USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Beskattningsrätt och skattskyldighet för kyrkan i Finland

Inom rättsvetenskap saknas grundforskning om kyrkoskatt. Det kan ha många orsaker. En renodlad skatterättslig forskning utan exkurser till andra vetenskaper är minst sagt otänkbar. Forskning inom ett gränsområde mellan teologi och rättsvetenskap måste inkludera drag av sociologi, politologi och ekonomi. Etik från teologi och moral genom lag kan vara självklart. Förankrad i liberal rättsfilosofi kan också beskattningen förstås på ett annat sätt om en förankring i ett historiskt perspektiv tas med. Den evangelisk-lutherska kyrkan och den ortodoxa kyrkan har skatterätt i Finland. Hur har det kommit sig och vilken rätt har andra trossamfund? Detta försöker vi här belysa genom skilda infallsvinklar. Laglig reglering av offentligrättsliga samfunds skatteintag, med kyrkans uppgifter med tydligt mindre lagbundenhet som utgiftsfält, ger de sociala aspekterna och barmhärtighet stort inflytande. En tidvis sekulär stat och de nationella bindningarna med historisk förankring ger ett konglomerat av skilda lösningsmodeller. Genom olika förankringar i skilda kulturer och språk kan en nationell kutym uppstå som skiljer sig mycket från andra. Dessutom kan speciella juridiska egenheter upptäckas. Vilken nationell modell man i en demokrati väljer, styrs av de politiskt stadfästa lagarna. Oberoende av kyrkans nationella ställning, ökar en större liberalism och fördragsamhet i en demokrati behovet av anpassning och nationell acceptans av andra religioner, vilket kan leda till ett behov av nya finansieringsmodeller för trossamfund.

Macrophages in regressed and progressed uveal melanoma

Uveal melanoma (UM) is the most common primary ocular malignancy in adults. In Finland, approximately 50 new cases are diagnosed yearly. Up to 50% of UM metastasize, mostly to the liver, although other organs are also affected. Despite improvements in the management of the primary tumour, the survival rates of patients with metastatic UM are poor. Until the 1970s, UMs were treated by enucleation i.e. removal of the eye. Currently, UM is usually treated by brachytherapy, which is known to influence tumour cells and blood vessels. UMs enucleated both primarily and secondarily after brachytherapy contain tumour-infiltrating macrophages, and a high number of macrophages in primary UM is associated with a shorter survival and a higher microvascular density (MVD) within the tumour tissue. The latter is independently associated with a shorter time to metastatic death. Macrophages have several diverse roles depending on their response to variable signals from the surrounding microenvironment. They function as scavengers, as producers of angiogenic and growth factors as well as proteases, which modulate extracellular matrix. Thus, tumour invasiveness and the risk for metastasis increase with increasing macrophage density. The aim of this study was to evaluate the effects of regression and progression of UM on macrophage numbers and microcirculation factors. Tumour regression is induced by primary brachytherapy, and tumour progression is evidenced by the development of metastases. Understanding the biological behaviour of UMs in the both states may help us in finding new treatment modalities against this disease. To achieve these aims case-control analyses of irradiated UMs and primarily-enucleated eyes (34 matched pairs) were performed. UMs were stained immunohistochemically to detect macrophages, extravascular matrix (EVM) loops and networks, and MVD. Following brachytherapy, a lower MVD was observed. The average number of macrophages remained unchanged. Considering that irradiated melanomas may still contain proliferating tumour cells, a clinically-relevant consequence of my study would be the reassurance that the risk for metastasis is likely to be reduced, given that the low MVD in untreated UMs indicates a favourable prognosis. The effect of progression on macrophages was studied in a paired analysis of primarily-enucleated UM and their corresponding hepatic metastases (48 pairs). A cross-sectional histopathological analysis of these pairs was carried out by staining both specimens in a similar way to the first study. MVD was greater in hepatic metastases than in corresponding primary tumours, and the survival of the patient tended to be shorter if hepatic metastases had a higher MVD. Hepatic metastases had also more dendritic macrophages than the primary UMs. Thus, the progression to metastasis seems to alter the inflammatory status within the tumour. Furthermore, determining MVD of biopsied hepatic metastases may serve as a supplementary tool in estimating the prognosis of patients with metastatic uveal melanoma. After irradiation, the majority of treated eyes have been clinically observed to have pigmented episcleral deposits. A noncomparative clinical case series of 211 irradiated UM eyes were studied by recording the number and location of pigmented episcleral deposits during follow-up visits after brachytherapy. For the first time, the study described pigmented episcleral deposits, which are found in the most UM eyes after brachytherapy, and proved them to consist of macrophages full with engulfed melanin particles. This knowledge may save patients from unnecessary enucleation, because episcleral pigmented deposits might be mistaken for extrascleral tumour growth. The presence of pigmented macrophage-related episcleral deposits was associated with plaque size and isotope rather than with tumour size, suggesting that, in addition to tumour regression, radiation atrophy of retinal pigment epithelium and choroid contributes to the formation of the deposits. In the paired (the same 34 pairs as in the first study) cross-sectional study of irradiated and non-irradiated UMs, clinically-visible episcleral deposits and migrating macrophages in other extratumoral tissues were studied histopathologically. Resident macrophages were present in extratumoral tissues in eyes with both irradiated and non-irradiated UM. Irradiation increased both the number of CD68+ macrophages in the sclera beneath the tumour and the number of clinically-observed episcleral macrophages aggregates. Brachytherapy seemed to alter the route of migration of macrophages: after irradiation, macrophages migrated preferentially through the sclera while in non-irradiated UMs they seemed to migrate more along the choroid. In order to understand the influence of these routes on tumour progression and regression in the future, labelling and tracking of activated macrophages in vivo is required.

