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Neural Population Dynamics Underlying Encoding of Time in the Cortico-Striatal Circuit
No full textInterval timing, the ability to accurately measure elapsed time between events, is critical for generating appropriate behavioral responses. Research conducted over the past decades has implicated the cortico-striatal-thalamic loop in time encoding,suggesting that the cortex utilizes high-dimensional dynamics to represent time. The dorsal lateral region of the striatum (d1St) is a key structure involved in interval timing,where neurons exhibit dynamic firing rate patterns that encode elapsed time, enabling animals to make perceptual judgments about time intervals. However, it is unlikely that this striatal activity is self-generated, pointing towards cortical afferent inputs as the driving force behind it. This thesis aims to investigate cortical activity responsible for time encoding that can drive striatal activity. To achieve this, we developed a 2-alternative force choice head-fixed sensory timing task in mice. The mice were trained to perceive and categorize two different time intervals by licking the corresponding waterspout, demonstrating their ability to learn the task. Challenge sessions introduced two additional time intervals within each category to assess generalization of the learned task. Initially, we explored the necessity of d1St activity in perceiving and accurately categorizing time intervals. Using bilateral activation of the inhibitory opsin GtACR to suppress d1St activity during time perception, we observed impaired performance of the sensory timing task. Subsequently inhibiting d1St activity after the delivery of time interval stimuli resulted in more variable effects on the animals\u27 performance. These experiments confirmed the crucial role of d1St neural activity in executing the sensory timing task accurately. To investigate the cortical inputs to the d1St, we employed a calcium indicator (GCaMP) to label cortical neurons projecting directly to the d1St. Under two-photon microscopy, we recorded their neural activity while the animals performed the sensory timing task. Most of the labeled neurons were found in the secondary motor cortex (sMO-d1St). We discovered that this cortical neuron population carries relevant information about perceived time intervals by forming coordinated neuronal ensembles, referred to as timing ensembles. Importantly, these neuronal ensembles exhibited dynamic engagement in neuronal activity on a trial-by-trial basis, rendering the timing ensembles as more reliable time encoders than individual neurons alone. Notably, timing ensembles exhibited linear adjustments in activity according to the timed interval, while individual neurons contributed to both linear and non-linear changes in their activity patterns. In conclusion, this thesis advances our understanding of interval timing by highlighting the essential role of cortical activity, particularly within the sMO-d1St circuit, in driving striatal encoding. The involvement of d1St neural activity in accurate time perception underscores its significance within the cortico-striatal circuit. Furthermore, the discovery of dynamic timing ensembles within the sMO-d1St population provides valuable insights into the coordinated and flexible nature of time encoding. These findings contribute to our understanding of how the brain tracks elapsed time and lay the groundwork for future research exploring the integration of timing information into complex cognitive processes and behavioral responses
Strain Variation as a Window Into the Neural Logic of Drosophila Mate Choice
No full textDrosophila males perform robust, stereo typed courtship routines which have been studied for over a century to understand the genetic and neural underpinnings of flexible, hard-wired behavior. Courtship–particularly on the crowded wild food patches where flies congregate to mate–is a dynamic interaction between a male and surrounding potential partners that requires he integrate multiple sensory cues to direct his efforts efficiently and appropriately. While the behavior follows a similar basic pattern across Drosophila, it can contain elements that are higly species-specific. Here, we described marked variation in one aspect of male courtship preference among inbred lines of the widely studied model species, D. melanogaster, and use it as a platform from which to explore how genetically specified differences in a chemosensory circuit give rise to diversity in behavior. Males from many species have been reported to show a preference for courting conspecific females. Here, we summarize past work and introduce new data to show that these preferences