Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. H1 also plays an important role in regulating gene expression. H1 has been described as a transcriptional repressor as it limits the access of transcriptional factors to DNA. Linker histone binds to DNA that enters or exits the nucleosome. Several crystal structures have been published for the nucleosome (histone core/DNA complex), and the interactions of the core histone proteins with DNA are well understood. In contrast the location of the linker histone and its interactions with ds-DNA are poorly understood. In this study we have used isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and CD spectropolarimetry to determine the thermodynamic signatures and structural changes that accompany H1 binding to ds-DNA. The thermodynamic parameters for the binding of intact linker histones (H1.1, H1.4, and H10) to highly polymerized calf-thymus DNA and to short double stranded DNA oligomers have been determined. We have also determined the thermodynamics for binding of H10 C-terminal tail (H10-C) and globular domain (H10-G) to calf-thymus DNA. The real surprise in the energetics is that the enthalpy change for formation of the H1/DNA complex is very unfavorable and that H1/DNA complex formation is driven by very large positive changes in entropy. The binding site sizes for H1.1, H1.4, and H10 were determined to be 36bp, 32bp, and 36bp respectively. CD results indicate that CT-DNA is restructured upon complexation with either the full length H1 protein (H10) or its C-terminal domain (H10-C). In contrast, the structure of H10 is largely unchanged in the DNA complex. Temperature dependence of enthalpy change, osmotic stress and ionic strength dependence of Ka were tested using ITC. These results indicate that the entropy driven H1/DNA complexes are a result primarily from the expulsion of bound water molecules from the binding interface. This study provides new insights into the binding of linker Histone H1 to DNA. A better understanding of the functional properties of H1 and its interactions with DNA could provide new insights in understanding the role H1 in DNA condensation and transcriptional regulation.

Heart rate variability (HRV) and music have been demonstrated to have a relationship in previous literature. The primary objective of this study is to further investigate that relationship by observing HRV during periods of listening to relaxing and exciting music and comparing the results to a baseline as well as the other condition. The secondary objective of this study is to investigate the efficacy and potential usage of the Polar H10 chest strap monitor in measuring HRV parameters. The results of the Polar H10 will be compared to the iWorx TA-220 and iWorx-ECG12, the existing gold standard in HRV and ECG recording. The data will be exported to Matlab and Excel and analyzed to see if particular types of music display any trends for these HRV parameters, as well as heart rate (HR). Polar data will be gathered and analyzed using the EliteHRV app. Analysis included Fast Fourier Transform (FFT), Low Frequency/High Frequency Ratio (LF/HF), standard deviation of NN intervals (SDNN). Data was gathered in 10 minute intervals of No Music, Relaxing Music, Exciting Music. Results showed notable changes in LF/HF ratio in both directions. SDNN and Mean RR interval had moderate decreases in both relaxing and exciting music, with Total Power having a significant decrease in both. Comparison of Polar H10 and iWorx-ECG data showed strong agreement in heart rate and RR interval data, but significant differences in other data. This suggests differences in calculation by the software used.