Otoksen edustavuuden mittaus R-indikaattorin avulla Suomen uhritutkimuspilotissa

Tutkielmassa sovelletaan aineiston edustavuutta mittaavaa laatuindikaattoria Suomen uhritutkimuspilottiin tilanteessa, jossa ilmenee vastauskatoa. Vastauskato on kasvava ongelma tilastotutkimuksissa: jos tutkimukseen osallistuneet eivät edusta otosjoukkoa tutkittavan asian suhteen, voi vastauskadosta aiheutuva harha olla estimoiduissa tunnusluvuissa hyvinkin suuri. Tutkimuksissa näkee usein julkaistavan vastausasteen ikään kuin se kertoisi aukottomasti tutkimuksen laadusta. Pelkkä korkea vastausaste ei kuitenkaan välttämättä takaa estimaattien harhattomuutta, sillä se ei kerro mitään vastanneiden ja vastaamattomien eroista tutkittavan asian suhteen. Tarvitaan siis muita mittareita, joilla vastanneiden laatua voitaisiin paremmin arvioida, ja R-indikaattori tarjoaa yhden vaihtoehdon. R-indikaattori mittaa otosalkioiden vastausalttiuksien välistä vaihtelua. R-indikaattorin estimoiminen edellyttää siis vastausalttiuksien estimointia, mikä puolestaan edellyttää apumuuttujien olemassaoloa kaikille otosalkioille. Vastausalttiuksien estimoimiseen käytettiin linkkifunktiona sekä logistista mallia että ja Särndalin ja Lundströmin (2008) vastausvaikutusten mallia. Vastauskäyttäytymiseen vaikuttavan apumuuttujajoukon valinta tehtiin alan kirjallisuuteen perustuen (Groves & Couper 1998). Koska R-indikaattorin estimaattori on satunnaismuuttuja, täytyi sille estimoida varianssi ja mahdollinen harha (Shlomo ym. 2009). Estimoinnissa käytettiin Bootstrap-pseudotoistomenetelmää, jossa alkuperäisestä aineistosta poimitaan niin kutsuttuja pseudo-otoksia, joiden avulla R-indikaattorin estimaattorille voidaan laskea keskivirhe. Suomen uhritutkimuspilotti koostui kolmesta eri tiedonkeruumenetelmällä poimitusta otoksesta: CAPI-, CATI- CAVVIotoksesta. Vastausasteet vaihtelivat aineistoissa paljon, mutta R-indikaattorin estimaatit olivat kaikille aineistoille liki samat. Suurempi vastausaste ei siis merkinnyt parempaa edustavuutta. Lisäksi CAVVI-aineistossa muistutusviestein ja -kirjein suoritettu vastausasteen kasvattaminen huononsi edustavuutta R-indikaattorin näkökulmasta. Mielivaltainen vastausasteen kasvattaminen ei siis ole välttämättä perusteltua. R-indikaattorin estimaattorin ominaisuuksien osalta empiiriset tulokset vahvistivat RISQ-projektin aiempia tutkimustuloksia. Estimaattorin arvo oli sitä pienempi mitä enemmän vastausalttiuden mallissa oli selittäjiä, koska tällöin vastausalttiuksien varianssi kasvoi (Schouten ym. 2009). Otoskoko vaikutti merkittävästi varianssin suuruuteen: mitä pienempi otoskoko oli, sitä leveämmät olivat luottamusvälit ja sitä vaikeampi oli tehdä johtopäätöksiä edustavuudesta.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.