The widely accepted theory for how Earth’s moon formed begins with an impact to Earth by a Mars sized protoplanet. This impact creates a disk of debris around the Earth, and the moon is subsequently formed as the debris collides and coalesces. Previous studies have modeled the debris disk in a hydrodynamic environment, but results have found that moon formation is uncommon. This research project models the debris disk after it has cooled and condensed into a collection of solid particles using an N-Body simulation. These simulations are run using the ChaNGa code developed by the University of Washington’s N-Body shop and processed on the Hyak supercomputer. Simulations begin with an initial conditions file that we generate with different parameters, including particle resolution, angular momentum, and coefficient of restitution. From each initial conditions file the simulation runs time-progressions that model each particles position and velocity at every time-step, calculating the collective gravitational forces between all of the particles and recording any collisions that occurred. I analyze the data using plots detailing the eccentricity, mass, and semi-major axis of objects that form. Previous results from work I have completed on this project appear to show robust moon formation, with roughly lunar sized objects forming around the Roche limit. Future work on this project will include running more simulations with similar initial conditions to determine how common moon formation is, as well as analyzing the data using plots of semi-major axis vs. number of collisions/bodies accreted to determine if there are specific regions of the debris disk where the moons’ mass is originating from. While previous studies have found that a moon is only formed at very specific impact angles and sizes, this study looks to see if the moon formation mechanism may be more robust when modeled using N-Body simulations.

Glaucoma is a neurodegenerative disease that causes loss of retinal ganglion cells in the retina, resulting in incurable blindness. While current research efforts have largely focused on regenerating RGCs and other retinal neurons, it has been recently shown that inflammation resulting from immune responses to injury/degeneration can negatively impact retinal regeneration. Microglia are part of the innate immune system and are the only resident immune cell in the retina and brain; they are involved in inflammatory signaling, phagocytosis, and monitoring tissue for foreign pathogens. Previously, our lab has shown that microglia restrict regenerative capacity; microglia ablation improves neuronal replacement strategies in a mouse model. Here, we want to determine if we can develop a new method to ablate microglia from the retina via intraperitoneal (IP) injections, and observe if ablation affects invasion of the peripheral immune system (monocytes) during the inflammatory response post injury. To comprehensively ablate microglia from the retina, we test both dosage and treatment length (days) of IP injections of PLX5622, a drug which inhibits a receptor necessary for microglia survival. After specific treatment courses, mouse retinas are harvested and immunostained for microglia markers to determine the level of ablation. To determine the interaction between microglia presence and monocyte invasion in a glaucoma model, we use a CCR2-RFP mouse line to track monocytes. These mice will be injected intraocularly with NMDA, a toxin that kills RGCs. The number of invading monocytes at each time point post-NMDA-injury can be assessed with and without PLX5622 treatment to see if ablation of microglia impacts the invasion of monocytes. We expect monocyte recruitment to drastically decrease after microglia ablation. This work will allow us to investigate the role of microglia in developmental processes and identify immune targets that would be most amenable to immunomodulation interventions in future therapies for glaucoma.
 

COVID-19 is an infectious viral disease that is caused by the SARS-CoV-2 virus entering host cells through surface spike proteins that bind to surface ACE-2 receptors. Some anti-SARS-CoV-2 antibodies allow SARS-CoV-2 to also have antibody-mediated entry (AME) into immune cells, often via Fcγ receptors. This phenomenon has been correlated with cytokine release syndrome, which occurs when the immune system has a highly inflammatory response to infection and is implicated in severe COVD-19 cases. It has also been shown that other antibodies have demonstrated inhibition of SARS-CoV-2 entry. During binding and viral fusion, all three receptor binding domains (RBD) of the spike protein fold to an upward conformation, which is necessary for binding. Inhibitory anti-SARS-CoV-2 antibodies impede this process through mechanisms such as premature cleavage of the RBD or stabilizing a three-down conformation. I hypothesize that inhibitory antibodies can stop AME from occurring, but it is not yet understood which inhibitory mechanisms are most effective at preventing AME. To understand the dynamic between AME and inhibitory antibodies, I am testing the infection levels of monocytes by SARS-CoV-2 pseudo-virus in the presence of antibodies shown to induce AME combined with varying concentrations of inhibitory anti-SARS-CoV-2 antibodies that have different mechanisms of affecting the spike RBD conformation. Preliminary results suggest that high concentrations of multiple neutralizing antibodies inhibit AME. It has also been observed that an inhibitory antibody that activates the up RBD conformation increases entry at certain concentrations, whereas an inhibitory antibody that stabilizes the down RBD conformation does not enhance AME. This work will contribute to our investigation of the connections between AME, spike conformational regulation, and immune cell inflammation. Studies of this type can aid in continued development of safe vaccines and therapeutics, as well as help understand how antibodies affect SARS-CoV-2 spike conformational regulation and therefore viral entry.

