Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2006. / Includes vita. Advisory Committee: Lucy Liaw, Scientist, Maine Medical Center Research Institute, Advisor; Carla Mouta-Bellum, Scientist, Maine Medical Center Research Institute; Jeong Yoon, Scientist, Maine Medical Center Research Institute; Dorothy E. Croall, Professor of Biochemistry; Thomas Gridley, Senior Scientist, The Jackson Laboratory. Includes bibliographical references (leaves 97-112 ).

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Gamma-thujaplicin (GT) and beta-hydroxy thujaplicin (BHT) are two of the isopropyl derivatives of tropolone found in the heartwood of western red cedar (Thuja plicata D. Don). Several of the tropolones are effective inhibitors of the enzyme catechol-O-methyltransferase (COMT). In this work, the isolated, spirally-cut rabbit thoracic aorta preparation has been used to study possible COMT inhibition as well as other pharmacological properties of the sodium salts of GT and BHT. It was found that GT, BHT and pyrogallol, a known COMT inhibitor, all potentiated the response of the aortic strip to norepinephrine (NE). However, since it was shown that the chelating agent EDTA also potentiated the NE response, the potentiation by GT and BHT could not be attributed to COMT inhibition without further evidence. GT and BHT were found to have a stimulatory effect of their own on aortic smooth muscle tissue. BHT was a more potent agonist than GT. Phenoxybenzamine blocked the stimulatory effects of BHT and GT. This suggested alpha-adrenergic receptor involvement although the halogenoalkylamine blocking agents are not absolutely specific in their action. Cocaine produces a small potentiation of the contraction produced by GT and BHT. This potentiation suggests the involvement of endogenous NE. Cocaine does not cause relaxation of the GT and BHT responses which indicates that GT and BHT differ in mode and perhaps site of action from tyramine. BHT potentiates the tyramine response on the rabbit aortic strip. This may be due to COMT inhibition, increased NE release or merely additive effects. GT was found to produce relaxation of a histamine-induced contraction. This relaxant effect was not prevented by beta-adrenergic blockade and is likely due to a nonspecific depressant effect by GT. Both GT and BHT have produced non-specific blocking effects against NE and histamine as well as acetylcholine throughout this work. In reserpinized preparations, GT no longer produced a contraction of the aortic strip. Instead, a relaxation below normal tone was produced. It would seem that the presence of endogenous NE is required in the tissue stores before GT can cause the strips to contract. GT causes relaxation of tyramine-induced contractions in reserpinized strips. The isolated rabbit thoracic aorta was found not to be a suitable preparation for the pharmacological investigation of COMT inhibition by GT and BHT due to their own agonistic effects on this tissue as well as their nonspecific chelating properties. However, this tissue was useful in providing information on other pharmacological actions of these compounds. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Actin dynamics are precisely regulated by a large number of actin binding proteins which collectively alter the rates of actin filament assembly and disassembly. Spectrin, an actin cross-linking protein, forms lateral filamentous networks that are linked to the plasma membrane and are required for membrane stability and resistance to mechanical stress. Adducin binds to spectrin-actin complexes, recruiting additional spectrin molecules, thereby further stabilising the membrane. In addition, adducin can bundle and cap actin filaments, and its actions have been implicated in cytoskeletal rearrangement in a variety of cell types. In vascular smooth muscle there is evidence that rearrangement of the actin cytoskeleton is involved in contraction and transmission of force to the extracellular matrix which leads to tissue remodelling. In addition, cytoskeletal dynamics are involved in vascular smooth muscle cell migration, proliferation and membrane dynamics. Protein kinase C (PKC), Rho-kinase, calmodulin and myosin light chain phosphatase are signalling proteins that are involved in these processes in vascular smooth muscle, and adducin is regulated by these signalling proteins in platelets and epithelial cells. The current study provides evidence for regulation of the actin cytoskeleton by α-adducin in vascular smooth muscle. Both α-adducin and spectrin are associated with the cytoskeleton in vascular smooth muscle cells of rat mesenteric small arteries. In response to activation by noradrenaline (NA), α-adducin becomes rapidly phosphorylated on Ser 724, a site specific for PKC, and dissociates from the actin cytoskeleton and spectrin in a PKC-dependent manner. Longer exposure of vessels to NA results in dephosphorylation of α-adducin on Ser 724 and its Rho-kinase-dependent reassociation with the actin cytoskeleton. Concurrent with this reassociation is enhanced association between the two proteins and an increase in the proportion of spectrin associated with the actin cytoskeleton. In addition, a rise in filamentous actin is observed, which can be blocked by inhibition of PKC or Rho-kinase and also by delivery of the α-adducin antibody into vessels in order to inhibit the function of endogenous a-adducin. These data provide evidence for a model in which α-adducin functions as an actin capping protein in resting vascular smooth muscle cells. Upon vasoconstrictor activation α-adducin becomes phosphorylated by PKC, inducing its dissociation from the actin cytoskeleton allowing elongation of actin filaments and further rearrangement of the actin cytoskeleton. Following this reorganisation, α-adducin re-associates with the actin cytoskeleton, possibly in response to phosphorylation by Rho-kinase, and recruits additional spectrin molecules, thus strengthening the newly formed actin filament network. These data provide further insight into the regulation of the actin cytoskeleton in vascular smooth muscle.