correspond with a male\u27s response to sex-and species-specific hydrocarbons displayed on the fly cuticle. In particular, individuals from many species appear to discriminate females based on species-specific chemical cues. When given a choice between a conspecific and a female that produces a distinct hydrocarbon profile, they strongly prefer to court the conspecific. However, they show little preference between courting a conspecific and heterospecific female when the two display the same chemical profile. We showed further that males from the globally distributed species, D. melanogaster, are remarkably promiscuous, courting closely related species as strongly as they court conspecific females, regardless of hydrocarbon profile. We next revealed that among strains of D. melanogaster isolated from sub-Saharan Africa (the ancestral range of the species) promiscuity varies widely, because males from some strains avoid the main D. simulans cuticular hydrocarbon(7-T) whereas others do not. Correspondingly, a population of male-specific interneurons (P1) that promote and guide the courtship ritual is suppressed by 7-T only in selective strain animals. Rather than differences in hydrocarbon detection per se, we found that this difference arises from altered sensitivity in the connection between the sensory periphery and an inhibitory circuit element (mAL) that impinges on P1. Finally, we investigated the role of mAL neurons in shaping ongoing courtship behavior moment-to-moment, demonstrating that repeated transient activation of these neurons over minutes stochastically shapes mate preferences by re-directing a male\u27s pursuit. Thus, mAL activity introduces variation in behavior moment-to-moment without impacting a male\u27s drive toward vigorous courtship. Our results reveal how the same circuit element both drives flexibility in ongoing mating behavior and is subject to genetically specified behavioral diversity within a species. The work presented here showcases the potential power–and the limitations–of strain comparison as a platform for uncovering mechanisms of behavioral evolution and the general principles of circuit function underlying fundamentally important behaviors
On the Second Telomere Maintenance Machine: Structure, Recruitment, and Regulation of the CST–Polα/primase C-strand Fill-in Complex
Full text linkTelomeres are the terminal nucleo protein components of eukaryotic chromosomes that safeguard genome integrity. Human telomeric DNA consists of double-stranded (ds) 5\u27-TTAGGG-3\u27 repeats terminating in a 3\u27 single-stranded (ss) overhang of the G-rich strand.The sequence and structure of the telomeric DNA is recognized and bound by the six-subunit shelterin complex, which plays critical roles in telomere protection and maintenance. First, shelterin protects the genome by repressing aberrant activation of the DNA damage response at telomeres, which resemble broken DNA ends. Second, due to their location at the end of the chromosome, telomeres present a unique challenge to the canonical DNA replication machinery. The replisome cannot fully copy the terminal repeats, resulting in gradual telomere shortening over successive cell divisions. Once telomeres become too short, they lose protection, triggering the DNA damage response and downstream apoptosis or senescence. Unchecked excessive telomere shortening is pathological and can manifest as a number of telomere biology disorders, but routine telomere shortening in somatic cells is also a tumor suppressor pathway. Thus, telomere length must be carefully controlled and shelterin recruits two maintenance enzymes to the telomere to do so: telomerase and DNA Polymerase α (Polα)/primase bound to Ctc1/Stn1/Ten1 (CST). Decades of research have focused on telomerase, which extends the G-rich telomeric strand to counteract telomere shortening in germline and stem cells. More recently, CST–Polα/primase,which synthesizes the complementary C-rich strand, has emerged as a second critical regulator of telomere length. The body of work contained in this thesis describes our efforts to understand the molecular mechanisms underlying C-strand maintenance by this enigmatic complex.Chapter 1 serves as an introduction to telomere maintenance by shelterin and CST–Polα/primase.I contextualize the work presented in this thesis alongside commentary on findings from other groups and the field at large, discussing recent studies on the evolution, function, and regulation of CST–Polα/primase. I also introduce the telomere biology disorder Coats plus syndrome (CP), which is caused by mutations in CST,and in one case, the shelterin subunit POT1.This thesis provides a molecular basis for the pathogenesis of a subset of these CP