The retina is made of mostly neurons and glia. In mammals when neurons degenerate in the brain or retina, they are not replaced. Studying other species, the Reh lab discovered a way to stimulate Muller glia in the retina, using the pro-neural Ascl1 transcription factor, to regenerate retinal neurons. My project focuses on another glial cell in the human retina: the astrocyte. Retinal astrocytes come from different progenitors in the brain and migrate, through the optic nerve, to the retina during development. There are two questions about astrocytes that are addressed in this project. When do astrocytes enter the retina? And can human retinal astrocytes be reprogrammed into neurons using the Ascl1 transcription factor? For the first question, based on immunohistochemistry(IHC) stainings done on sections of the human fetal retina I conclude that astrocytes enter the retina between 62-72 days of fetal development. For the second question, I hypothesize the Ascl1 transcription factor can reprogram human retinal astrocytes and the unique development and migration will affect the types of neurons they regenerate. My IHC stainings on cultures of retinal cells confirms that a lenti-virus I added to the culture with an astrocyte marker(Pax2) sufficiently targets astrocytes. I am currently working on isolating astrocytes from other cells in culture. Afterwards a lentivirus with the pro-neural Ascl1 transcription factor will be added to the astrocytes to begin reprogramming trials. Human retinal astrocytes have not been reprogrammed successfully before. If we can reprogram retinal astrocytes into neurons, it would potentially have implications for neural replacement in the retina. This would contribute to research in gene therapies for neurodegenerative retinal diseases such as: glaucoma, AMD and retinitis pigmentosa.

The retina is a neuronal tissue located on the back of the eye that receives light information, and relays this information to the brain, allowing us to see. The light is received by cells called photoreceptors, which send information through a series of inner-retinal neurons before being relayed to the brain via ganglion cells. My project is investigating the effect of a protein called DICER on retinal cell fate in the developing human retina. DICER is important for the maturation of microRNAs, which influence gene expression of retinal precursors that play a crucial role in the progression from early cell types in the mouse, such as ganglion cells, to later cell types, such as photoreceptors and inner retinal neurons. To understand if DICER is also necessary in human development, I am culturing human retinal organoids derived from embryonic stem cells (ESCs), which recapitulate human development in gene expression and developmental timing. I then preferentially knock out (KO) DICER at different developmental time points depending on the timing of infection. I have knocked out DICER in retinal organoids at an early time point and observed that, like mouse studies, cells without DICER more frequently become the early born cell type, ganglion cells. I am now investigating the necessity of DICER in retinal development at later time points. If DICER plays a similar role in cell fate progression, I expect to see an enrichment of later born cell types in the DICER KO cells. By better understanding human retinal development, we will advance future research of regenerative therapies to mitigate the impacts of retinal degenerative diseases leading to vision loss, such as macular degeneration and glaucoma, which affect photoreceptor cells and ganglion cells, respectively.

Despite heavy exposure to Mycobacterium tuberculosis (Mtb), the bacteria that causes Tuberculosis (TB), some individuals show no evidence of infection and by defining these resistance mechanisms, we may identify novel treatment strategies. Among Mtb resistant individuals, our lab identified the Rab5a protein as differentially expressed as compared to controls with Mtb infection. By regulating vesicle trafficking, Rab proteins modulate a variety of cellular pathways including inflammatory signaling, antigen presentation, and autophagy, likely playing a role in Mtb clearance. We hypothesized that loss of Rab5a would alter IFN-êžµ gene expression. Monocyte-like THP-1 cells were electroporated with siRNA targetting Rab5a and yielded 70-90% knockdown at 24 hours versus scrambled siRNA control. Cells were then stimulated with DNA ligands for four hours before RNA analysis. Loss of Rab5a resulted in lower levels of IFN-êžµ gene expression after stimulation with Sheared Calf Thymus DNA (p=0.002, 53.9% reduction), Poly(I:C) (p=0.01, 42.8% reduction), supercoiled plasmid (p=0.03, 45.3% reduction), and cGAMP (p=0.008, 45.7% reduction). We conclude that Rab5a expression is required for Type I IFN production through the DNA-sensing pathway. By characterizing the pathways by which Rab5a modulates the macrophage Mtb response, we may identify host targets to augment protective responses that may serve as adjuncts to current TB treatments and vaccines.

