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THE EFFECTS OF TRISOMY AND GENE DOSAGE IMBALANCE ON SYSTEMIC ENERGY HOMEOSTASIS
No full textDown Syndrome is a complex multi-organ genetic disorder, most frequently resulting from an extra 21st chromosome in humans. Due to global dysregulation of gene expression, as many as 80% of conceptuses miscarry. Those fortunate enough to survive are faced with cognitive deficits, abnormalities in craniofacial development, and are at a significantly greater risk of developing congenital heart defects, gastrointestinal disease, and early-onset dementia. Due to the profound impact of intellectual disability in individuals with Down Syndrome, research has understandably prioritized the investigation of neurological deficits associated with trisomy 21. While significant strides have been made, our current understanding of how the presence of an extra chromosome in individuals with Down Syndrome impacts their entire body is limited. As early as the 1960s, researchers noted that individuals with Down Syndrome were at an increased risk of developing obesity, insulin resistance, and diabetes. Despite these early observations, the underlying fundamental causes of these metabolic issues have remained largely elusive and underexplored. In an attempt to bridge this gap in knowledge, we have thoroughly characterized the metabolic land-scape of the Ts65Dn and TcMAC21 mouse models. The Ts65Dn is trisomic for nearly all syntenic regions of the human 21st chromosome in mice. In contrast, the TcMAC21 model is the most genetically similar mouse model to human trisomy 21 currently available, with every cell containing a near complete human chromosome 21. Careful evaluation of several key whole-body metabolic parameters show that Ts65Dn mice on an obesogenic, high-fat diet develop impaired insulin sensitivity, low-grade inflammation in the fat depots, and key fibrotic gene signatures. However, TcMAC21 mice exhibit unique responses to high-fat diet, again underscoring the context dependence and multi-level complexity of trisomy. RNA-sequencing of multiple tissues across both mouse models in different nutritional states has allowed this work to peer into the molecular underpinnings of said metabolic dysregulation. These fundamental observations serve as a crucial foundation for future research aimed at determining the specific combination of orthologous genes on human chromosome 21 that, when triplicated, play a role in disrupted metabolism at the organismal, tissue, and molecular levels
THE ROLE OF CARDIAC TISSUE RESIDENT MEMORY T CELLS IN THE PATHOGENESIS OF ICI-MYOCARDITIS
No full textBackground: Immune checkpoint inhibitor (ICI) therapies are efficacious cancer treatments but also cause immune related adverse events, the deadliest of which is ICI-myocarditis. We have shown that ICI-myocarditis is mediated by cardiac myosin specific (MyHC) T cells. However, their cardiac location, capacity for long-term residency, and response to cardiac injury are unknown. We propose that MyHC T cells are tissue resident memory T (TRM) cells that drive ICI-myocarditis.
Method: We characterized cardiac TRM cells through intravascular labeling and transcriptional profiling. We investigated the effect of cardiac injury on autoreactive TRM cell recruitment by inducing modified experimental autoimmune myocarditis (mEAM) and ischemia reperfusion (I/R) injury. Lastly, we developed a novel two hit ICI-myocarditis mouse model to demonstrate that MyHC TRM cells drive ICI-myocarditis development. In this model, either mEAM or I/R was induced, and mice were allowed to recover before being treated with anti-PD-1 antibodies to induce ICI-myocarditis.
Results: We determined that cardiac TRM cells are present in naive mice hearts and have distinct phenotypic and transcriptional profiles, including expression of Runx3, CD69, PD-1, and CXCR6. Cardiac injury induces the recruitment of MyHC TRM cells, which co-express PD-1 and CD69. Employing the two hit ICI-myocarditis model, we discovered that mice who recover from mEAM or I/R are more susceptible to ICI-myocarditis development. We established that cardiac injury induces the recruitment of TRM cells into the atria and perimyocardial area in both mice and humans. We found that patients with various cardiovascular diseases have TRM cells expressing CD69, PD-1, and CXCR6 in their pericardial fluid. Lastly, we demonstrated that human pericardial fluid is enriched with IL-15 and that the concentration of IL-15 positively correlates with the number of patient pericardial TRM cells.