mutations.In Chapter 2,I set out to determine the structure of CST–Polα/primase using cryogenic electron microscopy (cryo-EM), a state-of-the-art structural technique well suited to large and flexible macromolecular complexes. Surprisingly, the structure I obtained was of an inactive complex where the Polα/primase bound to CST is in an auto-inhibited conformation in competent for enzymatic activity. Evolutionary conservation analysis showed that the interface between CST and Polα/primase in this structure is unique to metazoans and lost in unicellular eukaryotes. A CP mutation maps to the primary interface observed, supporting a physiological role for this conformation. I propose that this inactive state may represent a recruitment state that forms during recruitment of CST–Polα/primase to the telomere and exists prior to activation of the enzyme. Concurrent to my finding of the CST–Polα/primase recruitment complex (RC), another group determined the structure of CST–Polα/primase in a pre-initiation complex (PIC) competent for enzymatic activity. The existence of these two drastically different conformations raised the question of how this complex might be regulated. In Chapter 3, I turn my focus to studying how CST–Polα/primase is recruited to telomeres. POT1/TPP1 is the heterodimer in shelterin that interacts with the ss 3\u27 overhang, and it was previously implicated in CST recruitment.I determined cryo-EM structures of POT1/TPP1 bound to CST, which revealed the molecular basis for the recruitment of human CST by POT1 and for how CP mutations in Ctc1 and POT1 disrupt their interaction. Notably, the POT1-bound CST cannot accommodate Polα/primase in the active conformation observed with the CST–Polα/primase PIC but can accommodate the auto-inhibited enzyme in the RC conformation.This finding was confirmed with in vitro C-strand synthesis assays, showing that POT1 inhibits CST–Polα/primase activity when bound. I identified a region of POT1 that, when phosphorylated, promotes its interaction with CST. Together, these results suggest a new model for the phosphorylation-dependent temporal regulation of CST–Polα/primase recruitment and activity at the telomere.In the final chapter, I discuss the findings presented in this thesis as a whole and highlight avenues for further study of the complex telomere length regulation mechanisms that finely tune the balance between normal function and pathogenic states
New Insights into the Evolution of Learned Vocalization from the Australian Zebra Finch, Taeniopygia castanotis
No full textThe common goal of my thesis is to understand the causative events in evolution that produced a clade of song learning birds from non-song learning ancestors.This information is important for shedding light on the evolution of spoken language in our own human lineage, where evolutionary analyses are technically limited. The most recent common ancestor of humans and chimpanzees was presumably a vocal non-learning African ape, alive ~6 mya. At some point between this ancestor and the emergence of modern humans 0.5 mya we evolved more dexterous hand control, bipedalism, light colored eyes, larger brains, less hair, weaker muscles, higher intelligence, greater eusociality, novel sweat glands, and spoken language; to name a few of the traits which separate us from other apes. As there are no extant species internal to this branch lacking one or more of these human specific phenotypes, it is much more difficult to ascribe observed human genome variation to the evolution of specific traits, especially behavioral traits. Further, neutral mutations cannot be removed by looking for shared variance across species because we are the only extant species of vocal learning primates. Human language is also difficult to isolate from a neuroanatomical perspective. Our current interpretation of the literature is that the neurons responsible for the learned movements of speech are either directly adjacent to or intermixed with those moving the hand and face in all primates, making them more difficult to localize. None of these limitations apply in oscine songbirds. In oscines, the vocal learning brain system is more segregated from non-vocal movements; There are over 4000 oscines, and most if not all are thought to be vocal learners; their suboscine outgroup clade is also species rich and consists of many vocal non-learners. Since oscine vocal learning neural circuits are discrete, potentially causative genes can be identified based upon their specialized expression within these circuits under various conditions. A subset of these genes have been found to have similar specializations in human speech brain regions, and thus these potentially trait-causative genes from songbirds can then be studied in the context of human evolution for the same trait using comparative genomics, as we begin to do here. I hope that these experiments and analyses can serve as the beginnings of a framework for future experiments seeking to understand our own evolution through the study of human-convergent traits in non-human lineages