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<p>Smooth muscle cells from pig aortic media were grown in tissue culture, in medium containing 10% calf serum. Lipid biosynthesis from radioactive substrates 1-¹⁴C-acetate, U-¹⁴C-D-glucose, 1-¹⁴C-oleic acid and ³²P-phosphoric acid was measured. In addition, the influence of various sera, including pig serum, normolipemic human serum (NLHS), and hyperlipemic human serum (HLHS) on lipid biosynthesis from acetate and phosphoric acid was studied</p> <p>Compared to calf serum, all three test sera caused a stimulation of lipid synthesis in the lipid classes, phospholipid (PL), free fatty acids (FFA), triglycerides (TG) and cholesterol esters (CE), and an inhibition of cholesterol plus diglyceride (S + DG) synthesis. The extent of stimulation was least for pig serum and greatest for HLHS; the inhibition of S + DG was greatest for HLHS and least for pig serum. It was noted that the HLHS stimulation of CE synthesis was proportionately greater than the stimulation of the other lipid classes and that the HLHS inhibition of S + DG was significantly greater than that seen with the other test sera.</p> <p>The morphology of cultured aortic smooth muscle cells grown in 10% calf serum and 10% HLHS was examined by means of scanning and transmission electronmicroscopy. It was observed that HLHS caused degenerative alterations in the morphology of the cultured smooth muscle cells, such as an abundance of lipid droplets and cellular debris. The implications of these results in relation to the development of atherosclerosis are discussed.</p> / Thesis / Master of Science (MS)

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus. There are four Brucella strains of zoonotic importance in our domestic species, subdivided by their culture phenotypes: Brucella abortus (B. abortus, B. melitensis, B. suis (smooth strains) and B. canis (rough strain). Dogs can serve as hosts for all four of the zoonotic strains; however, routine serologic testing in dogs has been limited to the identification of antibodies to B. canis. The aim of our study was to identify a serologic test that can be utilized to identify smooth Brucella strain antibodies in canines. We hypothesize that the Brucella abortus Fluorescence Polarization Assay would be successful in identifying antibodies to smooth Brucella strain in canines. Ninety-five dogs, including forty-five hog hunting dogs were screened for circulating antibodies to any of the four zoonotic strains of the bacteria utilizing a combination of Canine Brucella Slide Agglutination Test (CBSA), Brucella canis Agar Gel Immunodiffusion II test (AGID), Brucella abortus Card Agglutination Test (BCA), and the Brucella abortus Fluorescence Polarization Assay (FPA). Test interpretation results yielded a 0% (0/95) smooth Brucella strain seropositivity rate, with 2% (2/95) of dogs yielding inconclusive rough Brucella strain serology results (0-2% rough strain seropositivity rate). Additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of Brucella by testing previously banked canine serum samples stored at Cornell's Veterinary Diagnostic Laboratory from 2018-2019 via Brucella abortus FPA. Of the 769 serum samples tested, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, 30/769 (4%) yielded a positive FPA test result, and 1/769 (0.1%) had to be excluded due to insufficient sample remaining to perform the diagnostic test. Of the 30 FPA positive canine serum samples, 97% (29/30) also tested positive on the CBSA test. Additionally, there was a statistically significant (p <0.0001) likelihood of altered (spayed/neutered) and mixed breed dogs to be FPA positive when compared to intact, purebred dogs. / Master of Science / Brucellosis is a bacterial disease that can cause severe reproductive, orthopedic and general illness in both dogs and humans. There are four different species of Brucella that can be transmitted between animals and people: Brucella canis, abortus, suis, and melitensis. Although Brucella canis is the species that is widely recognized and breeding dogs are routinely tested for this strain, we have vastly under recognized the ability for dogs to contract and transmit the other three (smooth) Brucella species. Of added concern is the fact that the test currently used to screen dogs for brucellosis only identifies Brucella canis infection. Thus, veterinarians may be missing cases where dogs are infected with other Brucella species. This study revealed promising evidence in identification of smooth Brucella strain antibodies in canines, particularly altered and mixed breed canines, via the Brucella abortus Fluorescent Polarization Assay. The contributions of this study are threefold. First, to heighten awareness that both smooth and rough strains of brucellosis infection in dogs are infectious diseases of zoonotic concern. Second, it demonstrates that smooth Brucella strain infection along with the traditional strategy of selectively screening dogs breeding dogs may be underestimating the prevalence of brucellosis among the canine population in the United States thus, supporting the need for broadened screening recommendations. Third, it reveals the need for a commercially-available, validated test for the smooth strains of brucellosis in dogs and offers direction for future research efforts to likely focus on the validation of the Brucella abortus Fluorescent Polarization Assay.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. [alpha] in title is the Greek character. Includes bibliographical references. Also available online through Digital Dissertations.

Theses (M.S.)--Marshall University, 2007. / Title from document title page. Includes abstract. Includes vitae. Document formatted into pages: contains vi, 74 pages including maps. Bibliography: p.68-69.