Conclusion: We discovered that cardiac MyHC TRM cells are present in naive hearts and accumulate in the pericardial area in mice and humans after recovering from cardiac injury. TRM cells express high levels of PD-1, making them susceptible to activation after anti-PD-1 blockade. ICI-myocarditis is more prevalent after previous cardiac injury, demonstrating that TRM cells drive disease. Thus, our results provide novel insights into risk factors and the pathogenesis of ICI-myocarditis
Computational Approaches for Discovery of Gene Regulatory Mechanisms
No full textTremendous advances in genome sequencing have facilitated the study
of genomes and gene regulation at an unprecedented scale. This thesis
addresses fundamental questions in biology using various genomic approaches.
Chapter 1 provides an introduction to eukaryotic gene expression
and regulation, and how genomics and computational methods can
be applied to study biological questions in those contexts. The work in
Chapter 2 stems from a rotation project with Rajiv McCoy wherein we develop
a method to investigate patterns of aneuploidy from single-cell RNAseq
of human pre-implantation embryos. The next three chapters describe
my primary thesis work in C. elegans piRNA biogenesis mechanisms and
gene regulatory roles. In Chapter 3, we introduce a method to quantify
piRNAs from small RNA-seq, discovering subclasses of piRNAs regulated by
novel sex-specific piRNA biogenesis factors. In Chapter 4, we characterize
DNA binding profiles of snRNA and piRNA biogenesis factors from ChIP-seq
and adapt CUT&RUN for chromatin profiling in C. elegans. Together, our
work in Chapter 3 and Chapter 4 demonstrates how a conserved snRNA
transcriptional complex has been coopted and diversified in C elegans for
transcription of piRNAs. In Chapter 5, we develop an approach to computationally
predict target mRNAs of piRNAs. Chapter 6 concludes this thesis
Computational Colonoscopy for Enhancing Mucosal Contrast and Surface Analysis
Get PDFColorectal cancer (CRC) is the second leading cause of cancer death in the United States. Since 1985, CRC incidence rates have steadily declined in the United States, coinciding with the widespread uptake of CRC screening. Screening colonoscopy seeks to identify and remove precancerous lesions, called adenomas, before progressing to CRC. Colonoscopy has long been revered as the gold standard for CRC detection and prevention. However, it is not as protective as initially thought; while initial studies associated colonoscopy with a 90% reduction in CRC incidence, more recent studies estimate this rate to be substantially lower (<50%). A significant contributor to the limited protective value of colonoscopy is missed adenomas, which can be caused by poor visual contrast between healthy and malignant tissues or incomplete visual examination of the colon. Toward improving the efficacy of colonoscopy, this thesis introduces novel optical and computational methods for increasing adenoma contrast and colonic surface visualization. The research presented in this dissertation advances the field of endoscopic imaging by contributing: (1) a multi-contrast laser endoscopy system for exploring the potential of engineered illumination to improve tissue contrast in vivo; (2) an adversarial deep learning framework for speckle reduction to facilitate the translation of laser endoscopy; (3) a hybrid simultaneous localization and mapping (SLAM) algorithm for generating 3D reconstructions of the colon and assessing quality metrics; and (4) a 3D video dataset with paired ground truth for validating computer vision algorithms applied to colonoscopy. These contributions lay the foundation for technologies that reduce CRC incidence and improve patient outcomes
TRANSFORMED BY NATURE: LESSONS FROM THE SMALL
No full textHumanity is just one small part of this planet. Our lives and stories mingle with those of the creatures and places around us, and, if we pay attention, we can walk away from encounters with nature, even nearly microscopic pieces of creation, profoundly changed. Over time, observation leads to impact, which beckons understanding and meaning. The essays and articles in this thesis explore science from a research perspective and also through a more playful lens—viewing the human story and experience through the lives of other creatures
PASS_E2E_TEST_SUBMISSION_JOURNAL:Thu May 02 2024 00:35:21 GMT+0000 (Coordinated Universal Time)
No full textDecrypting pMHC-I Diversity Through Noncanonical Translation Initiation
Get PDFMHC class-I antigen processing provides CD8+ T-cells with a window into cellular proteomic states in the form of peptide-MHC-I (pMHC-I). In previous work, we uncovered a set of ATG-like translation initiation codons that define noncanonical open reading frames (ORFs) of cryptic peptides presented by MHC-I. The model cryptic initiation codon CTG shares key translation initiation requirements with canonical ATG initiation yet incorporates N-terminal Methionine or N-terminal Leucine. Here, we aim to characterize the activity and peptide products associated with the remaining cryptic codons, shedding light on the contribution of cryptic ORFs to the peptidome accessible to MHC-I.
