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OPHIOLITIC CONSTRAINTS ON THE EVOLUTION OF SUBDUCTION ZONE MAGMA GEOCHEMISTRY
Get PDFOceanic crust fragments known as ophiolites preserve valuable records of past tectonic events. Studying ophiolites can provide insight into major plate movements, such as the transition from a passive to active margin during subduction initiation. The Appalachian-Caledonian Orogen, formed during the closure of the Iapetus and Rheic Oceans, preserves numerous ophiolites across its >6500 km length. However, variations in geochemistry and timing of formation among these ophiolites have made it challenging to correlate major tectonic events along the orogen. This research utilizes geochemical, mineralogical, and geochronological evidence, paired with a model of subduction initiation evolution developed in an active subduction zone, to investigate the origins of multiple ophiolite complexes along the Appalachian-Caledonian Orogen.
We first investigate the Leka Ophiolite Complex (LOC), an ophiolite from the Norwegian Caledonides. The results of geochemical and geochronological analyses indicate the LOC formed during subduction initiation in the Iapetus Ocean at 491.36 ± 0.17 Ma. Next, we investigate the Dadeville Complex of Alabama and Georgia, USA. Geochemical evidence reveals early-stage and evolved magmatic products preserved in the complex. High-precision U-Pb zircon dating of the early units constrains the timing of the magmatism to no later than c. 467 Ma, and mineralogical evidence suggests high-grade metamorphism via underthrusting during subduction.
Felsic rocks known as plagiogranites are commonly utilized to date ophiolite complexes and their associated tectonic processes, therefore, we collected plagiogranites from four ophiolite complexes along the Appalachian-Caledonian Orogen and combined multiple lines of evidence to discriminate between their petrogenetic origins. Two hypotheses for plagiogranite formation are coeval fractional crystallization of the same magma that formed the units of the ophiolite, or subsequent hydrous partial melting of mafic rocks. Our data show that the Baltimore Mafic and State Line Complexes of Maryland and Pennsylvania host at least four distinct types of felsic rocks with differing origins. Based on geochemical discrimination of the samples, we identify the processes associated with formation for each and predict their relative order of formation. We will test our predictions using high-precision U-Pb zircon dating on each of the samples
The Mechanisms of Ribosome Rescue in Bacillus Subtilis
Get PDFStalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria
Dissecting functional diversities of human sensory neuron subsets to develop a new humanized platform for pain therapy
No full textPain and itch are subtypes of somatosensation that can be evoked by a wide range of chemical, thermal, and mechanical stimuli and are sensed by nociceptive (pain-sensing) and pruriceptive (itch-sensing) neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG). Efforts to develop novel non-opioid analgesics have been largely unsuccessful despite the promise shown in heterologous expression systems and animal models. This is partly because these models do not recapitulate the activity of neurons in their native environment, and due to species-specific differences in ligand-receptor activity of key molecules involved in pain signaling. Human pluripotent stem cells (hPSCs) have emerged as a new cell source for pain treatment research because they produce large quantities of cells that are otherwise hard to obtain. Here, we report the generation of purifiable populations of human transient receptor potential cation channel subfamily V member 1 (TRPV1)+, sodium voltage-gated channel alpha subunit 9 (SCN9A)+, and Mas-related G protein-coupled receptor X1 (MRGPRX1)+ neurons from hPSCs. Our functional studies show distinct functional polymodality of the three subsets. Additionally, we have also constructed a transcriptomic continuum of developing hPSC-derived primary afferents and validated that mature hPSC-derived sensory neurons (hPSC-SNs) recapitulate key molecular signatures of human DRG neurons. By demonstrating the ability of hPSC-SNs to modulate endogenous neuronal activity after being grafted into immunocompromised animals, we provide the first evidence of long-term in vivo survival and function of hPSC-SNs. Lastly, by exploiting the differentially expressed pathway regulators and membrane receptors among these three subsets, we identified voltage-gated channel subfamily Q member 2 (KCNQ2) as a novel pain target and installed a protective mutation in this gene using adeno-associated virus (AAV) delivery of CRISPR-Cas9 constructs. Results demonstrate that this missense mutation confers robust nociception-resilient effects in mature hPSC-SNs. In summary, we have generated a humanized platform that exploits the distinct molecular profiles of human nociceptor subtypes and developed a gene therapy strategy for managing chronic pain
INTEGRATING GENOMIC AND TRANSGENIC STRATEGIES TO MAP NEURAL CONNECTIVITY
No full textIn the wake of Ramon y Cajal’s investigations into neuroanatomy, the mechanism
by which neurons forge precise connections in the brain has captivated researchers.
