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Development of Electroosmotic Sampling for the Investigation of Galanin-Degrading Ectopeptidase Activity in the Hippocampus

Rupert, Amy Elisabeth (2013) Development of Electroosmotic Sampling for the Investigation of Galanin-Degrading Ectopeptidase Activity in the Hippocampus. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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There are currently no methods that have been successfully used to sample the extracellular component of brain slice preparations with the precision developed for in vivo sampling methods. We developed a slice-compatible method that removes fluid by electroosmosis. Two designs were investigated: one utilizing a single capillary where flow originates below the tissue and fluid pulled upwards and one using two capillaries where flow is between the two capillary lumens. The two-capillary approach is analogous to push-pull perfusion wherein a source ‘push’ capillary with a tapered tip is inserted into the tissue and a collection ‘pull’ capillary is positioned at the surface in close proximity to the source capillary. Voltage is applied across proximal capillary ends, which invoked fluid flow from source capillary into the tissue then to the collection capillary. Damage studies addressed minimization of perturbation of tissue by electric fields in both single and push-pull models. Flow rates were quantified for the two-capillary model using HPLC analysis of collected fluid. Numerical simulations aided understanding of electric field distribution and fluid flow within the tissue.
We then investigated the hydrolysis of exogenous galanin in the extracellular space after ischemic pre-conditioning, a method in which mild, short ischemia creates resistance in the brain against a longer duration of ischemia 24-48 hours later. The efficacy of many neuropeptides, including galanin, is controlled by hydrolysis of active peptide into smaller active and inactive fragments by ectopeptidases. We used push-pull electroosmotic sampling and MALDI mass spectrometry to identify hydrolysis products created after passing exogenous galanin through tissue. We then quantified the hydrolysis of galanin after ischemic pre-conditioning. We showed that pre-conditioned cultures have decreased galanin hydrolysis in the CA1 and DG areas, but not the CA3. Finally, we treated cultures with inhibitors for metallo- and aminopeptidases and quantified the resulting relative changes in galanin hydrolysis. Results indicate that metallopeptidases, particularly those that use zinc, are likely responsible for galanin degradation in the CA1 region of the hippocampus. Neuron specific aminopeptidase may also be hydrolyzing galanin. The distribution of hydrolysis products determined with MALDI-MS also indicate aminopeptidase activity.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Rupert, Amy Elisabethaeh37@pitt.eduAEH37
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairWeber, Stephensweber@pitt.eduSWEBER
Committee MemberRobinson, Renarena@pitt.eduRENA
Committee MemberMichael, Adrianamichael@pitt.eduAMICHAEL
Committee MemberSandberg,
Date: 30 January 2013
Date Type: Publication
Defense Date: 5 October 2012
Approval Date: 30 January 2013
Submission Date: 31 October 2012
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 131
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: method development, hippocampus, sampling, electroosmosis, galanin, ectopeptidase, ischemia
Date Deposited: 30 Jan 2013 14:51
Last Modified: 15 Nov 2016 14:06


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