Aging is associated impaired endothelial function in the skeletal muscle vasculature which contributes to decreased ability to increase muscle blow during exercise. This endothelial dysfunction is mediated, primarily, by impairments in the nitric oxide (NO) pathway in the skeletal muscle vasculature. The major purpose of this dissertation is to determine the mechanisms that mediate age-related endothelial dysfunction in rat soleus feed artery (SFA) and determine whether exercise training ameliorates this impairment in endothelial function. Therefore in these series of studies we sought to test three major hypotheses: 1) That exercise training reverses age-related decrements in endothelium-dependent dilation in SFA and that this improved endothelium-dependent dilation is the result of increased NO bioavailability due to increased content and phosphorylation of eNOS and/or increased antioxidant enzyme content; 2) That age-related endothelial dysfunction in rat SFA is mediated in part, by NAD(P)H oxidase-derived reactive oxygen species (ROS); 3) and, that impaired endothelium-dependent dilation in senescent SFA is due to an impaired potential for p-eNOSser1177. To test these hypotheses, SFA from young (4 month) and old (24 month) Fischer 344 rats were isolated for either determination of endothelium-dependent and –independent dilations or biochemical analyses. Results from these investigations suggest that 1) exercise training reverses the detrimental effects of aging on endothelial function in skeletal muscle feed arteries by enhancing the capacity to scavenge superoxide, increasing the bioavailability of NO; 2) ROS contribute to impaired endothelium-dependent dilation in old SFA; whereas, ROS appear to play a role in ACh-mediated dilation in SFA from young rats; 3) and, that the PI3 kinase/protein kinase B (Akt)/eNOS pathway is preserved with age.

A limitation to investigations of homodimeric protein dissociation is that the signals produced from methods such as fluorescence and circular dichroism represent both dissociation and protein unfolding that may be occurring simultaneously within a sample. Although size exclusion chromatography examines the state of a protein’s quaternary structure, complicated overlapping peaks representative of oligomer and monomer can result. To address these limitations the mixed dimer system has been adopted to investigate the dissociation of a homodimeric protein. A mixed dimer is a species in which each subunit of a homodimeric protein is associated with a different affinity tag. The two tags used are the His6-tag and the Glu7-tag. Such a mixed dimer will bind to a metal chelating column such as Ni-NTA so long as the dimer is intact. Denaturant- or temperature induced dimer dissociation can be detected by the amount of Glu7-tagged subunit present in the unbound fraction after the protein is passed over an Ni-NTA resin. SDS PAGE and densitometry assess the amount of Glu7-tagged subunit present in those unbound fractions. The experimental conditions necessary to implement this method were developed, and then applied to mammalian inducible nitric oxide synthase (iNOS) and Staphylococcus aureus NOS (SaNOS). With respect to both urea and temperature, the stability of SaNOS is higher than that of iNOS in spite of the bacterial enzyme having a much smaller dimer contact surface. We have also used the mixed dimer method to estimate an equilibrium dissociation constant (KD) for iNOS dissociation of no greater than 2.3μM. This value is compared to the results obtained for iNOS by analytical ultracentrifugation, which can characterize protein complexes and their stoichiometry. / Thesis (Master, Chemistry) -- Queen's University, 2007-10-26 15:28:14.182

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 139-147). Also available on the Internet.

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains ix, 135 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 127-135).

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--Ohio University, November, 2008. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until December 1, 2010. Includes bibliographical references (leaves 140-162)

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.