Equally significant is how these synaptic connections shape behavior. In my research, I
focused on the habenula-interpeduncular pathway, a conserved neural tract connecting
the forebrain and midbrain in vertebrates, which modulates various processes, including
sleep, fear, pain perception, and reward.
Employing transcriptomic profiling on dissected dorsal habenulae (dHb) and the
rest of the brain from adult zebrafish, I identified transcripts enriched in the dHb
and confirmed their expression by RNA in situ hybridization. By defining gene
expression among overlapping cell clusters in a dHb single-cell transcriptomics dataset,
a hierarchical structure was used to distinguish dHb subtypes. This approach identified
promising genes for targeted integration, in which their cis-regulatory regions control
the expression of a transcription factor that functions in a bipartite transcriptional
activation system. In the course of this work, I adapted and modified different
strategies for genome editing to generate gene-specific transgenic driver lines. I also
analyzed chromatin immunoprecipitation and transposase-accessibility datasets for
different zebrafish organs and identified accessible genomic regions as safe harbors for
expressing transgenic reporters in the central nervous system.
Exploiting membrane-bound fluorescent reporters regulated by the upstream activating
sequence to which the transcription factor binds, I mapped axon terminals of
labeled dHb neurons to specific dorsoventral subdomains along the interpeduncular
nucleus (IPN). Building on this framework, a classification network was employed to
predict the IPN innervation domains of additional dHb neuronal subtypes. This model
produced a comprehensive projection map for the dHb-IPN pathway. Additional
transgenic labeling of dHb subtypes and RNA in situ hybridization on neuropeptide
receptors substantiated the model.
Exploiting membrane-bound fluorescent reporters regulated by the upstream activating
sequence to which the transcription factor binds, I mapped axon terminals of
labeled dHb neurons to specific dorsoventral subdomains along the interpeduncular
nucleus (IPN). Building on this framework, a classification network was employed to
predict the IPN innervation domains of additional dHb neuronal subtypes. This model
produced a comprehensive projection map for the dHb-IPN pathway. Additional
transgenic labeling of dHb subtypes and RNA in situ hybridization on neuropeptide
receptors substantiated the model.
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STEROID HORMONE DEPENDENT PATHOGENESIS IN VASCULAR EHLERS-DANLOS SYNDROME
No full textUnlike females, male patients with Vascular Ehlers-Danlos Syndrome (VEDS) show a dramatic increased risk of arterial rupture coincident with the onset of puberty. The Col3a1G938D/+ VEDS mouse model recapitulates this sexually dimorphic vulnerability. Given pubertal onset, we hypothesized that androgen signaling may modulate this increased risk. We found that both deletion of the androgen receptor (AR) and selective pharmacologic AR antagonism improve survival in male but not female Col3a1G938D/+ mice, suggesting that AR signaling accounts for most of the observed sex differences. We also show that treatment of Col3a1G938D/+ mice with the dual AR and mineralocorticoid receptor (MR) antagonist spironolactone affords near-complete protection in both sexes, with intermediate survival benefit of a selective MR antagonist. Single-nucleus RNA sequencing revealed that VEDS-dependent perturbations of gene expression are reversed by AR and/or MR inhibition in both aortic vascular smooth muscle cells and endothelial cells, with a smaller number of transcripts being modulated in aortic fibroblasts. Taken together, these data suggest that dual AR and MR antagonism is a potential therapeutic strategy in both sexes, with the option of selective MR antagonism in males to avoid sexual side effects
Targeting the intersection of molecular signaling pathways and tumor immunobiology in NF1-associated malignant peripheral nerve sheath tumors
Get PDFMalignant peripheral nerve sheath tumors (MPNST) are a leading cause of death among patients with neurofibromatosis type 1 (NF1). MPNST are aggressive soft tissue sarcomas with a tendency for recurrence and distant metastases. They are difficult to treat because of their relative insensitivity to conventional chemotherapy and radiotherapy. Currently, the only curative treatment is surgical resection with wide margins for localized disease; however, oncologic surgery is often unfeasible due to the location among nerve bundles, the size of the tumor, or the presence of metastasis. There is currently no FDA-approved chemotherapy drug for the treatment of MPNST. Thus, novel therapeutic approaches are urgently needed in this dire disease. Recent efforts have focused on the use of