Our project is mainly focusing on hemichordates, they are marine invertebrates, related to echinoderms. While no chordate is able to regenerate all regions of its central nervous system after deathly injury. Ptychodera flava, a hemichordate, exhibits an exceptional ability to regenerate its entire central nervous system and anterior structures in as little as two weeks. We are studying this process to understand the underlying gene networks involved in neural regeneration. I am currently investigating the expression of transcription factors involved in neural development with homeodomain-containing genes Pax6, Six3, DLX, Frizzled, Chordin, Pou, and Msx-2, which have been shown to be upregulated during P. flava regeneration through transcriptome analyses. I have previously cloned the isolated PCR products into plasmids to make RNA probes for in situ hybridization. I am generating RNA probes, performing in situ hybridization. I will use RT-PCR to examine when there is a high RNA expression at various stages of regeneration, then I will perform in situ hybridization on P. flava tissue samples at these different stages. I will also be sectioning these samples after in situ hybridization and staining to image the cellular and tissue structures to aid in seeing where these genes are expressed in specific tissues during regeneration. My research goal is to ascertain the gene networks underlying regeneration and then to compare these to the expression of the same genes during embryonic development in P. flava. Finally, we can ask whether these genes exhibit similar gene networks that have been reported during development in direct-developing hemichordates. Further experiments will be examining if these gene networks are necessary and sufficient in regeneration by knocking-out or overexpressing these specific genes at different stages of regeneration. By using P. flava as a model to study transcriptional regulation during regeneration, we are aiming to identify the genetic and morphological mechanisms to achieve sufficient central nervous system and whole-body regeneration in a stem deuterostome, which might also give us the insight into potential key regulator factors during human regeneration.

Molgulidae ascidians are marine invertebrate tunicates, that are the sister group to vertebrates by phylogenomic analyses. We are interested in molgulid ascidians because they have evolved larvae that have lost all of the chordate features. Two main types of molgulids comprise tailed (urodele) larvae and tailless (anural) larvae, and we are using them to understand the evolution of the loss of the tail. Molgulid species are found in at least four distinct clades, three of which have multiple species with tailless larvae. Our current project in the Swalla lab focuses on how three species M. ficus, M. verrucifera, M. complanata are phylogenetically related to the other Molgulid Ascidians. Polymerase Chain Reaction (PCR) was used to isolate the 18S ribosomal RNA genes and a variable D2 loop region of 28S ribosomal RNA genes in these three species. Further sequencing, analysis, and alignment of three species’ genomic DNAs are used to construct molecular phylogenies. Moreover, the observation of larval development and adult morphology are used to characterize each species. For instance, if two species both have tailless larval development and same branchial basket morphology, additional to the identical 18S and 28S rRNA sequences, that suggests these two species are actually one species. If there are more similarities in their development and their genome sequences, they are more likely related to each other, and vice versa. In summary, the phylogenetical relationship of three species M. ficus, M. verrucifera, M. complanata compared to the other molgulid ascidians were determined based on the data of 18S and 28S ribosomal RNA sequences. This enhanced phylogeny allows us to understand the evolution and development of molgulid ascidians.

This study, hosted by the Washington State Department of Natural Resources, uses passive acoustic monitoring (PAM) and forest habitat surveys to measure ecological diversity and responses of biota to habitat change caused by forest management. The management experiment takes place in the Olympic Experimental State Forest (OESF) on the western Olympic Peninsula in Washington State, and includes upland forest manipulations with assessment of stand and upland biotic responses. The specific objective of this study is to evaluate bird species as indicators of habitat quality and function in forest stands at different seral stages and subject to different regeneration practices. To establish occupancy models for each indicator species, PAM instruments are set in forest stands of various seral stage and management practices across OESF, and from their recordings, audio surveys are developed for processing into occupancy data. Automated detection using R programming is utilized for the processing of this data. I personally contribute to this project through manual verification of species detection data to assess accuracy of automated detection systems. Habitat characteristics are sampled in the field and derived from remote sensing data using ArcGIS. Results will inform future management decisions by offering analyzed data on the ecological effects of different forest management strategies. Results will additionally provide a more robust evaluation of certain songbirds as indicator species of habitat quality and function. My concurrent independent research objective, examined via literature review and occupancy data subsampling, is to assess indicator species efficacy and identify species alternatives, considering criteria relative to suitability for passive acoustic monitoring and suitability in anticipation of climate change. All of these results can be consulted in future research and management in the region by providing a more detailed understanding of the effects of management strategies and how best to evaluate them.

