diplomsko delo
Nika Boštic (Avtor), Nina Vardjan (Mentor)

Povzetek

Astrociti so heterogena skupina celic glije z mnogoterimi funkcijami, ki ohranjajo homeostazo osrednjega živčnega sistema (OŽS). Med drugim astrociti predstavljajo presnovno oporo nevronom in uravnavajo sinaptični prenos signala. Njihovo delovanje ni vezano le na fiziološke procese, temveč so vključeni tudi v odziv na patološke spremembe v OŽS. Za razliko od nevronov niso električno vzdražni, izkazujejo pa znotrajcelično vzdražnost, katere osrednji del sta sekundarna prenašalca ciklični adenozin-3',5'-monofosfat (cAMP) in Ca2+, ki uravnavata širok nabor celičnih procesov, kot so presnova, morfologija in medcelična komunikacija. Citosolni ravni Ca2+ in cAMP sta dinamično uravnavani z zunajceličnimi dražljaji preko z G-proteini sklopljenih receptorjev (GPCR), ki so prisotni na površini celic in ob aktivaciji povzročijo razpad trimernih G-proteinov na podenote. Slednje sprožijo sprostitev Ca2+ iz endoplazemskega retikuluma ter stimulirajo ali inhibirajo tvorbo cAMP preko interakcije z adenilat ciklazo. Latrofiline uvrščamo med adhezijske GPCR. V sesalskih celicah so poznani latrofilini tipa 1, 2 in 3, ki različno modulirajo sekundarne prenašalce – povišajo citosolno raven Ca2+ in znižajo ali povišajo citosolno raven cAMP. Latrofilini so prisotni tudi v OŽS in naj bi sodelovali pri nastanku ekscitatornih sinaps, aksonskemu privlaku in nevronski migraciji. Njihovo delovanje v kontekstu astrocitov pa je še slabo raziskano, zato je bil namen diplomskega dela ugotoviti, katere signalne poti se sprožijo v astrocitih po draženju z endogenim ligandom latrofilinov. V izoliranih podganjih astrocitih smo s fluorescenčno mikroskopijo v realnem času merili spremembe v znotrajcelični ravni cAMP in Ca2+, kot dražljaj pa smo uporabili ektodomeno mišjega tenevrina 2 (mTen2). Pred začetkom beleženja koncentracije Ca2+ smo astrocite obarvali s Ca2+-indikatorjem barvilom Fluo-4 AM, medtem ko smo za spremljanje ravni cAMP celice transficirali z nanosenzorjem Epac1-camps, ki temelji na Försterjevem resonančnem prenosu energije (FRET). Naši rezultati nakazujejo, da draženje astrocitov z mTen2 povzroči statistično značilen padec v znotrajcelični ravni Ca2+, medtem ko na znotrajcelično raven cAMP ne vpliva. Poleg tega smo opazili, da predhodno draženje celic z mTen2 zmanjša porast v znotrajcelični koncentraciji Ca2+ po draženju z noradrenalinom, agonistom adrenergičnih GPCR, glede na kontrolne celice, ki jih predhodno nismo dražili. Naši rezultati nakazujejo, da prisotnost mTen2 zmanjša z noradrenalinom posredovano Ca2+-vzdražnost astrocitov.

Ključne besede

astrociti;G-proteini;adhezijski GPCR;latrofilini;tenevrin 2;cAMP;kalcijevi ioni;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [N. Boštic]
UDK: 577.2(043.2)
COBISS: 67463683 Povezava se bo odprla v novem oknu
Št. ogledov: 350
Št. prenosov: 44
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Measurements of intracellular concentration of calcium and cAMP in astrocytes after addition of adhesion GPCR ligands
Sekundarni povzetek: Astrocytes are a heterogeneous group of glial cells with many functions that maintain homeostasis of central nervous system (CNS). Among other things, astrocytes provide metabolic support to neurons and regulate synaptic signal transmission. In addition to roles in physiological processes, they are also involved in the response to pathological alterations in the CNS. Unlike neurons, astrocytes are electrically silent, however, they display intracellular excitability, the central part of which are the second messengers cyclic adenosine-3',5'-monophosphate (cAMP) and Ca2+. These two regulate a wide range of cellular processes related to astrocyte metabolism, morphology and intercellular communication. Cytosolic levels of Ca2+ and cAMP are dynamically regulated by extracellular stimuli via G-protein coupled receptors (GPCRs) that are present on the cell surface and, upon activation, cause the dissociation of trimeric G-proteins into subunits. The latter trigger the release of Ca2+ from the endoplasmic reticulum and stimulate or inhibit production of cAMP through interaction with adenylate cyclase. Latrophilins are classified as adhesive GPCRs. In mammalian cells one can find type 1, 2 and 3 latrophilins, which modulate second messengers in various ways – they increase cytosolic Ca2+ concentration and decrease or increase cytosolic cAMP levels. Latrophilins are also present in the CNS and may be involved in excitatory synapse formation, axonal attraction, and neuronal migration. Their activity in the context of astrocyte function is still poorly investigated, so the aim of the thesis was to determine which signaling pathways are triggered in astrocytes after stimulation with endogenous latrophilin ligand. In isolated rat astrocytes, changes in the intracellular levels of cAMP and Ca2+ were measured by real-time fluorescence microscopy, and the ectodomain of mouse teneurin 2 (mTen2) was used as a stimulus. Prior to intracellular Ca2+ recordings, astrocytes were stained with the Ca2+-indicator dye Fluo-4 AM. To monitor intracellular cAMP levels cells were transfected with Förster Resonance Energy Transfer (FRET)-based cAMP nanosensor Epac1-camps. Our results suggest that stimulation of astrocytes with mTen2 results in a statistically significant decrease in intracellular Ca2+ levels, whereas intracellular cAMP levels are unaffected. In addition, pre-stimulation of cells with mTen2 reduced the increase in intracellular Ca2+ concentration triggered by adrenergic GPCR agonist noradrenaline compared to control non pre-stimulated cells. Our results suggest that mTen2 reduces noradrenaline-mediated Ca2+-excitability of astrocytes.
Sekundarne ključne besede: astrocytes;latrophilins;teneurin 2;cAMP;calcium;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 1000371
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Strani: 52 str.
ID: 12977445