RNA Therapeutics for Fibrolamellar Hepatocellular Carcinoma
No full textFibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer characterized by a recurrent fusion of the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A (PRKACA). Due to limited efficacy of conventional treatments such as chemotherapy and radiation my work explored new therapeutic options. As DNAJB1::PRKACA is known to initiate tumorigenesis, I reasoned that it would be an ideal target for a therapeutic. However, it was not known if FLC tumors are still dependent on DNAJB1::PRKACA or if cells achieved independence of the initial oncogene. To test if FLC is dependent on the oncokinase I screened short hairpin RNAs (shRNAs) that tile over the fusion junction of the DNAJB1::PRKACA mRNA. I identified a shRNA that achieves strong knockdown of the fusion gene with minimal effect on the wildtype fusion partners.Using this shRNA, I demonstrate that specifically knocking down DNAJB1::PRKACA results in the cell death of FLC cells in vitro and in patient derived xenograft models of FLC in mice. These results show that DNAJB1::PRKACA has a pivotal role in tumor formation, maintenance, and progression and that FLC is oncogenically dependent on DNAJB1::PRKACA. This validates DNAJB1::PRKACA as a therapeutic target. To translate this finding into a potential therapeutic, I collaborated with Ionis Pharmaceuticals to synthesize small interfering RNAs (siRNAs) with state-of-the-art chemical modifications. Multiple siRNA therapeutics targeting genes in the liver have been FDA approved. The majority of these use a carbohydrate group, GalNAc, that binds to the asialoglycoprotein receptor (ASGR), allowing targeted delivery to hepatocytes. I hypothesized that ASGR might still be sufficiently expressed in FLC tumors to deliver siRNAs to cells in vivo. I demonstrated that indeed ASGR is still expressed across different stages of the FLC in patient samples and PDX models. Using PDX cells of patients, I show that GalNAc-conjugated siRNAs can be internalized via ASGR in vitro and result in knockdown of DNAJB1::PRKACA and cell death. In two different FLC PDX lines of subcutaneous and splenic tumor models, siRNAs targeting DNAJB1::PRKACA are effectively taken up in vivo with strong and durable target knockdown and tumor growth inhibition. To further enhance the delivery of RNA therapeutics to FLC cells, I developed lipid nanoparticles(LNPs) based on the ionizable lipid C12-200. These LNPs effectively delivered mRNA encoding luciferase to cells in vitro, demonstrating their potential for delivering therapeutic RNA payloads. However, delivery to FLC cells in vivo remains challenging. Additionally, I performed cell surface proteomics using a proximity-based labeling approach to identify potential targets for antibody-based targeting strategies. Comparative analysis revealed distinct surface protein profiles between FLC and hepatocellular carcinoma (HCC) spheroids. Validation of selected targets in patient samples confirmed their differential expression and localization in FLC tumors compared to healthy liver tissue, providing a valuable resource for developing targeted therapies for FLC. The successful knockdown of this fusion gene through inducible shRNA and siRNA strategies offers a promising treatment avenue for FLC patients, underscoring the potential of mRNA degrading modalities such as antisense oligonucleotides, siRNAs, and possibly PROTACS for targeted intervention. This work establishes a foundation for the development of precision therapies against FLC, highlighting the importance of the DNAJB1::PRKACA fusion kinase as a therapeutic target and serving as a model for targeting other fusion genes in pediatric and possibly adult cancers, paving the way for more effective treatments
Cellular and Circuit Consequences of a Genetic Locus for Attention
