Embryonic development is a process of regulated growth by which all cells are initially established. Previous studies suggest that there may be differential metabolic regulation in developing embryonic tissues. However, little research has been conducted to determine the specific metabolic factors that are differentially active during development and the key regulatory elements behind these metabolic processes, such as glycolysis. Hypoxy-inducible factor 1-alpha, hif1α, is a stress-induced transcription factor that is known to regulate glycolysis under hypoxic conditions. This project aims to investigate the role of hif1α in regulating the expression of glycolytic genes in the developing axial tissues of the Xenopus tropicalis embryo. X. tropicalis is a unique model for studying embryonic development. Due to their complete early-stage embryo cleavages, inhibitors can be restricted to one side of the embryo allowing for direct comparison with an internal control on the other side of the embryo. Preliminary data from in situ hybridization suggests that the inhibition of hif1α using translation-blocking morpholinos reduces glycolytic gene expression in early-stage X. tropicalis embryos. Based on these results, we plan to further test the regionalization of glycolysis, by determining what causes hif1α to activate a glycolytic gradient in certain tissues in the X. tropicalis embryo. This research implicates hif1α as a potentially important transcriptional regulator of glycolytic gene expression during embryonic development, and can lead to discovering new ways in which differential metabolic regulation can contribute to the form and function of embryos.

The eyecup consists of retinal pigment epithelium (RPE), choroid, and sclera. Eyecup tissue is used as a proxy for RPE. RPE metabolism is assumed to be dominant in eyecup preparations, but this has yet to be proven rigorously. In this study we probe the contribution of retinal pigment epithelial cells to eyecup metabolism. We approach this question by determining metabolic flux in eyecup tissue from control and mice injected with 50 mg/kg of the selective RPE cell toxin sodium iodate (NaIO₃). Seven days later we sacrificed mice and dissected eyecup tissue into physiological buffer, then quantified extracellular flux of glucose and lactate using spectrophotometric assays or intracellular flux using gas chromatography-mass spectrometry. We compared glucose flux in eyecups from saline-injected control mice to eyecups from NaIO₃-injected mice. NaIO₃ treated eyecups released 42% less lactate from media than controls (p<0.05), despite negligible glucose consumption. Surprisingly, most glycolytic and tricarboxylic acid (TCA) cycle metabolite levels were unchanged by NaIO₃ injection. NaIO₃ treatment did however significantly decrease levels of lactate and the TCA cycle metabolites malate and fumarate. Flux from glucose to lactate and malate was also decreased by NaIO₃ treatment. Our results suggest that lactate export in the eyecup is partly due to retinal pigment epithelium metabolism. However, metabolite levels and flux were partly maintained, implying that RPE metabolism may not be dominant in the eyecup. Remaining glucose metabolism may be from endothelial cells, or microglial cells recruited to the eyecup after NaIO₃ treatment. These findings may give insight into diseases affected by RPE metabolism, including some forms of retinitis pigmentosa and age-related macular degeneration. However, further analysis is still required to fully understand the role of RPE metabolism in the eyecup.

Unlike mammals, western clawed frog (Xenopus tropicalis) tadpoles are able to completely regenerate their spinal cord after tail amputation. This complete spinal cord regeneration is due to the ability of their neural progenitor cells (NPCs) to differentiate into neurons successfully. Our research focuses on two transcription factors—Meis1 and Pbx3– that are upregulated by regenerating neurons and are necessary for successful regeneration. We aim to elucidate how these two proteins are working together to guide successful spinal cord regeneration in X. tropicalis tadpoles. I am specifically investigating Meis1 and Pbx3 splice variant expression during neural regeneration. Previous work in mice found that different known splice variants of Pbx3 have different expression patterns. While X. tropicalis has two predicted splice variants each of Meis1 and Pbx3, nothing is known about their individual expression or function. I sought to fill in this gap by looking at Meis1 and Pbx3 splice variant expression in different tissues and over regenerative time. Based on previous research in mice, I hypothesize that both splice variants of Meis1 and Pbx3 have different gene expression patterns in different cell types over regenerative time. I aimed to investigate this hypothesis by doing two experiments. My first experiment was to study the expression of each splice variant over regenerative time by performing qPCRs in order to look at the presence of splice variant mRNA in uninjured, 24, and 72 hours post-amputation (hpa). For my second experiment, I am making in situ hybridization probes specific for each splice variant to identify their tissue-specific expression patterns. The experiment is being performed over regenerative time to observe how expression in tissues changes at the same time points hpa used in the first experiment.