Hypoxic-ischemic encephalopathy (HIE), a brain injury that occurs when infants do not get enough blood flow or oxygen to the brain, is a leading cause of neonatal mortality and morbidity worldwide. Therapeutic hypothermia (TH) is the current standard of care for newborns with HIE, but TH is only available in high-resource settings and only provides partial neuroprotection. Thus, the search for additional neuroprotective treatments is critical. The ferret provides an excellent model for investigating novel treatments for HIE as, unlike rodents, it has a gyrified brain and gray-to-white matter ratio that is like humans. Previous research has shown that the ferret brain is resilient to brain injury, requiring additional hypoxia periods and increased pro-inflammatory stimuli to create the same injury as in rats. However, no previous studies have evaluated why the ferret brain is so resilient. This study will use live rat and ferret organotypic brain slices to investigate this resiliency. Whole hemisphere brain slices from postnatal day (P)10-12 rats and P21-23 ferret, equivalent to term gestation in humans, will be collected. The slices will be randomized to oxygen-glucose deprivation (OGD) injury, to mimic HIE, or control groups. OGD slices will be in 0% oxygen for 2 hours, resulting in partial, but not total, cell death. Subsequent analyses will assess transcriptomics using NanoString nCounter technology, which provides a neuropathology panel of 770 genes, as well as cell-specific regional death in brain regions affected by HIE: hippocampus, cortex, corpus callosum, subcortical white matter, basal ganglia, and thalamus. Preliminary results show that genes such as UCHL1 and TLR4, both associated with injury, are upregulated in ferret OGD slices, but rat data are currently incomplete. Identifying the pathways associated with the resiliency of the ferret brain to injury at the transcriptome level could inform future therapies to treat infants at risk for HIE.

The period around birth is when neonates are at the highest risk of neurological injury or death. A common neonatal neurological injury is hypoxic-ischemic encephalopathy (HIE), which occurs after the brain does not receive enough oxygen or blood flow. There is a large disparity in the severity and long-term neurodevelopmental outcomes of HIE between high-income countries (HICs) and low-and-middle income countries (LMICs). In HICs, HIE occurs in 1-4 neonates per 1,000 births. In LMICs, the instance of HIE is at least 2-3 times higher. Furthermore, cases of HIE seen in LMICs suggest a different type of injury - a more prolonged intermittent injury resulting in white matter injury - compared to HIE in high-income countries that is more acute and affects the deep grey matter. Therapeutic hypothermia (TH) has been the standard of care for HIE in HICs; however, TH is not an effective treatment for HIE in LMICs. Thus, the creation of alternative and accessible therapies for HIE in LMICs is crucial. This study will seek to model HIE as seen in LMICs through an in vitro ferret model that may be used to pilot therapies before applying them to in vivo models. Organotypic brain slices from postnatal day (P) 21 ferrets, equivalent to a term neonate, will be cultured and randomized to receive increasing intervals of oxygen glucose deprivation (OGD), with and without serum deprivation. Serum deprivation is defined as culturing in 2.5% serum as opposed to the standard 5% to mimic certain aspects of malnutrition that may be more common in LMICs. Cell death and white matter injury will be assessed 24 hours after OGD. We hypothesize that slices with more rounds of intermittent OGD and serum deprivation will display relatively more cell death and white matter injury, thus serving as a model of HIE in LMICs.

Nature uses only four nucleobases to store genetic information in DNA. However, additional synthetic bases which use Watson-Crick pairing have been developed and are known as non-standard bases (NSBs). NSBs P, Z, B and S incorporated alongside standard bases A, G, C and T compose DNA strands using a new genetic alphabet. Nanopores offer the potential capability for direct single-molecule sequencing of DNA containing non-standard bases (NSBs). Using a voltage gradient, DNA strands were directed through a nanopore, the biological membrane protein MspA, while we measured the ion current through the pore over time. In studying the effect of NSBs on the ion current through the pore, we observe current measurements corresponding to the Z base have a different noise profile compared to other bases. We hypothesize this noise may be associated with pH-dependent protonation of the base. To test this hypothesis, we conducted experiments with identical sequences in buffers of pH 8 and pH 7, as Z is known to have a pKa of 7.8. I analyzed the noise from the ion current signals to look for signs of protonation. I found increased current noise values associated with the Z NSB in pH 7 compared to pH 8, while the canonical A base had no change in noise values from pH 7 and pH 8, supporting the hypothesis that the increased current noise is due to protonation of the Z base. In addition to indicating potential sensing abilities of nanopores for probing protonation kinetics of DNA, this research contributes to a better understanding of the fundamental mechanisms that control the currents in nanopore sequencing of DNA.