No public access
master's thesis
Development of Dual Reporter Luciferase system for in vivo detection of miRNA target gene downregulation

Kristina Žuža (2016)
University of Rijeka
Department of Biotechnology
Metadata
TitleDevelopment of Dual Reporter Luciferase system for in vivo detection of miRNA target gene downregulation
AuthorKristina Žuža
Mentor(s)Serena Zacchigna (thesis advisor)
Antonija Jurak Begonja (thesis advisor)
Abstract
Most frequent global death causes are cardiovascular disorders such as cerebrovascular diseases, high blood pressure, congenital heart disease and coronary artery disease. Cardiovascular diseases often result in development of heart failure syndrome which in most cases leads to death within 5 years after diagnosis. Similar to heart failure, acute myocardial infarction is a silent murderer of today’s developed society due to sedentary life style and unhealthy habits such as smoking and unhealthy diet. Both disorders are characterized by heart cell remodeling and depletion in number of viable cardiomyocytes what is considered to be main obstacle in finding efficient therapy. Heart is an organ unable to respond to damage by self-renewal and therapy provided up to date is insufficient to increase number of viable CMs. Although many pharmaceuticals and surgical interventions are developed, all of them temporarily mend the heart. For this reason development of novel therapeutics is required among which miRNAs show to be very promising in boosting heart proliferation rate. MiRNA-199a-3p and 590-3p showed to significantly increase cardiomyocyte proliferation in vitro and in vivo and to preserve cardiac function after injury cause by mimicking myocardial infarction. Homer1 and CLIC5 genes are showed to be most down regulated by miRNA-199a-3p and 590-3p. To measure level of this downregulation more precisely and to show that this targeting is limited specifically to cardiomyocytes, not to other heart cells (cardiac fibroblasts, endothelial cells…) we decided to develop dual luciferase reporter model of monitoring miRNA targeting in vivo. System would take advantage of adeno-associated virus (AAV) vector tissue tropism, specifically transducing cardiomyocytes. Vector would carry transgene consisted of a 3’UTRs of Homer1 or CLIC5 gene together with luciferase (Renilla or firefly) sequence. Gene downregulation would be assesed by luciferase bioluminescence decrease after miRNA mimic intracardiac injection.
Parallel title (Croatian)Razvoj dvostrukog luciferaznog reporter eseja za in vivo detekciju miRNA genskog utišavanja
Committee MembersAntonija Jurak Begonja (committee chairperson)
Elitza Petkova Markova - Car (committee member)
Sandra Kraljević Pavelić (committee member)
GranterUniversity of Rijeka
Lower level organizational unitsDepartment of Biotechnology
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeBiotechnology in medicine
Academic title abbreviationmag. biotech. in med.
Genremaster's thesis
Language English
Defense date2016-09-26
Parallel abstract (Croatian)
Kardiovaskularne bolesti kao što su visoki krvni tlak, cerebrovaskularna te koronarna oboljenja predstavljaju jedan od najčešćih uzroka smrti na globalnoj razini. Takva oboljenja često rezultiraju sindromom zatajenja srca koji najčešće dovodi do smrti pet godina nakon uspostavljanja dijagnoze. Slično kao i zatajenje srca, infarkt mokarda je tihi ubojica današnjeg razvijenog društva te je posljedica sjedilačkog načina života i nezdravih navika. Oba poremećaja su karaterizirana remodeliranjem srčanih stanica te smanjenim brojem funkcionalnih kardiomiocita što se smatra glavnom preprekom u pronalasku učinkovite terapije. Srce je organ kojeg karakterizira nemogućnost samo-obnove uslijed ozljede te su terapije razvijene do danas neučinkovite u povećanju broja funkcionalnih kardiomiocita. Razvijeni su mnogi korisni lijekovi i kirurški zahvati, ali oni često samo privremeno zaliječe srce. Sukladno tomu, nužan je razvoj novih terapija u liječenju srčanih oboljenja među kojima su se mikro RNA molekule pokazale kao vrlo obećavajuće u poticanju proliferacije kardiomiocita. Jedan od primjera su miRNA-199a-3p i miRNA-590-3p koje su pokazale da značajno povisuju razinu proliferacije kardiomiocita kako in vitro tako i in vivo. Također, pokazale su bitnu ulogu u očuvanju srčane funkcije nakon oštećenja izazvanog srčanim udarom. Dva gena koji su bili utišani u najvećoj mjeri od strane miRNA-199a-3p i miRNA-590-3p su Homer1 i CLIC5. Da bi u buduće što preciznije mjerili to utišavanje te kako bi dokazali da je ono ograničeno samo na kardiomiocite, a ne ostale srčane stanice (fibroblaste i endotelne stanice), odlučili smo razviti dvostruki luciferazni esej za in vivo detekciju miRNA genskog utišavanja. Sistem bi koristio prednost tkivne specifičnosti adeno-združenog viralnog vektora transducirajući isključivo kardiomiocite u srčanom tkivu. Vektor bi nosio transgen koji se sastoji od 3'UTR regije Homer1 ili CLIC5 gena zajedno sa sekvencom gena za luciferazu (Renilla ili firefly). Utišavanje gena bi se mjerilo smanjenjem luciferazne bioluminiscencije nakon intrakardijalne injekcije miRNA-199a-3p i miRNA-590-3p.
Parallel keywords (Croatian)Cardiovascular diseases Heart renewal Cardiomyocytes MicroRNA Homer1 CLIC5 Dual luciferase reporter system Kardiovaskularna oboljenja Obnova srca Kardiomiociti MicroRNA Homer1 CLIC5 Dvostruki luciferazni esej
Resource typetext
Access conditionNo public access
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:078017
CommitterIvana Dorotić