While humans cannot regrow a limb after amputation, animals like the Xenopus tropicalis tadpoles can fully regenerate their limbs. How metabolic pathways are reprogrammed to support the demand for anabolic building blocks needed for regeneration is still not understood. Our group has found that while regeneration increases glucose intake, tadpole tail regeneration does not depend on glycolysis for precursors to support proliferation. This result suggests a redirection of glucose flux to pathways other than glycolysis, specifically the pentose phosphate pathway (PPP). Previously, I inhibited glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in the PPP, with the pharmacological antagonists dehydroepiandrosterone (DHEA) and G6PDi and showed that inhibitor-treated tails are much shorter compared to dimethyl sulfoxide (DMSO) controls. From these results, I predicted that the PPP is required throughout regeneration to support increased cell proliferation rates. To investigate if the PPP is required for increased cell proliferation during regeneration, I performed phosphohistone-H3 (pH3) immunohistochemistry to label mitotic cells. Histone-H3 is phosphorylated at the start of mitosis, making pH3 a marker for actively dividing cells. The results showed that amputated tadpoles with inhibited PPP have reduced cell proliferation rates compared to controls, confirming that the PPP is required to support rapid cell proliferation during regeneration. To determine if the PPP has a shorter critical activation window during regeneration, I treated amputated tadpoles with PPP inhibitors while varying the length and start-time of treatments. PPP-inhibited tadpoles have significantly shorter tails as treatment length increases, regardless of the start time for PPP inhibition. This result suggests that PPP activity must be sustained throughout regeneration to fully regrow the tail. My work has therefore identified the PPP as a previously unknown but critical metabolic pathway promoting tadpole tail regeneration. This insight advances our understanding of how metabolic reprogramming provides the carbon building blocks for regeneration.

Mammalian retinas function in a more hypoxic environment than most tissues. The retina thus does not exclusively use oxygen as a terminal electron acceptor in the electron transport chain (ETC). As a result, expression of proteins involved in energy metabolism may be affected. We determined the levels of metabolic proteins of various tissues, and found that compared to eyecup (consisting of retinal pigment epithelium and choroid vasculature), kidney, and cerebellum tissue, the retina expresses higher levels of Hexokinase I. This suggests that glycolysis may occur faster in the retina than in other tissues. We also observed higher levels of the ETC proteins cytochrome c and subunit 4 of cytochrome c oxidase levels compared to both the eyecup and cerebellum. This implies an increased capacity for electron transport in the retina, despite a lower O2 tension. We also investigated post-translational protein modifications that could be affected by a hypoxic tissue microenvironment. Lysine succinylation is one such modification, and is controlled by regulators of energy metabolism such as SIRT5. I observed that succinyl-lysine intensities were higher in the retina than in the cerebellum, kidney, liver, and eyecup. Further investigation will be necessary to determine the role that lysine succinylation plays in the retina. Through these experiments we show that retina tissue is well-suited for rapid energy metabolism in spite of its hypoxic environment.

Small heat shock proteins (sHSPs) fall into a class of proteins known as protein chaperones, molecular tools that help prevent aggregation, an often-unhealthy phenomenon in which proteins clump together. This protection against aggregation plays a critical role in helping to prevent diseases such as cataracts, and may play a role in preventing neurological diseases associated with protein aggregation such as Alzheimer's disease or Parkinson's disease. Yet little is known about the mechanism by which sHSPs prevent misfolded proteins from aggregating. The study of this mechanism is significantly complicated by the dynamic nature of sHSPs; rather than having just one structure, these protein chaperones fluctuate between several different stable structures. The purpose of this research project was to see what insights into sHSP structure and function could be gleaned by simplifying this dynamic structure. We focused on the central region of a small heat shock protein, known as the alpha-crystallin domain (ACD). Individual ACDs associate to form dimers in an antiparallel fashion, and in doing so form a central fold known as the dimer-interface groove. In a typical wild type small heat shock protein, these ACDs would continuously slide against each other along this groove. We created three distinct cysteine mutations in the ACD of the small heat shock protein HSPB5, producing the R116C, E117C, and F118C mutants. We expected each of these mutant dimers to form a disulfide bond tethering the two ACD subunits of the dimer together at the dimer interface groove, preventing them from sliding against each other, and therefore locking the dimer's central region into one of three distinct conformations. Additionally, we expected the affinities of other proteins for the mutants' dimer-interface grooves to differ between mutants, giving us insight into how the conformational state of the ACD affects its ability to interact with other proteins.

