Aktivnost palmitoil-protein tioesteraze 1 u homogenatu leukocita mjerena fluorimetrijskom metodom

master's thesis

• Title (english): The activity of palmitoyl-protein thioesterase 1 in leukocyte homogenate measured by fluorimetric method
• Author: Andrea Hulita
• Mentor: Ksenija Fumić (mentor)
• Committee member: Ksenija Fumić (predsjednik povjerenstva)
• Committee member: Lovorka Vujić (član povjerenstva)
• Committee member: Anita Somborac Bačura (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2020-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Neuronalne ceroidne lipofuscinoze su skupina genetski nasljednih lizosomskih neurodegenerativnih bolesti karakteriziranih unutarstaničnim nakupljanjem skladišnog materijala autofluorescentnog lipopigmenta koji uzrokuje progresivnu neurološku degeneraciju u svim dobnim skupinama. Pripadaju skupini lizosomskih poremećaja skladištenja. Klinički tijek uključuje progresivnu demenciju, napadaje, progresivno zatajenje vida (osim u obliku koji se javlja u odrasloj dobi) te često poremećaje kretanja. Sveukupno gledajući, ovi poremećaji su najčešći uzročnici nasljednih dječjih neurodegenerativnih bolesti i sve se češće prepoznaju u oblicima koji se javljaju kasnije u životu i u odrasloj dobi. Klasična infantilna bolest CLN1 uzrokovana je mutacijama u genu PPT1. To je genetski poremećaj s pojavom simptoma u dobi između 6 i 24 mjeseca, no zabilježene su i kasnije prezentacije CLN1 bolesti, a to su maloljetnička (juvenilna) i rana odrasla (adultna). Simptomi mogu uključivati znakove intelektualnog i motoričkog zaostajanja, uključujući zaostajanje u razvoju, trzanje mišića (mioklonski trzaji) i napadaje.
Rana dijagnoza CLN1 (neuronalna ceroidna lipofuscinoza tipa 1) bolesti je presudna kako bi se osigurala optimalna skrb za bolesnike i njihove obitelji. CLN1 bolest je moguće dijagnosticirati mjerenjem aktivnosti enzima PPT1. Ovaj enzim je neophodan za pravilno funkcioniranje lizosoma. Funkcija ove lizosomske tioesteraze je da ukloni masne kiseline vezane tioesterskim vezama na cisteinske ostatke u proteinima.
U ovom radu je cilj uvođenje metode za mjerenje aktivnosti enzima palmitoil proteina tioesteraze 1 u uzorcima leukocita zdrave populacije izoliranih iz pune krvi. Koristila se in house fluorimetrijska metoda određivanja aktivnosti enzima palmitoil protein tioesteraze 1 u leukocitima izoliranima iz EDTA pune krvi. Metoda se pokazala dovoljno pouzdanom za primjenu u selektivnoj laboratorijskoj dijagnostici nakon postavljene kliničke sumnje na CLN1. Međutim, kako sama fluorimetrijska metoda ima mnogo ograničenja i nedostataka, kao prva metoda probira preporučuje se metoda mjerenja aktivnosti PPT 1 iz uzorka suhe kapi krvi na filter papiru.

Abstract (english)

The neuronal ceroid lipofuscinoses (NCLs) are a group of genetically inherited lysosomal neurodegenerative diseases characterized by the intracellular accumulation of autofluorescent lipopigment storage material that causes progressive neurologic degeneration in all age groups. They belong to the group of lysosomal storage disorders. The clinical course includes progressive dementia, seizures, progressive visual failure (except in the adult-onset forms), and often movement abnormalities. Collectively, these disorders are the most common cause of an inherited childhood neurodegenerative disease and are increasingly recognized in late-onset or adult-onset forms. Classic infantile CLN1 disease is caused by an alteration in the PPT1 gene. It is a genetic disorder with an onset of symptoms between 6 and 24 months of age, with recorded later presented CNL1 diseases, juvenile and early adult. Symptoms may include signs of intellectual and motor decline including delays in reaching developmental milestones (development delays), twitching or jerking of muscles (myoclonic jerks), and seizures.
Early diagnosis of CLN1 (neuronal ceroid lipofuscinoses type 1) disease is crucial to ensure optimum care for patients and their families. CLN1 disease is diagnosed by measuring the activity of the enzyme PPT1. This enzyme is necessary for the proper functioning of lysosomes. The function of this lysosomal thioesterase is to remove the fatty acids bound by thioester bonds to cysteine residues in proteins.
In this study, the aim is to introduce a method for measuring the activity of an enzyme palmitoyl protein thioesterase 1 in a sample of leukocytes isolated from whole blood. An in house fluorimetric method was used to determine the activity of an enzyme palmitoyl protein thioesterase 1 in leukocytes isolated from EDTA whole blood. This method has proven to be sufficiently reliable for use in selective laboratory diagnostics after clinical suspicion of CLN1 has been established. However, as the fluorimetric method itself has many limitations and drawbacks, it is recommended that the first method of screening is the method of measuring PPT 1 activity from a dry drop of blood sample on filter paper.

• Keywords: neuronalna ceroidna lipofuscinoza; neuronalna ceroidna lipofuscinoza tipa 1; CLN1 bolest; PPT1; Battenova bolest; lizosomi
• Keywords (english): neuronal ceroid lipofuscinose; neuronal ceroid lipofuscinose type 1; CLN1 disease; PPT1; Batten disease; lysosomes
• Language: croatian
• URN:NBN: urn:nbn:hr:163:592151
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije i laboratorijske medicine (magistar/magistra medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-04-27 09:22:03