molecularly targeted therapeutics against dysregulated signaling pathways which have been identified as critical to MPNST tumorigenesis. In parallel, the tumor immune microenvironment (TIME) plays a critical role in advanced cancers and there is little known about the interaction of activated RAS signaling pathways, small molecule inhibitors, and the tumor-infiltrating immune cells in MPNST. To address this, we conducted a number of studies aimed at bridging the intersection between molecularly targeted therapy and immunobiology in NF1-MPNST. First, we tested the combination of SHP2 and CDK4/6 inhibitors and demonstrated profound anti-tumor efficacy in a diverse panel of patient-derived cell lines and in vivo xenograft models. Second, we performed an in-depth interrogation of the tumor immune microenvironment (TIME) in the spectrum of human NF1-peripheral nerve sheath tumors in order to identify critical tumor-infiltrating immune cells that propel the progression to malignancy. Immunophenotyping confirmed increased immunosuppressive inflammation on malignant progression with a notable predominance of intra-tumoral myeloid cells, particularly tumor-associated macrophages (TAM). Additionally, MPNST specimens demonstrated elevated levels of immunosuppressive TAM, as indicated by their heightened PD-L1 expression. The immunosuppressive TIME correlated with poorer patient outcomes. Lastly, we optimized a syngeneic mouse model of MPNST to investigate the interplay between RAS signaling pathway inhibitors and the immune-modulating cells within the TIME. A deeper insight into the relationship between pre-existing immunity and molecular pathways will inform the design of potential clinical trials using rationally designed therapies
DETERMINING THE IMPACTS OF INDEL MUTATIONS ON TARGET RECOGNITION BY A MULTI-SUBUNIT CRISPR SYSTEM
No full textCRISPR-Cas systems are part of the bacterial adaptive immune system that provides ‘memory’ of past infection by foreign nucleic acids. The type I-E CRISPR system found in E. coli consists of the ribonucleoprotein surveillance complex, Cascade, loaded with a transcribed and processed CRISPR RNA (crRNA), and the Cas3 endonuclease. During immune response, Cascade identifies repeat infections by complementary binding between the crRNA and the invader sequence (protospacer), leading to recruitment of Cas3 for DNA cleavage. However, invading genetic elements can evade the immune system by introducing mutations into their sequence. Previous studies examined point mutations' effects on CRISPR-Cas interference, but the impact of indel mutations remains unknown. To address this gap, in vitro binding and interference assays were conducted using supercoiled DNA libraries with sequentially introduced deletions and insertions across two protospacer sequences. Activity assay plasmids were subsequently analyzed using next-generation sequencing to quantify depletion or enrichment of library members. Overall, results showed high tolerance of indels by Cascade, with efficient binding and cleavage of nearly all indel targets outside of the seed. However, activity was notably impaired for targets with indels in the protospacer seed, a region where sequence complementarity is crucial for stable R-loop formation and Cas3 degradation. Select exceptions of indel seed mutations that did not significantly hamper Cascade targeting were PAM, spacer sequence, and position dependent. We also determined select cryo-EM structures of Cascade bound to biochemically confirmed indel off-targets and our structures thus far have shown that insertion mutations outside of the seed region are accommodated via a base-extrusion and/or mismatch mechanism while deletion mutations outside of the seed are accommodated via a base-skipping mechanism. These findings demonstrate that indel mutations in the seed region impact Cascade's target recognition, while non-seed mutations are well tolerated and can be structurally accommodated. Future work includes exploring the length limits of indel tolerance by Cascade as well as expanding these studies to include additional spacers, which may help clarify whether there are universally shared patterns underlying indel tolerance
Estimation of upstream instability waves from wall-pressure measurements in separated high-speed flows
Get PDFFlow separation and reattachment have a profound impact on the performance of flight vehicles, yet are probed using only a small number of discrete wall sensors. As the flow crosses the onset of separation, the spectra of the incident disturbances change significantly. As a result, the accuracy of interpreting wall-pressure data is sensitive to sensor placement, whether it is positioned upstream of the separation or within the reverse-flow region. This research investigates the challenges of flow estimation within a separated high-speed flow.