Altered cellular metabolism is intimately linked to cancer, both by supporting the increased metabolic demands of cell proliferation and through changes in metabolism that initiate tumorigenesis. For example, loss of function mutations to the metabolic enzymes Succinate Dehydrogenase (SDH) or Fumarase (FH) of the tricarboxylic acid cycle are sufficient to promote renal cell carcinomas (RCC). Normally, these enzymes fulfill metabolic roles, where SDH converts succinate to fumarate, which is then converted to malate by FH. Inactivating mutations to SDH and FH cause the accumulation of their substrates succinate and fumarate, respectively, which can drive cancer relevant signaling changes. Recently, studies have shown that perturbations to SDH cause resistance to ferroptosis, an iron-dependent form of nonapoptotic cell death. Inversely, another study has shown that FH-inactivation causes ferroptosis sensitivity. Considering SDH and FH are metabolically adjacent and have similar oncogenic consequences, it is unknown why they have opposing effects on ferroptosis induction. Cell death by ferroptosis occurs in response to the accumulation of oxidized polyunsaturated fatty acid-containing membrane lipids. I hypothesize that FH and SDH mutations have different effects on RCC cell lipid metabolism that cause divergent responses to ferroptosis induction. To test how SDH and FH alterations affect ferroptosis induction, I am performing a series of dose response assays with pro-ferroptosis treatments and measuring the effect on proliferation and lipid peroxidation in wild type, SDH-impaired, or FH-impaired cells. To inhibit SDH and FH, I will use two different patients derived RCC cell lines, UOK269 and UOK262, which have endogenous SDH and FH mutations, respectively. Each cell line will be compared to their respective SDH or FH addbacks. Ferroptosis induction is a potential opportunity for cancer treatment. Thus, it is essential to understand how different metabolic alterations affect ferroptosis sensitivity to identify conditions that may be most amenable to ferroptosis induction therapy.

The exact cellular and molecular mechanisms for the progression of amyloidogenic diseases such as Alzheimer’s and Parkinson’s disease are elusive. During amyloidogenesis, soluble protein monomers aggregate to form soluble oligomers, which further aggregate to form insoluble, êžµ-sheet rich fibrils. The Daggett lab investigates the involvement of a nonstandard secondary structure called É‘-sheet in the aggregation pathway of amyloidogenic proteins. The É‘-sheet hypothesis states that É‘-sheet is formed in the soluble oligomeric species during protein aggregation. This soluble, oligomeric form of the protein with É‘-sheet secondary structure is implicated to be the toxic species in amyloidogenesis and a driver of disease pathology. One amyloidogenic protein is É‘-synuclein, the major constituent of Lewy bodies, which are a pathological hallmark of Parkinson's disease. Preliminary data reveal the presence of É‘-sheet in Parkinson's disease patient samples, showing promise for detection of Parkinson’s disease earlier than previously possible utilizing É‘-sheet secondary structure. I am working on connecting the É‘-sheet hypothesis with the amyloidogenesis of É‘-synuclein by using synthetic É‘-synuclein to link together data from different types of experiments. Experiments I conducted include the purification of É‘-synuclein from E. coli, thioflavin T aggregation assays to measure the progression of the protein from monomer to fibril, circular dichroism spectroscopy to measure the predominant secondary structure in a peptide solution, and a soluble oligomer binding assay to detect the amount of É‘-sheet content in patient or synthetic peptide samples. The purpose of my experiments is to demonstrate that É‘-sheet is formed during the amyloidogenesis of É‘-synuclein prior to fibril formation by linking together the above experiments, supporting the idea that detection of É‘-sheet in patient samples may lead to methods of catching the progression of amyloid diseases earlier than previously possible.

