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Effects of Brain NAT8L Knockdown on Myelin
Neurological disorders such as multiple sclerosis and Alzheimer’s disease are a result of decreased levels of N-acetylaspartate (NAA) in the brain. NAA is a brain-specific mitochondrial metabolite that plays a role in lipogenesis and myelination within the central nervous system. NAA is synthesized by an N-acetyltransferase enzyme that is encoded by the gene N-acetyltransferase-8-like (NAT8L) and metabolized into acetate and aspartate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. Oligodendrocytes are neuronal cells responsible for myelinating the central nervous system axons. This study is sought to examine the changes in myelin lipids using brains of postmortem NAT8L knockout mice, which were unable to synthesize NAA. Western blotting and immunohistochemistry will be performed to measure the effects of NAA deficiency on myelin.
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Working Backwards: Enhancing Forest Restoration by Reversing Effects of Reclamation on Soil Bulk Density
Cuyahoga Valley National Park is readily accessible and located only 15 miles from Kent State University. My study focuses on one of more than 40 legacy mines in the park. Before the 1970’s, these mines were used for gravel to build nearby highways. After mining, reclamation efforts were minimal. Industrial rollers were used to compress the soil to stop nutrient runoff and prevent further pollution. Although this brought life back to the sites, the compaction made it nearly impossible to grow woody plants due to inability to spread roots. Recently the National Park Service and Kent State has become interested in reintroducing hardwood trees to return these sites to their natural form. The national park adopted a soil ripping method to reduce the soil bulk density to allow roots to expand and grow. Large shanks were dug into the ground and “ripped” through the soil in a 2x2 meter grid pattern throughout the field site. To determine its effectiveness, we dug pits and took soil bulk densities at varying depths to 60cm. The bulk density of the soil was generally lower in the rips than in the non-rips at depths deeper than 20cm. This helped to reverse the previous compaction resulting from land remediation. The lower bulk densities lead us to believe that the roots of hardwood trees will penetrate soils easier in the rips than in the non-rips. We hope that this soil ripping technique will have a lasting positive effect on the root penetration of our trees.
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Full Spectrum Hemp Extracts and Black Tea Extract Inhibit Ovarian Cancer Cell Proliferation
Isolated cannabinoids, catechins and theaflavins exhibit cytotoxicity toward many cancer cells, including those derived from ovarian cancer. However, other phytochemicals in whole plant extracts may potentially modulate each other’s effects via the “entourage effect” as has been observed with Δ9-tetrahydrocannabinol (THC) versus whole plant extracts (Biochem Pharmacol 157:285-293, 2018). Therefore, we investigated if these extracts inhibit the proliferation and migration of ovarian cancer cells. All experiments were performed in serum-free DMEM:F12 medium in the presence or absence of broad and full spectrum hemp extracts and a standardized black tea extract. For cell survival studies, the extracts were tested on subconfluent cells. Cell migration assays were performed on confluent cells. The activity of matrix metalloprotease-2 (MMP-2) was assayed by zymography on gelatin gels. Data were analyzed by one-way ANOVA and Bonferroni post-hoc test. The results of our studies suggest that both full spectrum hemp extract, which contains multiple cannabinoids and terpenes, and the black tea extract with theaflavins are cytotoxic to SKOV3 ovarian cancer. Broad spectrum hemp extracts did not exhibit any antiproliferative activity, while the full spectrum and black tea extracts inhibited the proliferation of the cells. The cells rounded up, swelled, and detached from the substratum in the presence of the hemp extract alone, but remained attached and swollen when treated simultaneously with both extracts. The migration of cells was inhibited primarily by the black tea extract. The activity of MMP-2, a gelatinase associated with tumor cell migration and metastasis, was also markedly inhibited by the black tea extract.
