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Item Gap junctions in the development of the nervous system(Montana State Univeristy, 2017-04) Warthen, TheodoreGap junctions are intercellular ion channels composed of two intermembrane protein complexes called connexons, which are bound together forming an intercellular pore. These pores then allow for passive transport of small molecules and ions. Calcium along with other ions that move through these gap junctions are known to be involved in intercellular communication. We hypothesize that intercellular signaling through gap junctions helps begin, direct, and end developmental processes such as convergent extension in the embryonic stages of gastrulation and neurulation. I have begun to determine at which stages gap junction proteins (connexins) are expressed. To do this cDNA (DNA reverse transcribed from RNA isolated from blastula, gastrula and neurula Xenopus laevis embryos) was used as a template for PCR with primers specific to each connexin. The PCR products were analyzed by gel electrophoresis (the primers had been tested previously and were known to be functional). By using this form of PCR we were able to determine what genes were expressed at what stages. The connexin 46 and connexin 43.4 genes were found to be expressed at stages 18 and 20 (late neurula and early tailbud), while connexin 30 was found to be expressed at stages 12-20 (late gastrula through early tailbud). There were several connexin genes, which were not expressed at the stages that were tested. The next step is to determine the expression patterns of the expressed genes through in situ hybridization of gastrula and neurula Xenopus embryos.Item Characterization and Comparison of Vertebrate Voltage Sensing Phosphatases(Montana State Univeristy, 2017-04) Stengel, KarenThe voltage sensing phosphatase (VSP) is a transmembrane protein which regulates the phosphatidylinositol phosphate (PIP) signaling pathway in a voltage dependent manner. The membrane potential is an important signal in normal cellular processes controlling neuronal signaling, muscle contractions, and immune responses while PIPs regulate many different processes in the cell, including membrane trafficking, promoting cell death, and cell growth. When either pathway is compromised, many serious diseases can occur, including autism, epilepsy, and cancer.Interestingly, VSP has been found to be expressed in non-small cell carcinoma and hepatobiliary cancers, suggesting it may also play a role in cancer and could indicate an unexplored role of voltage in cancer cell propagation. The majority of VSP research has focused on the tunicate Ciona intestinalis (sea squirt) species of the protein (Ci-VSP) and very little is known about the vertebrate VSPs. I have been studying the vertebrate VSP species Gallus gallus (chicken, Gg-VSP) and Danio rerio (zebrafish, Dr-VSP) in order to compare the functions of these vertebrate species to Ci-VSP, focusing on the 210 and 212 equivalent sites of Ci-VSP. Dr-VSP has been successfully mutated for voltage clamp fluorometry (VCF) experiments. VCF is a technique that allows us to monitor protein motions through a fluorescent tag on the VSP, all in a live cell. Several of the Dr-VSP mutations have expressed and display voltage-dependent fluorescence changes that vary from the equivalent Ci-VSP mutation suggesting that the different species of VSP do not all function similarly. The rest of the vertebrate species being studied are still being mutated to include labeling sites.Item Investigation of the nervous system in a mouse model for Familial Dysautonomia(Montana State Univeristy, 2017-04) Shchepetkina, VeronikaFamilial dysautonomia (FD) is a genetic disorder affecting the development and maintenance of the nervous system, and is prevalent in those of Ashkenazi Jewish descent. FD is caused by a point mutation in the Ikbkap gene, resulting in a decreased amount of the IKAP protein. FD patients experience symptoms such as decreased sensitivity to pain or temperature, dysfunction of the autonomic nervous system, incoordination, hypotonia, various dysfunctions of the organs, and even death. In addition, FD patients experience progressive blindness due to the loss of retinal nerve fiber layer. In order to study the role of IKAP in the retina, we developed the retina-specific Ikbkap conditional knockout (CKO) mouse model. We used this model to quantify retinal ganglion cells in the retinal nerve fiber and found decreased cell numbers in the mutant mouse at different ages. Data from our lab has revealed evidence of CNS deficits in mice with FD, such as behavioral alteration, a reduction in specific neuronal populations, reduction in spinal motor neuron innervation, and alteration in cortical morphology. In order to further investigate the implications of FD on the CNS, we generated another mouse model (Tuba1α) in which Ikbkap is deleted in all neurons. Our data show that both adult and embryonic Tuba1α mice have enlarged lateral ventricles in the brain, a symptom occurring in other degenerative and developmental disorders. We used this mouse model to investigate proprioceptive and nociceptive neurons in the embryonic DRG in order to compare development in the DRG to the brain. Neither population was altered in the mutant mouse, suggesting that these cell types are resilient to the disease during embryogenesis.Item CD103 regulation in human dendritic cells using retinoic acid in the gastric microenvironmnet(Montana State Univeristy, 2017-04) Sewell, Marisa A.CD103 (αE integrin) is an important marker for dendritic cells (DCs) in the human mucosa. Iliev et. al (2016) showed that CD103+ DCs display tolerogenic behavior in the human gut and