We use the subfemtomole sensitivity of the hyperB3Z reporter T-cell hybridoma to interrogate the identity of an OVA peptide variant (SIINFEHL) reporter in a standardized cryptic initiation context. Transfection of mammalian cell lines with reporter constructs enabled the purification of peptides from cell extracts, followed by Reverse Phase HPLC to distinguish N-terminally extended and processed SIINFEHL intermediates. Our findings indicate that CTG and ACG initiation incorporate N-terminal Methionine. At the same time, N-terminal Leucine was not observed under CTG initiation despite coexpression of the H2-Db allotype known to bind and protect N-terminally extended OVA peptide. Peptide reporters with a P2 satisfying the optimal (CACCxxxG) Kozak sequence (i.e., glycine and aspartic acid) revealed that bulky residues at P2 enhance recovery of the total reporter, suggesting that peptide stability from cryptic reading frames largely depends on P2 residue. Furthermore, our model replicates the loss of endogenous antigens resulting from TAP deficiency and is amenable to studying the effects of nonsense-mediated decay on antigen presentation. These tools expand our understanding of cryptic initiation products, providing insights into defining the immune self and uncovering new sources of pMHC-I unique to tumor immunology, autoimmunity, and viruses
A NOVEL MACROCYCLIC ACTIVATOR OF YAP/AP-1 WITH POTENTIAL IN REGENERATIVE MEDICINE
No full textAdult mammalian organs lack the intrinsic ability to regenerate; therefore, when organ failure or severe injury occurs, the only option available is organ transplantation. Yes-associated protein (YAP) is a member of the Hippo pathway which regulates organ growth and size. Activation of YAP, a transcriptional coactivator which promotes growth, wound healing, and regulates stemness in both terminally and non-terminally differentiated cells, is an appealing strategy for promoting organ regeneration where none was possible before. We screened our novel library of macrocyclic molecular glues, called rapafucins, and discovered an activator of YAP. This molecule, WL5A5, activates YAP activity by signaling through the Rho/ROCK pathway to induce cytoskeletal changes that drive YAP to activate transcription. Through a yet unknown target, WL5A5 not only activates YAP, but also induces activity of MAPK pathways to drive AP-1 transcription. We observed upregulation of YAP target genes, as well as AP-1 target genes Il-6, Il-11, and other members of the senescence-associated secretory phenotype (SASP) which, when transiently expressed, are known to have proregenerative effects. We show that WL5A5 drives spheroid and organoid formation, as well as upregulates YAP and AP-1 target genes in zebrafish, our first in vivo model of regeneration. We ultimately aim to study the effects of WL5A5 on colon regeneration in IBD models. To identify the direct molecular target of WL5A5, we have employed multiple probe-based methods to enrich potential protein binders of WL5A5, which is still ongoing. Taken together, we describe preclinical development WL5A5, a potential regenerative therapeutic functioning as an activator of YAP/AP-1 and showing promise in models of regeneration
The immunological effects of Tofacitinib nanodelivery in the treatment of type 1 diabetes
No full textType 1 diabetes (T1D) is still an uncurable autoimmune disease characterized by the destruction of pancreatic beta cells by the immune system. T1D is heterogeneous, with both genetic and environmental components contributing to its development. Immunotherapies have been investigated for T1D, however, long-term improvements of glycemic control and the restoration of insulin independence remain elusive. Interestingly, among the genetic risk factors for T1D, there are loci of genes of several inflammatory cytokines that use Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathways. Consequently, we tested the viability of exerting immune regulation