The vesicular stomatitis virus glycoprotein G is responsible for low pH mediated membrane fusion induced by the virus. Four linker insertion mutants (H2, H5, HIO, A4) of the G ectodomain were found to disrupt fusion and yet maintained all the requirements for proper folding and cell surface expression (Li et al., 1993). Site specific mutagenesis of residues 123 to 137, surrounding the H2 mutant, either blocked or shifted the pH optima and threshold of fusion to more acidic values with a concomitant reduction in cell-cell fusion efficiency (Zhang and Ghosh, 1994; Fredericksen and Whitt, 1995). The region is highly conserved among vesiculoviruses and was found to insert into lipid membranes by hydrophobic photolabelling (Durrer et al., 1995) suggesting a possible role for this domain as the fusion peptide. Site-directed mutagenesis of residues 190 to 210, surrounding the H5 insertion mutant, did not significantly affect fusion (Fredericksen and Whitt, 1995). Surrounding the H10 and A4 insertion mutants is a conserved region, residues 395 to 424, that does not interact with target membranes (Durrer et al., 1995). To determine the functional importance of this region, site-directed mutagenesis was employed. Substitution of conserved Gly 404, Gly 406, Asp 409, and Asp 411 with Ala, Ala, Asn, and Asn, respt:.ctively, both reduced fusion and caused a shift in the pH of fusion threshold to more acidic values (tested by Y. He as published in Shokralla et al., 1998). In this study, the Hl0/A4 region is further mutagenized and tested for fusion. Cell surface expression was examined by indirect immunofluorescence and lactoperoxidase catalyzed iodination. Rates of transport from the endoplasmic reticulum and oligomerization into trimers were tested by resistance to endoglycosidase H and sucrose density gradient centrifugation, respectively. Low-pH induced conformational changes were assayed by resistance to proteolytic digestion. Residues Gly 395, Gly 404, Gly 409 and Ala 418 were substituted with Glu, Lys, Asp, and Lys, respectively. All mutants, with the exception of A418K, were expressed at levels similar to or above wild-type. Mutants G404K and D409A completely abolished fusion. Mutant G395E reduced cell-cell fusion efficiency by 82% and shifted both the pH threshold and optimum of wild type fusion. Although all mutants were capable of trimer formation, alterations in the structure of mutants G404K, D409 A, and A418K were detected by slower transport rates. All Hl0/A4 mutants were more susceptible to trypsin than wild-tyr,e at the pH of6.5, and mutant G404K was completely susceptible at this pH Reductions in the extent of fusion, along with shifts in the pH optima and thresholds of fusion suggest that the Hl0/A4 region (residues 395 to 418) of vesicular stomatitis virus G protein is important for G mediated fusion. The region may influence low-pH induced conformational changes. Double mutants of the H2 and HI0/A4 regions were also tested for their effects on fusion. The extents of fusion mediated by double mutant G proteins were severely reduced with levels ranging from 28% wild-type fusion to complete fusion deficiency. Only mutant Gl31A G404A was capable of 83% wild-type fusion. Mutants Gl31A G395E, Gl31A G404A, Gl31A D4LIN, Dl37N G404A, and the fusion defective D137N D411N were expressed at levels above wild-type G protein at the cell surface. Mutants Fl25Y D411N and Pl26L D411N, although capable of very low levels of fusion were not detectable at the cell surface by immunoflorescence and were detected at low levels by lactoperoxidase catalyzed iodination of cell surface proteins. These two mutants, along with Gl31A G404A, also showed slower transport rates than wild-type G. All double mutants showed increased sensitivity to trypsin at the pH of 6.5 with mutant Fl25Y D411N showing complete susceptibility. They were also all capable of trimer formation by sucrose density gradient centrifugation. In comparing the fusion profiles of double mutants with those of their component single mutants, it was found that in most cases the pH threshold of fusion by double mutants was greater than the sum of the single mutants and that the pH optimum of fusion corresponded to that of the constituent H2 single mutant. Although, the regions are functionally independent, they may indirectly affect one another through alterations in protein structure. / Thesis / Master of Science (MS)

The influenza virus, a member of the Orthomyxoviridae family, causes regular, large-scale morbidity and mortality in birds and humans and significant human suffering and economic loss. The primary aim of this study was to develop a novel influenza vaccine. Vaccines are an essential tool for the control of influenza because they increase resistance to infection, prevent illness and death and help to limit virus transmission to other birds and mammals, including humans. By reducing the environmental contamination of influenza virus in global poultry stocks, the risk of a new pandemic virus being generated by the human-avian link is diminished. Marek’s Disease is a common lymphoproliferative disease of poultry that is readily controlled worldwide using the live attenuated vaccine, CVI988. The Marek’s Disease Virus (MDV) CVI988 viral genome, available as a Bacterial Artificial Chromosome (BAC), forms viable infectious viral particles when transfected into Chicken Embryo Fibroblast (CEF) cells. Using BAC mutagenesis, two non-essential genes in the MDV CVI988 BAC (UL41 and US10), were identified and replaced by the low pathogenic influenza haemagglutinin 10 (H10) gene. These live recombinant MDV-H10 vectors will allow simultaneous vaccination against both pathogens. In addition, the non-essential genes were also replaced with GFP creating MDV-GFP constructs. Both genes were expressed initially using a CMV promoter, although this disrupted the MDV CVI988 BAC; a second promoter, PGK-1, proved more successful. A third MDV gene (UL50) was deleted, but severe attenuation prevented the incorporation of H10 into this open reading frame. Future work to test the MDV-HA constructs in vivo will be carried out in collaboration with the Istituto Zooprofilattico Sperimentale delle Venezie in Italy. In addition, development of MDV constructs containing multiple HA genes (H10 and H5) linked by the 2A polyprotein can be developed with the goal of establishing heterosubtypic immunity.