induce Treg cell development. However, not much is known about the regulation of its expression, though it is widely used as a delineator of DC populations. Previous work in my group shows that retinoic acid (RA) and toll-like receptor agonists contribute to the regulation of CD103 expression in human DCs (Roe et al, 2016). We postulate that CD103 functions to initiate DC binding to gastric epithelium, possibly to E-cadherin, in order to allow for antigen sampling through tight junctions by DC dendrites. Additionally, previous research in this lab has concerned the identification of gastric stromal factors using gastric stroma- conditioned media (SCM), which is a model for the gastric microenvironment. I have shown that gastric stromal factors are responsible for suppressing dendritic cell maturation in Helicobacter pylori infection. We have thus confirmed SCM-derived immunoregulatory factors as a suitable model for generating dendritic cells with a tolerogenic mucosal phenotype. Here, we’ve confirmed that (RA) induces CD103 expression in peripheral-blood monocyte derived dendritic cells (MoDCs). Additionally, we show that the addition of SCM increases the extracellular expression of CD103 in both RA and non RA treated conditions. Lastly, I show that intracellular concentrations of CD103 in the presence of SCM are lower than extracellular concentrations, whereas CD103 is predominantly located in the cytoplasm in the absence of SCM, indicating that SCM may be a catalyst for initiating redistribution of CD103 to the cell membrane.Item Therapeutics for rescue of Ikbkap deficient dorsal root ganglia neurons: An In-vitro Approach(Montana State University, 2017-04) Russell, MagdalenaThe Lefcort lab has developed a mouse model that recapitulates the hallmarks of Familial Dysautonomia by conditionally deleting the IKAP protein from neural crest cells, which gives rise to the neurons and glial cells of the peripheral nervous system, via the Wnt1-cre driver. The mouse shows significant reductions in neuronal number in the dorsal root, sympathetic, and parasympathetic ganglia. As such, it provides an excellent model system for determining the function of IKAP in embryonic development and for mimicking the Familial Dysautonomia found in humans. This research projects aims to assess the neuroprotective effects of two different drugs, Metformin and the GSK2606414 PERK inhibitor, on the Ikbkap deficient dorsal root ganglia (DRG) neurons within this mouse model. The ultimate goal of this study is to identify and target new cell signalling pathways and their neuroprotective potential as they relate to Familial Dysautonomia. An absolute cure for the disease is improbable due to the widespread devastation it causes. However, determining a mechanism to rescue neuronal death could lead to valuable therapeutic approaches and new rescue strategies that could eventually be used to increase the quality of life and overall lifespan in the mice and, eventually, in humans.Item Characterization of Pseudomonas aeruginosa Antibiotic Resistance to Polymyxin(Montana State University, 2017-04) Narayan, MichellePolymyxin B is a cationic peptide antibiotic that has efficacy at killing the opportunistic pathogen, Pseudomonas aeruginosa. However, the efficacy of polymyxin B is reduced at high calcium concentrations, which are characteristic of in vivo environments. The goal of this study is to identify P. aeruginosa genes that impart calcium-dependent polymyxin B resistance. In previous research, a series of P. aeruginosa mutant strains was obtained that had increased sensitivity to polymyxin B when cultured at high calcium concentration. Some of the mutants have been complemented with P. aeruginosa DNA that restores polymyxin B sensitivity, and these genes have been sequenced. Sequence analysis has shown that the genes that effect resistance are ones that have not yet been identified previously, and unexpectedly are not related to lipopolysaccharide modification, as been shown in other research. In this project, I used a microtiter plate assay and antibiotic disk diffusion assays to determine the minimum inhibitory concentration (MIC) of the wild-type strain, the mutant strains, and the complemented strains, at low and high calcium concentrations. Unlike prior results using e-strip diffusion assays, the MIC results do not show a significant difference between the strains cultured differing calcium concentration. In future studies, I will modify the MIC methodology and perform e-strip analysis to determine if the difference observed here from prior studies are a result of differing methodologies for determining MICs.Item Determining the Place of Aqp3b in Noncanonical Wnt Signaling(Montana State University, 2017-04) Machado, OscarDuring Xenopus laevis gastrulation, convergent extension is required for the mesoderm to extend into the embryo and shape the embryonic body plan. Recent results from our lab suggest that the inhibition of aquaporin3b (aqp3b) prevents convergent extension of the mesoderm and that aqp3b acts through noncanonical Wnt signaling. Wnt signaling is a key signaling pathway for embryo and tissue development. There are two types of Wnt signaling pathways, the canonical and the noncanonical pathways. There are three separate branches to noncanonical Wnt signaling. Our lab has shown that aqp3b acts through the noncanonical Wnt/Ca2+ pathway and that it acts upstream of the cytoplasmic Wnt signaling pathway member Disheveled (Dsh). The Frizzled7 (fzd7) membrane receptor is part of the noncanonical Wnt/Ca2+ pathway and also acts upstream of Disheveled (Dsh). I will test, whether in this signaling cascade, aqp3b acts upstream or downstream of fzd7. Thus, I will test whether fzd7 