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Assessing Plant Species’ Diversity with the Whittaker Plot Method in Queen Elizabeth National Park, Uganda
Uganda’s Queen Elizabeth National Park (QENP) has historically contained diverse vegetation for wild animals. New prevalence of the invasive woody plant Dichrostachys cinerea has caused rapid biodiversity loss and forced these animals into private pastureland that worsens human-wildlife conflicts. Consequently, QENP staff have devoted countless hours relocating animals back into the park and assisting the community. As an intervention designed to mitigate woody plant encroachment, habitat patches within QENP have been clear cut to reset vegetation succession and remove invasive species. QENP’s long-term goal is to restore the grasslands, bring back the grazer populations, and reduce human-wildlife contact. Whittaker’s plant diversity sampling method consists of a pattern of taped off quadrats that are compounded to project the species richness. Kent State University students laid out Whittaker plots in QENP with the help of students and staff from the Uganda Wildlife Research and Training Institute. The purpose was to compare species richness in a cleared habitat patch with a patch of similar size that had not been cleared of the woody invasive plants. Results project that the cleared habitat patches had higher plant species abundance than the uncleared patches. There were numerous juvenile plants in the recently cleared area; high species competition can be inferred from this. This was preliminary research to assess the effectiveness of clear cutting as a conservation practice for native flora. Future monitoring should be done to conduct the Whittaker plot method on a larger scale and test the possibility of using alternative conservation methods that manipulate fire and grazing.
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Lichens of Two Kent State University Properties in Portage County, Ohio
Lichens are a diverse and speciose group of organisms that have colonized every continent on the planet. They occur on virtually all natural substrates, as well as numerous synthetic substrates, and play essential roles in healthy terrestrial ecosystem. Similar to other groups of inconspicuous organisms, lichens are underrepresented in almost every facet of biological research, and much about them, including diversity, ecology, distribution, medicinal uses, etc. remains hidden under a veil of obscurity. In this study, two comprehensive surveys of lichens were conducted at two different properties owned by Kent State University. A specimen of each species found was collected for each site in order to increase lichen representation in local herbaria. A total of 91 species were found, all of which were included in a dichotomous key in order to provide identification capability for a novel combination of lichen species.
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Generation of Tissue-specific Huwe1 Knockout Mice
Huwe1 is a ubiquitin E3 ligase that plays a role in regulating the DNA damage response, apoptosis, metabolism, and autophagy in various cell types. The objective of this study was to determine the impact of Huwe1 deletion in different tissues by generating transgenic mouse models. We crossed a tissue-specific Cre mouse line with the Huwe1flox/flox mouse line in which exon 11 of the Huwe1 gene is flanked by two loxP sites. When expressed in the same cells, Cre recombinase would excise exon 11, resulting in a frameshift that generates a premature stop codon. By choosing different tissue-specific Cre mouse lines, this method allowed us to create tissue-specific Huwe1 knockout mice. First, lymphocyte-specific Huwe1 knockout mice were generated using a mouse line that expressed Cre recombinase under the Rag-1 promoter. Second, liver-specific Huwe1 knockout mice were created utilizing an Alb-Cre mouse line that expressed Cre recombinase under the albumin promoter. Lastly, inducible whole-body knockout mice were generated through the Cre allele fused to the modified estrogen receptor (ER), which translocates from the cytoplasm to the nucleus upon binding to the estrogen antagonist, tamoxifen. In this Cre-ER model, injection of tamoxifen induced acute deletion of the Huwe1 gene. In all the knockout mouse models, we performed PCR to confirm their genotypes. We also carried out western blot to analyze the depletion of Huwe1 protein in the tissues. We will discuss phenotypes of each tissue-specific Huwe1 knockout mouse model.