Full text linkAnimals are constantly bombarded by any number of sensory inputs but have a limited capacity with which to process them. A mechanism for filtering, prioritizing, and directing mental assets is required to prevent sensory overload, enable meaningful comprehension, and allow for further action. Attention is the process of directing cognitive resources toward specific stimuli, which can be dispensed in a top-down manner to carry out higher-order cognitive functions. However, despite extensive and careful study at the molecular, cellular, and, circuit scales, unifying principles have been challenging to elicit. In this thesis, I aimed to provide a new perspective by taking a forward genetics approach to identify genes with prominent contributions to attentional performance. We studied 200 mice from a highly genetically diverse, multi parent mouse population on measures of pre-attentive processing and through genetic mapping identified a small locus on chromosome 13 (95%CI: 92.22-94.09 Mb) driving substantial variation (19%) in this trait. After identifying the parental genomic contributions driving this variation, we validated that the locus also drove variation in attention, but not other related cognitive processes, using similarly diverse mice homozygous for the appropriate founder haplotypes. Further characterization of the locus revealed Homer1, encoding a synaptic protein, as the causative gene. Further analysis determined that down-regulation in the prefrontal cortex (PFC) only during a developmental critical period of two short, activity-dependent isoforms Homer1a and Ania3 led to significant improvements in multiple measures of attentional performance in the adult. Subsequent single-cell RNA seq experiments revealed that prefrontal Homer1 down regulation in excitatory neurons is associated with GABAergic receptor upregulation in those same cells. Moreover, physiological studies demonstrated that this increase in GABAergic receptors corresponded to strong inhibitory tone in PFC. This enhanced inhibitory influence, together with dynamic neuromodulatory coupling, led to strikingly low PFC activity at baseline periods of an attention task but targeted elevations at cue onset, predicting short-latency correct choices. Notably high-Homer1, low-attentional performers, exhibited uniformly elevated prefrontal activity throughout the task. We thus identify a single gene with a large effect on attention–Homer1 –and find that it improves prefrontal inhibitory tone and signal-to-noise (SNR) to enhance attentional performance. Complementary to older models focused mainly on uniformly amplifying PFC activity, this work provides a new paradigm of attentional control–one in which reduced prefrontal activity can improve SNR
Recall Germinal Center and Antibody Responses
Full text linkRepeated exposure to viruses or their antigens elicits anamnestic antibody responses that produce antibodies that are of greater magnitude and affinity compared to those induced after primary exposure to antigen.The anamnestic nature of the response is a result of the recall of memory B cells (MBCs) that have undergone clonal expansion and affinity maturation in germinal centers(GCs) during the primary response. Upon antigen re-encounter ( boosting ), MBCs, aided by enhanced help from memory T cells, efficiently differentiate into antibody-secreting plasma cells. At the same time, boosting induces recall GCs that could in principle either further affinity mature primary-derived MBCs or engage naïve B cells with potentially new epitope-specificities. The balance between these two possibilities is important for the rational design of vaccine strategies that can induce broadly neutralizing antibodies against mutating viruses like HIV-1, influenza A virus and SARS-CoV-2. Moreover, it is important in the context of a serum phenomenon termed antigenic imprinting or original antigenic sin (OAS), in which the antibody response is thought to repeatedly derive from the first cohort of B cells engaged by primary antigenic exposure, at the expense of inducing de novo antibodies against related antigens. Recall antibody responses therefore emerge from a complex interplay of B cell clones with varying degrees of specificity, affinity maturation and via intercalating MBC stages. The response is further affected by T cell help and potentially shaped by the competition of B cells with pre-existing antibodies for access to antigen (antibody-mediated feedback). Our studies, employing various prime-boost models in genetic fate-mapping mice, address multiple aspects of the recall B cell and antibody response. First, using cellular fate-mapping in which we mark the primary-cohort B cells and their MBC progeny with fluorescent proteins, we addressed the contribution of MBC clones to the recall response. We found that recall responses are characterized by a clonality bottleneck that restricts the responding clones to few dominant ones out of the vast number of MBC clones generated throughout the primary response. These selected winner clones were generally derived from relatively high-affinity germline precursors compared to the large diversity of MBC clones that was not reengaged detectably by boosting. We also found that further diversification of MBCs