Hemophilia A (HemA)—a severe genetic bleeding disorder affecting 1 in 10,000 people—is caused by mutations in the F8 gene. These mutations cause an inability to produce the coagulation factor eight protein (FVIII) necessary to stop bleeding after a wound. Current treatment- repeated FVIII replacement, is costly and frequently ineffective, as around 30% of patients develop inhibitor antibodies causing the immune system to reject the foreign protein. Alternatively, our lab hopes to utilize gene therapy to restore the functional gene and allow the body to continue producing the essential FVIII protein itself. 45% of human HemA cases are caused by a mutation of the human F8 gene where a large portion called Intron 22 (In22) is inverted. The In22 inversion halts translation of the rest of the gene, and the resulting FVIII protein is truncated and non-functional. To address the mutation, our lab aims to use a CRISPR-based knock-in approach to the DNA following In22, upstream of the mutation site. We expect that this strategy can restore endogenous production of missing FVIII and potentially provide curative treatment for affected patients. To test this treatment’s efficacy, this project utilizes a HemA mouse model (E16) in which a neo cassette insertion at the 3’ end of exon 16 disrupts FVIII expression. We propose using the same strategy to integrate the missing DNA upstream of the mutation and restore FVIII function in HemA mice. I use molecular cloning to construct and evaluate different versions of the CRISPR-Cas9 plasmid containing sgRNA, and a plasmid containing the donor DNA. This research allows us to determine the safety and efficacy of our gene therapy strategy, and evaluate how to maximize recovery of FVIII production. This project aims to eventually contribute to treatment of human HemA patients, without the expensive and unreliable replacement of the protein.

The first-in-class capsid (CA) binding antiretroviral, Lenacapavir (Len), inhibits viral spread at multiple steps in the viral life cycle. Structural studies show that Len interacts with an FG-binding pocket between the N-terminal and C-terminal domains of adjacent CA monomers resulting in destabilization of the CA core lattice. Three key binding functional groups within Len that interact with CA were identified. Subsequently, six Len analogues (Lenalogs) were designed and synthesized. These Lenalogs vary by the removal or replacement of one of the identified functional groups. My work investigates the impact of Len and Lenalog binding on CA assembly rate, as well as, the structure of the assembled protein. Using an IPTG  E. coli expression system and ion exchange chromatography, I have expressed and purified CA protein. I induced in vitro assembly of the purified CA protein by the addition of inositol hexakisphosphate (IP6) in both the presence and absence of Len or the Lenalogs. Relative to Len, LL-10.4 and LL-15 promoted assembly, LL-14 was similar, while LL-11, LL-19 and LL-20 promoted assembly to a lesser extent. Samples with LL-10.4 and LL-15 were chosen for cryo-EM analysis as these promoted assembly to a greatest extent. CA was assembled on lipid vesicles (templated CA-like particles or CLPs) by the Dick lab (Emory University, Atlanta, GA), and these were subjected to cryo-EM data collection and analysis. Both LL-10.4 and LL-15 bound to the FG-binding pocket like Len. Negative stain transmission electron microscopy and light scattering will be used to further assess the effect of Len and Lenalogs on assembly kinetics. My work will be used to inform the design of next generation CA-targeting antiretrovirals.

The Pacific sand lance, Ammodytes personatus, is an ecologically important forage fish in the Salish Sea. Adult sand lance bury themselves head first into sandy substrates to avoid predation and hibernate in colder winter waters, whereas juveniles remain pelagic and do not burrow until their first winter. Many head-first burrowing species exhibit cranial skeletal adaptations that facilitate substrate penetration, yet the specific skeletal modifications that enable A. personatus to burrow efficiently remain poorly understood. This study investigates how vertebral mineralization patterns change over development and how these changes may contribute to burrowing efficiency. We analyzed over 345 vertebrae of preserved A. personatus from 20-80 mm SL using a Bruker SKYSCAN 1273 micro-CT scanner. Using hydroxyapatite reference phantoms (25% and 75%) to calibrate grayscale intensity values, we quantified vertebral mineral density. We compared mineralization across three vertebral regions (cranial, mid-body, and caudal) and over ontogeny. We hypothesized that cranial vertebrae would be the most mineralized and vertebral mineralization over ontogeny would increase linearly. Contrary to our initial hypothesis, caudal vertebrae were 1.5x more mineralized than those in the mid-body or cranium, but cranial vertebrae were still more mineralized than those in the middle of the body. This suggests that the tail may play a more significant role in burrowing mechanics than we previously assumed. We identified a significant negative correlation between mineralization and body length in both mid-body and caudal vertebrae. Our data show that as these fish grow, their vertebral regions become less mineralized. This pattern challenges our expectation that adults would exhibit greater skeletal reinforcement for burrowing and instead suggests that juvenile sand lance may experience stronger selective pressures for vertebral mineralization or that adults employ alternative physiological or behavioral adaptations for substrate penetration.

