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"Development of Analytical Tools for the Quantific

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(Total Views: 848)
Posted On: 10/05/2018 11:12:39 AM
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Posted By: Murphalot
"Development of Analytical Tools for the Quantification of MANF and CDNF in Disease and Therapy"

Author: Galli, Emilia
Contributor: University of Helsinki, Faculty of Pharmacy, Division of Pharmaceutical Biosciences
Doctoral Programme in Drug Research
University of Helsinki, Institute of Biotechnology
Thesis level: Doctoral dissertation (article-based)
Belongs to series: Dissertationes Scholae Doctoralis ad Sanitatem Investigandam Universitatis Helsinkiensis - URN:ISSN:2342-317X

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are expressed ubiquitously in the body and show protective effects on neurons and other cell types. Although the effects of MANF and CDNF administration have been studied in various animal disease models, such as neurodegenerative diseases, ischemia, and diabetes, it is largely unknown whether their endogenous levels and expression patterns are changed in relation to the pathology of these diseases.

This thesis describes the development and validation of enzyme-linked immunosorbent assays (ELISAs) for the specific and sensitive quantification of MANF and CDNF in biological samples including serum, tissues, and in vitro cell samples. Since MANF removal leads into a clear diabetic phenotype in mice, we studied the circulating MANF levels in relation to human type 1 diabetes. The aim was to find out whether MANF expression and/or secretion is differently regulated before and at the onset of clinical symptoms of the disease, thus giving insights into its association to type 1 diabetes development in humans.

Multiple studies have demonstrated neuroprotective and neurorestorative effects of MANF and CDNF. Since they are unable to penetrate the blood brain barrier, they need to be delivered within the central nervous system. In this thesis, the effects of intracranial gene therapy of human CDNF were studied in animal models of Parkinson’s and Alzheimer’s disease. Transgene expression depends on multiple variables, such as transduction efficiency, stability, and virus titer. Thus, the quantification of expressed protein is important in the interpretation of its effects. The species-selectivity of the developed ELISAs was utilised when analysing human CDNF levels in rodent brain.

In addition to gene therapy, long-term protein delivery can be implemented by encapsulated cells that secrete the therapeutic protein to the target site. The developed ELISAs were used for the quantification of secreted CDNF from cells. Despite the successful production of several cell clones with high CDNF expression, its secretion was found to decrease markedly once the cells were confluent in cell culture plates or in polymeric microcapsules. This thesis describes modifications that were done to the CDNF coding sequence to improve its secretion, aiming at efficient delivery of CDNF by encapsulated cell therapy.

The developed sensitive, specific, and thoroughly validated ELISAs for the quantification of MANF and CDNF can be utilised in future studies aimed at understanding the regulation and functions of these proteins in health and disease. In addition, the ELISAs hold the potential for analysis of these proteins as biomarkers.

Date: 2018-10-13

https://helda.helsinki.fi/handle/10138/243247


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