MCB Industrial Affiliates Program Fall 2020 Symposium

The question I am addressing: How do small molecules and nutrients regulate mTORC1 (related to cancer, epilepsy) and autophagy (cancer/neurodegeneration/aging)? Techniques used: Protein Purification from Mammalian and Insect Cell Cultures (AKTA-FPLC). In vitro, cellular, and in organello biochemical and biophysical assays (IP, Western Blot, Immunofluorescence). Confocal microscopy, qRT-PCR.

We used nanofabrication to create a set of substrates with circular tops, diameter 50-1000 nanometers, which mimic the stages of Clathrin-mediated endocytosis. These curved substrates induce sites of endocytosis and shorten the dynamics of productive endocytic sites. Curved substrates also rescue the knockdown of clathrin, which is consistent with the model of Clathrin acting as a Brownian ratchet during endocytosis.

Chemotherapy drug-induced peripheral neuropathy (CIPN) affects a proportion of chemotherapy patients and causes pain as a symptom. To identify candidate molecules involved in the development of chemotherapy drug-induced mechanical hypersensitivity, I am utilizing natural variation in distinct wild-type strains of mice and investigating gene expression differences that correlate with the development of mechanical hypersensitivity due to chemotherapy treatment.

Weavers are a family of sparrow-like birds with 117 species distributed throughout sub-Saharan Africa and parts of Southeast Asia, that occupy a wide array of habitats and exhibit marked variation in plumage color, behavior, as well as a diversity of nesting structures. The purpose of my doctoral research is to combine morphological, ecological and character trait data with whole genomes to understand the evolutionary processes that have driven species diversification in the family. My research talk will provide a snapshot of the methods I've employed to address my research questions, current progress, and I will end with commentary about the future utility of museum collections in the genomic era.
 

Dyskeratosis congenita (DC) is a rare, severe bone marrow failure syndrome caused by defective telomere maintenance. By introducing a known DC mutation via CAS9-mediated genome editing, we have established a human pluripotent stem cell model of DC and further demonstrated a genome editing strategy that can rescue the telomere shortening phenotype to restore the mutant cells’ replicative potential. Currently, we are testing this potential therapeutic strategy in a humanized mouse model of DC to study its efficacy in the human hematopoietic system.

Stimulation of glucocorticoid (GR) and vitamin D receptors (VDR) inhibits neonatal mouse cardiomyocyte (CM) proliferation in vitro. However, GR and VDR in CMs are dispensable for neonatal mouse CM cell-cycle arrest and polyploidization in vivo. Furthermore, loss of GR or VDR signaling does not enhance CM proliferative potential in mice with CM-specific thyroid hormone signaling deficiency.
 

Characterization of a distinct mode of gene regulation occurring during viral host shutoff and programmed cell death, which reveals direct feedback between mRNA synthesis and decay
 

The mammalian retina is comprised of neural circuits that detect specific features in the visual scene. These features include color, edges, contrast and motion, among many others. I study how the onset of visual experience, when an animal opens its eyes, influences the development and organization of motion-detecting circuits.
 

Cellular identity in the early embryo of the fruit fly is determined by unique combinations of transcription factors that bind to regulatory DNA sequences called enhancers. Decades of careful dissection of gene regulatory networks have identified the molecular players and regulatory DNA sequences that specify developmental fates. Yet,  we still lack the predictive power to calculate output gene expression levels from input transcription factor concentrations and regulatory DNA architecture. Here, we generate theoretically tractable enhancers and utilize quantitative live imaging to build and test theoretical models based on statistical mechanics.

I've found that emerging tumors driven by RasV12 oncogene expression extrude from a Drosophila epithelial tissue. This extrusion seems to be driven by interaction with the neighboring wildtype cells and results in protection of the overall tissue from overgrowth.
 

RNA velocity is the time derivative of gene expression state, estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. It predicts the future state of individual cells on a timescale of hours, and reveals the rate and direction of change of the entire transcriptome during dynamic processes. In this study RNA velocity analysis is performed on the mouse dentate gyrus cells to study aging, by exploring cell developmental paths and gene transcriptional dynamics that cannot be captured in static single-cell RNA sequencing data.

Collagen, the major component of the extracellular matrix, is the most abundant protein in animal bodies, making up ~30% of our dry weight. However, how this structural protein evolved remains unclear. I am investigating the function of collagen proteins in choanoflagellates, aquatic microbes that are animals' closest living relatives, in order to shed light on how collagen evolved in our single-celled ancestors.
 

With the advent of new live imaging techniques, the in vivo dissection of transcriptional regulation at high spatiotemporal resolution has become possible. Nevertheless, new computational techniques are needed to extract quantitative insights from these data. We developed a method that combines theoretical modeling with statistical inference to quantitatively characterize the transcription cycle and apply it to uncover new relationships, such as a correlation between mRNA transcript cleavage and polymerase density.
 

Insulin resistance leads to metabolic diseases like type 2 Diabetes and fatty liver. However, humans with mutation in Growth hormone signaling are protected from these metabolic diseases in an unknown fashion. My research in mice identifies a specific pathway in adipose tissue that is controled by Growth Hormone and promote insulin sensitivity, thus, shading light on new therapeutic angle to fight metabolic disease.

Most important information about structural transitions in biomolecules, such as drug-binding, intermolecular communication, or folding, lies in rare fleeting events when it is caught in the act of switching between stable conformational basins. Recent advancements in single molecule experiments, computing hardware, and data analysis methods have allowed measurement of these so-called transition paths in protein and nucleic acid folding studies. Such measurements have revealed important limitations in our current understanding of biomolecular dynamics and, in my talk, I will present how certain low-dimensional dynamical models offer to reconcile those limitations.

In retinal degeneration, the progressive death of photoreceptors in the outer retina causes pathophysiological remodeling of inner retinal neurons, which become spontaneously and intrinsically hyperactive, degrading vision even further. We have discovered that hyperactivity is caused by retinoic acid receptor and blocking it in-vivo dramatically improves vision in visually-impaired mice.
 

Regeneration of damaged tissues is a complex process that requires the surviving cells to respond, proliferate, and repattern to replace what was lost. By investigating gene expression changes at single-cell resolution, we observed diverse cellular responses at different distances from the site of tissue damage. In addition, we observe the upregulation of many novel genes in the regenerative blastema that we are now testing for function during regeneration.