After infecting a cell, HIV-1 reverse transcribes its genome resulting in a double-stranded viral DNA (vDNA) copy of the viral RNA (vRNA) genome. The vDNA is then integrated into the host genome, establishing infection. Upon activation, the integrated vDNA is transcribed resulting in unspliced, partially, or fully spliced vRNA. Fully spliced vRNAs are exported via the canonical host mRNA pathway involving NXF1/T1. Partially and unspliced vRNA require the viral protein Rev, which binds the RNA Rev response element (RRE) sequence, and recruits host CRM1/RanGTP to facilitate nuclear export. Replacing the RRE sequence with a constitutive transport element (CTE) sequence can mediate vRNA export via NXF1/T1, however at a slower rate than RRE-RNA export. This may be due to differences in RRE-RNA and CTE-RNA sizes caused by CTE-RNA having more, and variations in associated proteins. My study aims to identify and characterize the proteins packaged into viral-like particles (VLPs) made from RRE- and CTE-expressing HIV-1. VLPs containing RRE-RNA, CTE-RNA, and synthetic Gag (SG - no packaged RNA) were prepared by the Hu lab (NIH, MD). I was involved in preparing the samples for mass spectrometry (MS) which involved VLP lysis, protein reduction and alkylation, TCA precipitation and tryptic digestion. The samples were then analyzed via LC-MS/MS by the Moritz lab (Institute of Systems Biology, WA). Preliminary results indicate 78 proteins, not present in the SG control, overlap between RRE-RNA and CTE-RNA samples. Many of these are involved in procentriole subcellular localization. Furthermore, 201 unique proteins were identified for RRE-RNA samples and are primarily involved in cell cycle regulation. A total of 109 unique proteins were identified for CTE-RNA samples and are primarily involved in cell adhesion proteins. Additional analysis aims to further assess trends within the sample sets and take into account differences in relative abundance.

Self-initiated mobility has multi-faceted implications for early development, influencing cognitive, social, and physical growth. Children with Down syndrome experience delayed motor milestones—learning to walk much later than their neurotypical peers—potentially resulting in a delay of their overall development. Currently, limited research describes the impact of mobility aids on the muscular development of young children, particularly those with Down syndrome. Our study aims to address this gap by comparing and analyzing muscle activation patterns in children with Down syndrome aged 12-36 months,  both with and without mobility aids. I hypothesize that mobility aid use will result in an increase of muscle activation during play. Participants engaged in 30-minute exploratory play sessions in an enriched environment with and without mobility aids. During these sessions, data was recorded using surface electromyography sensors on the legs. The data was then analyzed to identify the nuances in muscle activation across different methods of movement—both aided and unaided. Preliminary results show that muscle activity may be similar regardless of the use of mobility aids. By identifying key muscle movement patterns, this analysis could inform future designs and protocols for motor skill development in all children, including those without Down syndrome. These findings could have implications for physical therapy and the recommendation of mobility aids for pre-ambulatory young children.

Self-exploration and mobility are crucial parts of a child’s development. Young children with Down syndrome experience movement delays compared to typically developing peers. The use of mobility aids, such as gait trainers and orthotics, has been shown to support these children with increasing their mobility. However, there remains a distinct lack of research on children with Down syndrome’s use of mobility aids. Therefore, this study examines children’s exploration in the Permobil Explorer Mini, a powered mobility device meant to facilitate self-exploration. In particular, this study compared changes in exploration as measured by distance traveled when using an Explorer Mini with a standardized rigid seat and a dynamic soft seat. During play sessions their movement was tracked using synchronized video cameras and a region-of-interest movement-tracking algorithm. This data, combined with annotations from the sessions, was used to determine if there is a significant difference in exploration between the rigid and dynamic seats. I expect there to be a significant increase in distance traveled with the dynamic seat than with the rigid seat due to its increased flexibility, comfort, and adjustment for children. The results of this study will help to expand research on mobility aids in promoting self-autonomy for young children with disabilities. These results can also aid in improving future mobility aid designs to ensure greater comfort for the children using them.

Neuroendocrine bladder cancer (NEBC) is a rare and aggressive urothelial tract cancer. NEBC is characterized by high metastatic potential and poor clinical prognosis. Neuroendocrine cancers often exhibit characteristic genetic changes including loss of tumor-suppressing genes like TP53 and RB1 and amplification or activating mutations in proto-oncogenes, e.g., MYC. However, not all bladder cancers with these characteristic mutations progress to NEBC, suggesting other occult genetic or epigenetic drivers of disease progression. To investigate the clonal origins of NEBC tumor heterogeneity, our lab developed a genetically engineered mouse model by introducing orthotopic mutations observed in human tumors (TP53, RB1, and MYC) in murine bladders by lentiviral delivery of Cre recombinase. We found some of the resulting tumors had high levels of the pioneer transcription factor, FOXA2. To further explore the role of this gene in NEBC development, we conducted an overexpression experiment in which FOXA2 was expressed in mouse-derived bladder cancer cell lines. We performed RNAseq (RNA sequencing) analysis in a panel of syngeneic murine NEBC lines, including samples with FOXA2 over expression and parental controls. In the course of this work, we developed an informatics pipeline to interrogate clonal heterogeneity at the transcriptional level in genetically identical syngeneic tumor lines – a method which we termed clonal phylogenies from RNAseq (CPR) data. My role in this project involved designing and implementing a bioinformatics pipeline to analyze both single-cell and bulk RNAseq data. By integrating cross-species comparisons with computational analysis, we aim to uncover novel molecular mechanisms driving NEBC emergence. While our research is ongoing, this approach highlights a new bioinformatics method allowing deeper insights into human cancer biology.