Cannabis is one of the most commonly used recreational drugs in the United States. Its effects are mediated by cell surface receptors binding to cannabinoids found in cannabis, and expression varies with location and cell type. Endocannabinoid receptor 1 (CB1) expression occurs primarily in the nervous system while endocannabinoid receptor 2 (CB2) expression is commonly associated with immune cells. Cannabinoids can affect the immune response, however their specific effects on the immune response to pulmonary viral infection is unknown. In these experiments we use a mouse model to study the effects of cannabis smoke inhalation on subsequent influenza virus infection. C57BL/6 wild type mice or CB2 knock-out mice were exposed to a sub-acute level of smoke for 2 to 4 weeks using a TE Manual Smoking Machine. Cages of mice were exposed every other day to two rounds of smoke from six cannabis cigarettes. Each smoking round lasted 30-60 minutes with a 10-15 minute recovery period. Tetrahydrocannabinol (THC) levels, an active component in cannabis, were assayed by mass spectrometry in urine and blood. THC levels (0.74 +/- 0.17 ng/mL in urine, 14.3 +/- 0.94 ng/mL in blood) indicate an effective smoking regimen. Mice were subsequently infected with 10-20 plaque forming units of mouse adapted Influenza A virus and harvested 9 days post infection. Data includes bronchoalveolar lavage (BAL) cell count, daily weight change, and flow cytometry analysis of lung cells. No statistical significance in BAL cell count of mice air or cannabis smoke exposed was seen. Air exposed female mice exhibited significantly more weight loss than cannabis exposed female mice, indicating a more severe response to the virus. Cannabis exposed mice exhibited less weight loss on days 5 to 9. Continuing studies include a more detailed flow analysis of cannabis smoke exposed immune cells and its impact on viral infection.

Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that is involved in the signaling pathways of Interleukin-1 receptor (IL-1R) and Toll-like receptors (TLR). Previous studies have shown that single nucleotide polymorphisms in the TOLLIP gene are associated with mortality and outcomes in idiopathic pulmonary fibrosis, however, the biology of TOLLIP in lung injury and repair is unknown. While animal studies have shown that TOLLIP modulates acute inflammation, the effect of TOLLIP deficiency in the mouse model of lung injury and fibrosis is unknown. In this study, our group investigated the effect of TOLLIP deficiency in the bleomycin model of lung injury. Previously, we observed that TOLLIP deficiency attenuated acute lung injury in the lipopolysaccharide (LPS) model of lung injury. We hypothesize that TOLLIP deficiency leads to increased lung injury in bleomycin-induced lung injury. To study this, we treated wild-type (WT) and TOLLIP knockout (KO) mice with bleomycin through intratracheal instillation (IT). At 14 days post-treatment, we collected bronchial alveolar lavage fluid (BALF) and lung tissues to evaluate the degrees of lung injury and fibrosis. Throughout this study, I helped maintain the mouse colonies, genotype the mice, process the samples during the harvests, collect BALF cell differentials, and analyze the data. Preliminary analyses of weight loss, BALF total protein, and cell differentials suggest TOLLIP deficiency results in worse lung injury at 14 days post-bleomycin. This work provides insight into the role of TOLLIP deficiency as an attenuator in long-term lung injuries and how it may be used as a potential treatment for inflammatory disorders and infections.