GTPases that remodel cellular membranes. Membrane fusion likely proceeds through two distinct steps, first tethering two organelles and second lipid mixing; however, much of the mechanism is poorly defined. Previous studies have solved crystal structures of a partial construct of the mitofusins, revealing a GTP dependent conformational change ; however, this is not a complete analysis as at least two states in the catalytic cycle are missing. Our project aims to quantify the conformational changes of Mfn2 throughout the entire mechanism of GTP hydrolysis. To achieve this, we are utilizing a novel transition metal Förster Resonance Energy Transfer (tmFRET) developed by Dr. Gordon and Dr. Zagotta. This system utilizes a noncanonical amino acid as the donor and a transition metal as the acceptor to measure changes as small as 3Å. Currently, we’re mutating the cystines to develop a single donor-acceptor pair, while keeping the stability and GTPase function of Mfn2. Our main approach is to introduce targeted mutations in key cysteine residues and analyze their effects on the protein’s enzymatic activity. Using molecular biology, we design DNA plasmids encoding the mutations,and express and purify the mutant proteins.  Finally we measure the GTPase activity using malachite green assays. Our current findings suggest some mutations have trivial impact on MFN2’s GTP hydrolysis, suggesting that it’s viable. The further goal of our project is to keep only one solvent accessible cysteine while maintaining protein function. This research will further elucidate the mechanism of mitochondrial fusion and its role in disease pathogenesis. Explanding the biophysical understanding of membrane remodeling.

Protein Kinase Inhibitors (PKIs) are a family of heat stable, high-affinity inhibitors of the catalytic subunit of Protein Kinase A (PKAc). In the presence of Mg-ATP, the three isoforms—PKIα, PKIβ, and PKIγ—bind to PKAc with very low dissociation constants: 0.758nm, 1.875nm, and 0.4142nm respectively. In vitro studies have shown that PKIs can translocate PKAc from the nucleus to the cytoplasm, suggesting a role for PKIs in terminating nuclear cAMP-driven PKA activity. Previous research, including studies from our lab, has found that dysregulated PKAc mutants play a significant role in Cushing’s syndrome, a rare and potentially fatal metabolic disorder caused by excessive cortisol production. Building on these findings, we hypothesized that increasing PKI expression could counteract the hyperactivity of PKAc mutants and reduce cortisol production. To test this, we expressed each PKI isoform in adrenal cell lines and assessed their steroidogenic capacity using biochemical assays such as western blots, RNA-seq, qPCR, and ELISA-based cortisol assays. We observed that PKIα and PKIγ led to a general suppression of steroidogenic associated proteins such as StAR, Cyp11a1 and SF1. This altered proteome was accompanied by significantly suppressed cortisol synthesis only in the PKIα and PKIγ expressing cells. The difference between PKIα/γ and PKIβ was surprising given that all PKI isoforms are postulated to potently inhibit PKAc. Thus, we questioned whether PKIα/γ effects are mediated through PKAc. To answer this, we have cloned mutant PKI isoforms that do not bind PKAc, and confirmed the mutant PKIs do not inhibit PKAc through kinase assays. Our next step is to express the mutant PKI isoforms in adrenal cells and assess their effect on steroidogenic capacity of the cells. Our findings suggest that PKIα and PKIγ play key roles in cortisol regulation and may have broader implications for gene regulation in adrenal cells.

Under acute genotoxic stress, such as chemoradiation, stem cells can undergo cell cycle arrest at the G1/S phase to avoid apoptosis. This protective state, called quiescence, is reversible once stress-free conditions allow re-entry into the cell cycle to regenerate daughter cells. We have previously demonstrated a common mechanism by which two types of stem cells—Drosophila germline stem cells (GSCs) and human-induced pluripotent stem cells (hiPSCs)—enter quiescence. Recently, we found Cyclin E (CycE) associated with the outer mitochondrial membrane (OMM) in both GSCs and hiPSCs. We are interested in studying the interaction between CycE mitochondrial localization domains and mitochondrial proteins responsible for CycE localization.To map the CycE mitochondrial localization domain, I have generated four CycE truncations tagged with GFP: ΔN-terminus, ΔCyclin Box_N terminus, ΔCyclin Box_C terminus, and ΔC-terminus. I have tested these constructs in various cell lines, including Rcc4, HCT116, MCF10A, HEK, and HeLa, and found that HCT116 exhibits mitochondrial localization of CycE. I will compare the localization of wild-type CycE-GFP versus mutant CycE using immunofluorescent staining of CycE and mitochondria in HCT116, as this cell line is well-suited for transfection studies. We have shown that mitochondrial CycE is degraded in quiescent stem cells through PINK1/PARKIN-mediated mitophagy. We propose that CycE degradation is necessary for quiescence entry. In Drosophila GSCs, we observe that upon irradiation, cells overexpressing non-degradable CycE continue cell division, whereas control cells undergo quiescence. Understanding the mechanism by which Cyclin E localizes to the OMM will enhance our knowledge of how it prevents quiescence entry, thereby contributing to the development of anti-cancer treatments.