The kinetochore is a protein complex responsible for transmitting force between microtubules and the centromeric region of DNA on chromosomes. The OA and Mif2 protein subunits of the kinetochore make direct attachments to the chromosome. They both bind to another subunit called MIND, which forms a bridge to the microtubule-binding elements of the kinetochore. Preliminary research shows that OA and Mif2 exhibit different binding preferences for the MIND complex. OA binds constitutively, regardless of whether MIND is in an open or closed conformation, whereas Mif2 strongly prefers the open conformation. However, the binding affinities for both OA and Mif2 for MIND have not yet been measured. This study developed the experimental methods for performing immunoprecipitation assays with OA, Mif2, and MIND. By performing phosphomimetic and truncation mutations to promote the MIND open conformation, the binding affinities of OA and Mif2 will be quantified. It is important to quantify the subunit binding affinities to gain a deeper understanding of kinetochore assembly and force transmission between microtubules and chromosomes. Ultimately, this research has applications in cellular division and cancer research.

During mitosis, the kinetochore plays a central role in ensuring the proper segregation of chromosomes in the parent cell. It does so by forming attachments to spindle microtubules, which facilitate the equal distribution of chromosomes to daughter cells. In budding yeast, the Dam1 and Ndc80 complexes are essential protein complexes that bind the kinetochore to spindle microtubules. The Ndc80 complex functions as the direct contact between the kinetochore and the dynamic microtubule tip and it is required for the Dam1 complex to associate with kinetochores. The Dam1 complex strengthens the kinetochore-microtubule attachment. In the presence of microtubules, Dam1complex oligomerizes into a sliding ring. This self assembly has been observed to occur with nanomolar concentrations of the complex in the presence of microtubules but in the absence of microtubules, appreciable oligomerization occurs at concentrations of the complex in the micromolar range. Dimers of the complex appear to predominate in high salt concentrations (500 mM NaCl) in comparison to monomers. This is thought to be due to electrostatic interactions between the monomers. When yeast histones were swapped for human histones, several mutations occurred in the Dam1 complex, and one mutation in the Ndc80 complex, that rescued the yeast cells from defects in mitosis. Preliminary characterization of the mutant Dam1 complexes lead to the hypothesis that the mutations that allow the yeast cells to adapt to the humanized histones changed the monomer-dimer equilibrium for the Dam1 complex. To measure the affinity of Dam1 complex monomers for each other, I will purify the protein complex and use size-exclusion chromatography and western blotting to quantify the relative abundance of the monomer and dimer at different concentrations of the complex. This will contribute to a greater understanding of mitosis and in turn cancer because it focuses on the dynamics that control proper chromosome segregation in cells.

The goal of our research is to understand how a nervous system integrates multiple sensory inputs, such as vision, touch, and olfaction, to direct the behavior of an animal. We use the nematode C. elegans as a model system because it has a well-defined neural circuit that can process multisensory information for its survival. Here, we set up an experimental paradigm to examine how two sensory inputs interact in the living neural circuits. C. elegans is given a primary stimulus – an attractive odor – and then a secondary stimulus – a gentle touch to its body. Our hypothesis is that the reflex avoidance response to touch is modulated by the attractive drive for animals to pursue favored odor. To provide the touch stimulus, we expressed the light-sensitive ion channel ChR2 in touch receptor neurons via the mec-4 promoter and presented the worms with blue light. As a response to touch, animals reliably carry out a reversal behavior. We find that while the worm is traversing to the attractive odor, it suppresses its natural response to touch, as quantified by the proportion of worms responding to the touch. Thus, our results suggest that C. elegans is a promising system to study the integration of multiple senses. Using this system, we will determine how neurons process complex sensory signals in living animals.

Designing binders for single beta strand and beta-hairpin peptides could be useful for a wide range of biomedical applications. This project aims to design peptide binders that force unstructured peptides into a beta strand conformation. The computationally designed peptide-binder pairs were expressed and purified utilizing Immobilized Metal Affinity Chromatography (IMAC) and characterized via Size-Exclusion Chromatography (SEC) and biolayer interferometry. Successful binders were redesigned to bind pathogenic fragments of Amyloid-beta, alpha-synuclein and tau. These fragments are known to form beta strand mediated fibrils in the brain that are associated with the development of neurodegenerative diseases like Alzheimer’s and dementia. I am currently characterizing the redesigned binders for Amyloid-beta and expect higher-affinity binding with these designs. Success of this project could provide a new tool in studying the fibril formations and allow for future drug therapies.