activates aqp3b, if aqp3b activates Fzd7, or if aqp3b is bypassed and fzd7 activates disheveled. When fzd7 is active, GFP-labeled protein kinase C (PKC-GFP) relocates from the cytoplasm to the plasma membrane. Thus, I will inject either PKC-GFP alone, PKC-GFP + fzd7, or PKC-GFP + fzd7 +aqp3bMO (morpholino oligonucleotide, which inhibits aqp3b) into two-cell Xenopus embryos and examine under a fluorescence microscope whether the PKC is bound to the membrane (active Wnt signaling: PKC + fzd7 injection or if aqp3b acts upstream of fzd7) or remains in the cytoplasm (no Wnt signaling: PKC injected alone or if aqp3b acts downstream of fzd7). With this procedure the place of aqp3b within the Wnt/Ca2+ signaling pathway will be determined.Item Tryptophan Metabolites and Their Role in IL10-R1 Expression(Montana State Univeristy, 2017-04) Corpron, JacobWithin the gut lives a diverse microbial flora. Intestinal epithelial cells (IEC) need to maintain a functional barrier in order to protect the body from this antigenic luminal environment. The immune system’s inflammatory response to invasion of antigens in the IEC layer can lead to disruption and damage to the mucosal lining. This compromises the barrier function of the epithelium, resulting in an increase of bacterial diffusion across the intestinal epithelia as well as an increase in inflammation. Regulatory T cells (Treg) can mediate the pro-inflammatory response through the signaling of the cytokine interleukin-10. Il-10 and its receptor IL-10R1 play key roles in suppressing inflammation resulting from a disruption of intestinal epithelial homeostasis and epithelial barrier. Recent research has shown the activation of the aryl hydrocarbon pathway induces IL-10R1 IECs. There is currently a gap in our knowledge regarding the specific tryptophan metabolites implicated in the upregulation IL10-R1 in intestinal epithelial cells via the aryl hydrocarbon receptor pathway. Based on previous findings, we hypothesize that specific tryptophan metabolites including indole-3-carboxaldehyde (IAl), indole-3-proprionate (IPr) and indole-3-acetic acid (IAa) upregulate IL-10R expression through the AhR pathway in intestinal epithelial cells.Item Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum(2012-06) Valenzuela, Jacob J.; Mazurie, Aurélien J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Fields, Matthew W.BACKGROUND: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome hasbeen sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis hasbeen reported under different growth conditions. To elucidate P. tricornutum gene expression profiles duringnutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P)and whole-genome transcripts were monitored over time via RNA-sequence determination.RESULTS: The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, theincrease in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphatewas depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be anearly trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genesassociated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulationafter growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbonreduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbonassimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon(DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed(2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth.Alternative pathways that could utilize HCO-3 were also suggested by the gene expression profiles (e.g., putativepropionyl-CoA and methylmalonyl-CoA decarboxylases).CONCLUSION: The results indicate that P. tricornutum continued carbon dioxide reduction when population growthwas arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels.Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up ofprecursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actualenzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellularresponses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.Item In situ gene expression profiling of the thermoacidophilic alga C yanidioschyzon in relation to visible and ultraviolet irradiance(2014-06) Skorupa, Dana J.; Castenholz, R. W.; Mazurie, Aurélien J.; Carey, Charles C.; Rosenzweig, F.; McDermott, Timothy R.Ultraviolet and high-intensity visible radiation generate reactive intermediates that damage phototrophic microorganisms. In Yellowstone National Park, the thermoacidophilic alga Cyanidioschyzon exhibits an annual seasonal biomass fluctuation referred to as 'mat decline', where algal viability decreases as ultraviolet and visible irradiances increase during summer. We examined the role irradiance might play in mat decline using irradiance filters that uncouple ultraviolet and visible effects along with custom microarrays to study gene expression in situ. Of the 6,507 genes, 88% showed no response to ultraviolet or visible, implying that at the biomolecular level, these algae inhabit a chemostat-like environment and is consistent with the near constant aqueous chemistry measured. The remaining genes exhibited expression changes linked to ultraviolet exposure, to increased visible radiation, or to the apparent combined effects of ultraviolet and visible. Expression of DNA repetitive elements was synchronized, being repressed by visible but also influenced by ultraviolet. At highest irradiance levels, these algae reduced transcription of genes encoding functions involved with DNA replication, photosynthesis and cell cycle progression but exhibited an uptick in activities related to repairing DNA damage. This corroborates known physiological responses to ultraviolet and visible radiation, and leads us to provisionally conclude that mat decline is linked to photoinhibition.