in recall GCs does occur, but at very low frequency. Instead, recall GCs are composed predominantly of a clonally diverse repertoire of naïve-derived B cells that did not undergo prior affinity maturation. Despite this potential of recall GCs to induce de novo antibody responses, we found that these antibodies were suppressed in the serum of newly-generated molecular fate-mapping mice repeatedly boosted with the same antigen. Instead, we found that recall antibodies were almost exclusively derived from the primary cohort of B cells, even after three or four antigen exposures. However, this primary addiction , with OAS-type suppression of de novo responses, decreased upon boosting with variant antigens as a function of antigenic distance. These de novo antibodies targeted variant-specific epitopes not covered by primary-derived antibodies, consistent with antibody-mediated feedback. Thus, whereas recall GCs contain mostly naïve-derived B cells, these only contribute to serum antibody when the antigenic distance is sufficient, and instead recall antibodies tend to result from primary addiction. Finally, we set out to elucidate the potential effects of antibody-mediated feedback and memory T cell help on the differential reliance that recall GC and antibody responses have on MBCs. We show that this schism is at least partly explained by a marked decrease in the ability of recall GC B cells to detectably bind antigen. Variant priming and plasma cell ablation experiments show that this decrease is largely due to suppression by pre-existing antibody, whereas hapten-carrier experiments reveal that increased memory T cell help allows B cells with undetectable antigen binding to access GCs. We propose a model in which antibody-mediated feedback steers recall GC B cells away from previously targeted epitopes, thus enabling tailored targeting of viral escape epitopes. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens
Developing EasySci, a High-Throughput and Low-Cost Single-Cell Genomic Technique, to Study Aging and Alzheimer\u27s Disease in Human and Mouse Brain
Full text linkPrevious single-cell methods are limited by their relatively low throughput and high costs. This prevents most laboratories from performing large-scale cell profiling, complicating the understanding of rare cell type contributions to aging and disease. Moreover, these methods often focus on the 3\u27 end of genes. Without capturing the complete gene body coverage, these technologies have limited detection of specific isoforms. To address these limitations, we extensively optimized a combinatorial indexing-based framework to develop EasySci. We used EasySci to study cell population dynamics associated with aging and Alzheimer\u27s disease in the mammalian brain. This dataset recovered approximately 1.5 million single-cell transcriptomes and 400,000 chromatin accessibility profiles from 20 mouse brains. An optimized clustering framework identified more than 300 distinct cell subtypes. This extensive cataloging revealed the molecular characteristics and spatial distribution of these subtypes, providing new insights into the cellular landscape of the brain. For instance, we identified the spatial locations of astrocyte and neuron subtypes, mapping them to distinct anatomical regions. We observed cell population shifts of rare cell types during aging, i.e. an expansion in inflammatory subtypes of microglia and oligodendrocytes. Contrary, neuronal and oligodendrocyte progenitor cell populations declined. In exploring the cellular response to Alzheimer\u27s disease associated genetic mutations, we investigated the cell population dynamics of both established early-onset and novel late-onset Alzheimer\u27s disease models. This comparison highlighted the late-onset Alzheimer\u27s disease model, APOE*4/Trem2*R47H, having cell population dynamics reminiscent of the 5xFAD early-onset model. This suggests the novel model\u27s potential utility in Alzheimer\u27s disease research. For instance, the expansion of septal nuclei neurons were observed in both models. This neuron subtype had increased axonogenesis gene module expression. Contrary, the reduction of a choroid plexus epithelial cell subtype, marked by the enrichment of neuroprotective mitochondrial genes, were detected in both models. On a molecular level, a novel isoform of theTrem2gene was consistently upregulated in both mouse models. Furthermore, we profiled 118,240 cells from human brains. We detected region-specific transcriptomic changes in Alzheimer\u27s disease patients, as well as conserved trends between mouse models and human patients. This dataset also identified novel, sensitive and specific histological markers for pericytes, i.e. SLC6A12 and