The impact of separation on the accuracy of flow estimation from wall measurements is first quantified in a compression ramp configuration with a six-degree ramp angle. At freestream Mach , this configuration produces a sufficiently strong compression which leads to separation upstream of the corner and downstream reattachment on the ramp. An ensemble variational (EnVar) data assimilation technique is used to perform two flow estimations: the first is conducted with sensor observations taken upstream of the separation, and the second with sensor observations taken from within the separated region. This study adopts numerical observations in lieu of experimental measurements. Whether the sensor data are extracted upstream or within separation, the non-linear optimization improves the error in the initial estimate of the boundary layer instability waves. However, to a lesser extent when the flow estimation utilizes observations from within the separated region. A comparison of the two flow estimations reveals increased errors in the disturbance spectra, as well as in instantaneous wall-pressure observations for the estimate derived from observations in the separated region.
Sensor sensitivity to flow disturbances directly impacts to the efficacy of the EnVar procedure. A lack of sensitivity results in an inaccurate or inconclusive assimilation. The difference in the accuracy of the two estimations is interpreted/explained in terms of sensor sensitivity. The sensor sensitivity analysis is conducted using two methods: an ensemble-based approach termed the normalized gradient and an adjoint-based method. Results from both approaches are consistent and reveal a decrease in sensor sensitivity within the separated region to the most unstable boundary layer modes. This decrease leads to increased errors in reconstructing the upstream spectra.
The compression-ramp study is the proof-of-concept prior to conducting a first-of-its-kind flow estimation over a cone-flare configuration, using experimental observations. The same EnVar data-assimilation technique is employed, with sensors placed throughout the domain: upstream, within, and downstream of the separated region. The outcome of the flow estimation is consistent with the results from the compression-ramp study. The EnVar technique is successful in improving the initial estimate, and reduces the error in peak spectral content and intensity at each sensor location. However, residual errors remain, predominantly at the downstream sensor locations. Sensor sensitivity analysis highlights adequate sensitivity to the most unstable planar waves that diminishes at higher frequencies. The sensitivity of the sensors to harmonics of the most unstable modes, generated nonlinearly within the domain, is adequate prior to the onset of separation. However, post separation and reattachment, the sensitivity degrades, leading to larger errors in the estimated harmonic modes
TISSUE INJURY AND BIOMATERIAL TREATMENT MODULATE MURINE TUMOR GROWTH AND RESPONSE TO IMMUNOTHERAPY
No full textDespite remarkable advancements in immunotherapy that have transformed cancer care, clinical outcomes remain complicated by vast interpatient heterogeneity in cancer progression and therapeutic response. In addition to tumor-intrinsic factors, such as genetic mutations, emerging research is unveiling how various tumor-extrinsic host factors can influence cancer outcomes. Surgical and traumatic tissue injuries induce substantial physiological stress with a concomitant cascade of local and systemic immune responses that can impact the tumor microenvironment (TME). Prior studies show that tissue injuries can contribute to cancer recurrence and metastatic spread, but less is known about their potential to affect tumor response to immune checkpoint blockade (ICB) therapy.
In this doctoral dissertation, we investigate how a distal tissue injury and corresponding biomaterial treatment can alter the TME immune landscape, which may influence cancer progression and ICB therapy response. We established a concurrent tissue injury-tumor murine model that couples a hindlimb volumetric muscle loss (VML) injury with subcutaneous inoculation of syngeneic cancer cells (CT26 colon adenocarcinoma and B16F10 melanoma). We demonstrated that a concurrent VML injury can accelerate tumor progression and reduce tumor response to adjuvant ICB therapy. The injury-induced accelerated CT26 tumor growth coincided with a decreased intra-tumoral density of tumor-reactive CD8+ T cells, which are potent cytotoxic effector cells involved in anti-tumor immunity. Additionally, the distal VML injury only impacted CT26 tumor growth kinetics when it shared a primary draining lymph node–a central location for immune cell priming and activation–with the tumor. We implanted a pro-regenerative biological scaffold into the muscle defect to facilitate injury repair, which abrogated the accelerated tumor growth in an interleukin-4 dependent manner.
Collectively, this work suggests that injury driven immune dysfunction may contribute to cancer progression and adjuvant ICB therapy resistance. Treatment of the injury site with wound-healing biomaterials may offer a viable strategy for mitigating adverse cancer outcomes, thereby motivating future consideration of their utility in surgical cancer settings