Nitric oxide synthase expression and activity in cell models relevant to human pathophysiology

master's thesis

• Author: Kristina Radić
• Mentor: Jerka Dumić (mentor)
• Committee member: Jerka Dumić
• Committee member: Sandra Šupraha Goreta
• Committee member: Jasna Jablan
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2015-09-15, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Složene interakcije mitohondrijske i nuklearne DNA onemogućuju otkrivanje pravog uzroka nasljednih mitohondrijskih bolesti kao i pružanje učinkovite terapije. Leberova nasljedna optička neuropatija (LHON), jedna od najučestalijih nasljednih optičkih neuropatija, je posljedica točkastih mutacija mitohondrijske DNA koje dovode do smanjenja aktivnosti kompleksa I u respiratornom lancu. Tipična obilježja LHON-a, sa biokemijskog stajališta, su smanjenje proizvodnje energije na staničnoj razini i povećana proizvodnja reaktivnih kisikovih (ROS) i dušikovih (RONS) spojeva no, to ne objašnjava odgođenu pojavu simptoma bolesti i učinak samo na određene stanice. Naime, iako je mutacija prisutna u cijelom tijelu, štetan učinak iskazuje samo na ganglijskim stanicama mrežnice. Obzirom da je LHON karakteriziran deficitom oksidativne fosforilacije, a NO· je poznati negativni modulator mitohondrijskog respiratornog lanca istražili smo njegovu ulogu u razvoju LHON-a. Rezultati u ovom radu pokazuju da dugotrajna izloženost dušikovom oksidu ima veći utjecaj na smrtnost stanica LHON-a uspoređujući je sa smrtnosti kontrolnih stanica i pretpostavljaju da se ROS i RONS stvaraju u većim količinama u ovom patološkom stanju i vode ka staničnoj smrti. Vođeni tom mišlju, provjerili smo i količinu iNOS-a i eNOS-a na razini mRNA. Obzirom da rezultati nisu statistički značajni, pretpostavili smo da postoji razlika u ekspresiji jednog ili više NOS enzima kod pacijenata oboljelih od LHON-a ali ovo pitanje ostaje otvoreno za daljnja istraživanja. Buduća perspektiva je potpuno objašnjenje uloge NO· u specifičnim mitohondrijskim bolestima i rješavanje zagonetne ekspresije i aktivnosti samog NOS-a. Možda upravo metabolizam NO· može biti važan u patofiziologiji LHON-a te njegovo razumijevanje može pridonijeti razvoju efikasnijih terapija i preventivnih mjera.

Abstract (english)

Due to complex interactions of mitochondrial and nuclear DNA, the real cause of inherited mitochondrial diseases is still unclear and patients currently lack effective treatments. Leber’s hereditary optic neuropathy (LHON), one of the most common inherited optic neuropathies, is caused by point mutations in mtDNA that result in a defect of respiratory chain complex I activity. From the biochemical point of view the bioenergetic failure and the enhanced reactive oxygen species (ROS) production are typical hallmarks of LHON disease, but still they do not explain the late onset and the incomplete penetrance. Moreover it is still unknown why, although the mutation is present overall the body only the RGCs are affected. Since LHON is characterized by a deficit in the oxidative phosphorylation chain and NO· is an established mitochondrial respiratory chain negative modulator, we checked if it might have a role in the LHON development and progression. The results presented in this thesis prove that long–term exposure to NO· has a larger impact on LHON cells viability compared to controls and suggest that higher levels of reactive oxygen and nitrogen species (ONOO-) are produced in this pathological state, leading to cell death. In addition, careful though still statistically insufficient evaluations of the levels of iNOS and eNOS mRNA expression seems to suggest that there might be a difference in the expression of one or more NOSs in LHON patients: this question however remains open. Future perspectives are, indeed, to provide a complete picture about involvement of NO· in this specific mitochondrial disease and to solve the conundrum of the NOSs expression and activity. The feeling is that the NO· metabolism can be relevant to LHON pathophysiology, its comprehension opening to the development of more efficient therapeutic and preventive interventions.

• Keywords: dušikov oksid; sintaza dušikovog oksida; oksidativni stres; Leberova nasljedna optička neuropatija
• Keywords (english): nitric oxide; nitric oxide synthase; oxidative stress; Leber’s optic hereditary neuropathy
• Language: english
• URN:NBN: urn:nbn:hr:163:846232
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-01-22 16:23:43