of T1D development via localized release of the JAK inhibitor Tofacitinib (Tofa) through encapsulation into lipid nanoparticles (LNp). Preliminary data indicated that, following oral administration, the LNp accumulated into gut draining lymph nodes including pancreatic lymph nodes, the most probable physiological sites of T1D autoimmune response activations. A short-term oral administration of Tofa nanoparticles to non-obese diabetic (NOD) mice (a model of spontaneous disease development) delayed T1D onset and decreased its incidence. The preliminary investigation of the mechanisms behind this therapeutic effect pointed toward a localized and lasting accumulation of anergic CD4 T cells, functionally inactivated cells that, in healthy individuals, are formed as part of intrinsic mechanisms of immunological tolerance.
This thesis focused on understanding the mechanisms behind the therapeutic effect of Tofa-LNps and the work showed: 1. The existence of a novel immune regulatory function of JAK inhibition during restimulation of CD4 T cells that promotes hypo-responsiveness, while inhibiting dendritic cell (DC) maturation. 2.The localized accumulation of LNp and their uptake by various immune cells, making the LNp a desirable vehicle for regioselective delivery of immunotherapy. 3. A short course of Tofa encapsulating LNp delays the development of T1D, highlighting their therapeutic potential. 4. A fraction of anergic cells can convert into regulatory T cells (Treg) in NOD mice, suggesting they can act as a source of active immune modulators.
Overall, my thesis research demonstrated the potential of Tofacitinib encapsulated into nanoparticle as preventative therapy for T1D in the NOD mouse model and indicated that the underlying mechanism involves direct and indirect induction of two anergy states
GENETIC KNOCKOUT OF CREATINE KINASE BRAIN-TYPE IMPAIRS DRUG METABOLISM AND COGNITIVE FUNCTION IN MICE
Get PDFTenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection, and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can catalyze the formation of tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, and evaluate the impact of naturally occurring mutations on CKB activity, in vitro. To determine the extent CKB contributes to TFV activation in vivo, we generate a CRISPR/Cas9-mediated CKB knockout mouse strain, Ckbtm1Nnb. Using an ex vivo activity assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5% and 77.4% less TFV-DP than WT brain lysates of the same sex, respectively. Further, following oral dosing we reveal that TFV activation in Ckbtm1Nnb male livers decreases 22.8% when compared to WT male livers. Utilizing mass spectrometry-based proteomic analyses, we elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how the loss of CKB may be diminishing tenofovir activation in these tissues. To phenotype the novel mouse strain, we conduct behavioral studies on aged Ckbtm1Nnb mice and uncover a significant decline in hippocampal-dependent learning and memory. Next, we perform proteomic and metabolomic analyses on various regions of the brain to comprehensively understand the physiological changes that occur in Ckbtm1Nnb mice. Interestingly, carnosine and anserine are significantly downregulated in the hippocampus of Ckbtm1Nnb mice, indicating a potential mechanism underlying the cognitive deficits. Further, we perform proteomic analyses to determine how age, sex, and genotype may impact the landscape of nucleotide analogue-activating enzymes in blood, serum, colon, vagina, and prostate. Lastly, we optimize and validate a single cell proteomics workflow for the interrogation of cells dissociated from brain, with a proof-of-concept experiment that unveils a potential co-regulation of PKM, PGK1, and NME2. Together, this body of work covers the canonical and pharmacological roles of CKB in several tissues. The data presented here provides evidence that CKB dysregulation impairs drug metabolism and cognitive function