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Perilipin 3 Binding to Model Lipid Droplets Using Pendant Drop Tensiometry
Lipid droplets (LDs) are dynamic organelles that are critical for cellular energy regulation. LDs consist of a neutral lipid core and a phospholipid monolayer, which allows for protein localization and binding. The mechanism of protein-lipid interactions at this monolayer remains less understood compared to those at biological membranes. These LD-associated proteins are essential to the development and functionality of LDs. One of the most abundant families of mammalian LD-binding proteins is the perilipin family, consisting of five proteins, named perilipin 1-5. Perilipins 1 and 2 are found bound to LDs, while perilipins 3-5 exchange between the surface of LDs and the cell cytosol. We are interested in the mechanism by which these exchangeable, or cytosolic, proteins bind to LDs. Here, we study the protein perilipin 3. Langmuir monolayer data from our lab suggest that the α-helix bundle located on the C-terminus of perilipin 3 has strong interactions with the lipid interfaces. To obtain a more comprehensive picture of the function of this C-terminal domain, we compare the full-length perilipin 3 to a truncated sequence that contains only this domain. Pendant drop tensiometry is used to examine protein binding to simulated LD monolayers with lipids that have the same headgroup structure but varying acyl chain saturation, and with lipids with the same acyl chain saturation but varying headgroup size and charge.
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Tight Junction Proteins in Mice
The purpose of this study was to evaluate tight junction protein changes during different time points of cuprizone treatment in the corpus callosum and cortex of mice. Cuprizone is a copper chelator that causes demyelination and is used as an animal model of multiple sclerosis. Demyelination is the result of breakdown of the myelin sheath that insulates neurons and can cause nerve impulses to slow down or even fail. It is important to study demyelination because it causes neurological problems associated with multiple sclerosis and current treatments are lacking. Studies in our lab have shown early breakdown of the blood brain barrier during cuprizone treatment. Tight junction proteins are molecules that maintain the integrity of the blood brain barrier that protects cells in the brain. TJ proteins have barrier functions that hold cells together and facilitate signaling in the central and peripheral nervous system. Mice were given 0.3% cuprizone as a diet for two different time periods, three days and one week. The brains were extracted and sliced and the tight junction proteins were immunolabeled and imaged using a confocal microscope. Cuprizone fed and control mice staining patterns were quantified and compared in the corpus callosum and cortex at three days and one-week of treatment.
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Variation in Muscle Thermogenic Response to Predator Threat Stimuli in Mice
With the growing obesity epidemic, there is a drive to determine a better route for weight reduction. We are interested in mechanisms to increase caloric expenditure in mice and rats, including muscle thermogenesis. We discovered that muscle thermogenesis can be acutely induced by predator threat, specifically predator odor, in rats and mice. In mice, other stimuli may more potently activate this sympathetically- driven muscle thermogenesis. Here, we investigated the potential multimodal activation of muscle thermogenesis in the mice. We hypothesize that the mice will show a greater thermogenic activation when exposed to the multimodal predator stimulus (mobile robotic stimulus) compared to the predator odor because of the increased threat perceived. First, mice were habituated to the conditions of the study. Then, mice were presented with three stimuli in randomized order: control, ferret odor, and the multimodal robotic bug. There was a trend observed toward a significant effect of the multimodal predator threat on muscle thermogenesis with control inducing the lowest thermogenesis, then ferret odor and multimodal predator stimuli with higher activation of thermogenesis. This suggests that the multimodal approach may more potently activate muscle thermogenesis; however, more statistical power is needed. Additional combinations of stimuli can be examined in combination with the multimodal approach.
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The Search For Happy Chromatin: An Analysis of the Relationship Between Neurodegeneration and Serotonylation
Multiple sclerosis (MS) is an autoimmune disorder characterized by demyelination of the central nervous system (CNS) and neurodegeneration. Serotonin is a neurotransmitter that plays a role in numerous pathways throughout the body including sleep, happiness, and epigenetic modifications. Recent studies have found that serotonin levels are decreased in MS for reasons that are currently unknown. This is significant because serotonin plays a role in the post-translational modification of histone 3. Because serotonin is decreased, we have hypothesized that serotonylation is decreased which leads to subsequent aberrant gene expression in MS. The purpose of this experiment is to explore a new epigenetic mark and its role in neurodegeneration and MS. Using the cuprizone mouse model of MS, we isolated brain tissue and looked at changes in serotonylation. Because Alzheimer’s disease (AD) is also characterized by neurodegeneration, brain tissue from mice treated with APP (AD mouse model) was also analyzed. Analysis was conducted on nuclear fractions via Western Blotting, confocal imaging, and densitometry.
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