SLC19A1. Antibodies targeting these proteins were highly effective in staining pericytes in human brains. These experiments validated the efficacy of these markers and selected the SLC6A12 antibody as the most sensitive and specific markers of human pericytes. Furthermore these novel markers overperformed the traditionally used PDGFRB antibody. In conclusion, this dataset provided an extensive resource for understanding the cellular dynamics of aging and Alzheimer\u27s disease. Furthermore, we introduced EasySci, a novel, high-throughput, and cost-effective single-cell technique. This method significantly enhances the capacity of independent laboratories to profile millions of cells, and advances our understanding of normal and pathological aging
A Platform for Analyzing Force Sensitivity and Multivalency in Actin Networks
No full textThe physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks. In this thesis, I first present our work engineering filament linker complexes and combining them with photo-micropatterning of myosin motor proteins to produce an in vitro reconstitution platform for examining how force impacts the behavior of ABPs within multi-filament assemblies. Our system enables monitoring dozens of actin networks with varying architectures simultaneously using total internal reflection fluorescence microscopy, facilitating detailed dissection of the interplay between force-modulated ABP binding and network geometry. Secondly, I present data applying our system to study a dimeric form of the critical cell-cell adhesion protein α-catenin, a model force-sensitive ABP. We find that myosin forces increase homodimeric α-catenin\u27s engagement of filament bundles, particularly smaller bundles embedded within networks. This activity is largely abrogated in a force-sensing deficient mutant, whose binding is not increased to the same degree on tensed filament bundles and scales linearly with bundle size. I present our model to explain the relative differences in binding between larger and smaller bundles based on differences in per-filament loads, which could influenceα-catenin\u27s distribution across actin-myosin networks with varying sizes in cells. I discuss potential further work to substantiate that model and its implications. Finally, I discuss our progress designing and creating a fluorescence based force detection system intended to be embedded in the filament linker complexes we created. Such a setup would enable correlative measurement between ABP binding behavior and tensile forces on bound filament bundles. Collectively the work introduces a new approach for the in vitro analysis of F-actin networks and binding proteins which may prove useful in bridging the gaps between existing in vitro, single molecule and cellular approaches
Proteomic Analysis of WNT Signaling and Metabolism of Fibrolamellar Hepatocellular Carcinoma
No full textFibrolamellar hepatocellular carcinoma (FLC) is a rare form of liver cancer that primarily affects young adults and adolescents.Although characterized as a subvariant of hepatocellular carcinoma (HCC), we have shown it is distinct based on its pathogenesis, patient population and drug response. Our lab has identified a fusion gene DNAJB1::PRKACA that is found in almost all FLC tumor samples sequenced to date. The fusion gene when expressed in mouse liver is sufficient to generate FLC, and elimination of the fusion transcript kills human tumors. The fusion results from a ~400kb deletion on chromosome 19, beginning after the first exon ofDNAJB1, which encodes for a member of the DnaJ or Hrsp40 family of proteins, and ends before the second exon of PRKACA, the catalytic subunit of protein kinase A (PKAc). To date, there is no known successful treatment for this disease, and the five-year survival rate is around 40%. In this Thesis, I will present my work in investigating several areas in which the disease alters normal cellular pathways. First, I investigate the role Wnt signaling plays in the pathogenesis of FLC. It has long been reported that canonical Wnt signaling may play a role based on the transcriptomic analysis, and synergistic mutagenesis screens. I will next utilize proteomics, imaging, and drug screening data which shows that Canonical Wnt signaling through β-catenin does not play a significant role in FLC. In the following chapter I will show my work on researching the metabolism and molecular dynamics of the mitochondria in FLC.It has long been reported that there is an unusual increase in the number of mitochondria in FLC compared to normal hepatocytes. I use immunofluorescence as well as transcriptomics, proteomics, and phosphoproteomics analysis to investigate some of the ways mitochondria are altered in FLC