Istraživanje biokemijskih osobina mutiranih inačica dipeptidil - peptidaze III, R510W i V514M te njihovog utjecaja na regulaciju signalnog puta Nrf2/Keap1 ; Enzimi - primjer dobre nastavne prakse

master's thesis

• Title (english): Investigation of the biochemical properties of R510W and V514M mutant variants of dipeptidyl peptidase III and their impact on the regulation of Nrf2/Keap1 signalling pathway ; Enzymes - an example of good teaching practice
• Author: Domagoj Christian Kindl
• Mentor: Mihaela Matovina (mentor)
• Mentor: Draginja Mrvoš-Sermek (mentor)
• Committee member: Draginja Mrvoš-Sermek (predsjednik povjerenstva)
• Committee member: Jasmina Rokov Plavec (član povjerenstva)
• Committee member: Ines Radanović (član povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2023-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Diplomski rad sastoji se od eksperimentalnog i metodičkog dijela. U eksperimentalnom dijelu
ispitane su biokemijske osobine mutiranih inačica R510W i V514M humane dipeptidil–
peptidaze III (hDPP III) čije su genomske sekvence nađene u uzorcima zloćudnih tumora, te
je ispitan njihov utjecaj na regulaciju signalnog puta Nrf2/Keap1. Ciljanom mutagenezom
napravljeni su vektori za ekspresiju mutiranih inačica proteina te su one eksprimirane u
bakteriji Escherichia coli i pročišćene. Utvrđeno je da je aktivnost svih triju proteina viša u
prisutnosti kobaltovih iona, a analizom kinetike reakcije je utvrđeno da R510W ima oko 1000
puta, a V514M oko 2 puta nižu katalitičku aktivnost od divljeg tipa. Određivanjem razine
ekspresije gena utvrđeno je da prekomjerna ekspresija divljeg tipa i mutiranih inačica hDPP
III nemaju utjecaj na ekspresiju gena kontroliranih s Nrf2 u HEK293T stanicama.
U sklopu metodičkog dijela rada osmišljeni su pokusi na temu enzima, temeljem kojih su
oblikovani metodičko-didaktički materijali za jedan 90-minutni nastavni sat. Pri tome su
postavljeni odgojno-obrazovni ciljevi temeljeni na prethodnim učeničkim znanjima,
vještinama i sposobnostima.

Abstract (english)

The thesis consists of an experimental and methodological part. In the experimental part, the
biochemical properties of the mutant variants of human dipeptidyl peptidase III (hDPP III),
whose genomic sequences were found in malignant tumors, R510W i V514M, and their
influence on the regulation of the Nrf2/Keap1 signaling pathway were examined. The
expression vectors for the mutants were constructed using site-directed mutagenesis and
expressed in E. coli and purified. The activity of all three proteins was higher in the presence
of cobalt ions, and the kinetic parameters analysis revealed that R510W has about 1000 times
and V514M about 1 times lower catalytic activity than the wild type. Gene expression
analysis detected that overexpression of the wild-type and mutants of hDPP III had no effect
on the expression of genes controlled by Nrf2 in HEK293T cells.
In the methodological part of the thesis, experiments on the topic of enzymes as well as
methodical-didactic materials related to 90-minute lesson were designed. At the same time,
educational goals were set based on the students' previous knowledge, skills and
competencies.

• Keywords: enzim; humana dipeptidil-peptidaza III; ekspresija gena; Keap1; kinetika; mutageneza; mutirane inačice
• Keywords (english): enzyme; human dipeptidyl peptidase III; gene expression; Keap1; kinetics; mutagenesis; mutant variants
• Language: croatian
• URN:NBN: urn:nbn:hr:217:251991
• Project: Number: IP-2018-01-2936; Title: Biološka važnost dipeptidil peptidaze III i njezin utjecaj na zdravlje čovjeka; Acronym: DPP3BioRe; Leader: Sanja Tomić; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Study programme: Title: Integrated undergraduate and graduate university study of Biology and Chemistry Education; specializations in: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Course: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra edukacije biologije i kemije (magistar/magistra edukacije biologije i kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-10-27 12:31:07