MicroRNAs as novel regulators of skeletal homeostasis

The human skeleton is composed of bone and cartilage. The differentiation of bone and cartilage cells from their bone marrow progenitors is regulated by an intrinsic network of intracellular and extracellular signaling molecules. In addition, cells coordinate their differentiation and function through reciprocal cell‐to‐cell interactions. MicroRNAs (miRNAs) are small, single‐stranded RNA molecules that inhibit protein translation by binding to messenger RNAs (mRNAs). Recent evidence demonstrates the involvement of miRNAs in multiple biological processes. However, their role in skeletal development and bone remodeling is still poorly understood. The aim of this thesis was to elucidate miRNA‐mediated gene regulation in bone and cartilage cells, namely in osteoblasts, osteoclasts, chondrocytes and bone marrow adipocytes. Comparison of miRNA expression during osteogenic and chondrogenic differentiation of bone marrow‐derived mesenchymal stem cells (MSCs) revealed several miRNAs with substantial difference between bone and cartilage cells. These miRNAs were predicted to target genes essentially involved in MSC differentiation. Three miRNAs, miR‐96, miR‐124 and miR‐199a, showed marked upregulation upon osteogenic, chondrogenic or adipogenic differentiation. Based on functional studies, these miRNAs regulate gene expression in MSCs and may thereby play a role in the commitment and/or differentiation of MSCs. Characterization of miRNA expression during osteoclastogenesis of mouse bone marrow cells revealed a unique expression pattern for several miRNAs. Potential targets of the differentially expressed miRNAs included many molecules essentially involved in osteoclast differentiation. These results provide novel insights into the expression and function of miRNAs during the differentiation of bone and cartilage cells. This information may be useful for the development of novel stem cell‐based treatments for skeletal defects and diseases.

The Effects of Estradiol, Androgens and the Selective Estrogen Modulators: Ospemifene, Raloxifene and Tamoxifen on Explant Cultures of Human Breast Tissue

The impact of menopausal hormone therapy (MHT) on increasing the risk for breast cancer (BC) remains controversial. To understand MHT-elicited cellular breast effects and the potential risks, included with using this therapy, a further investigation into this controversy is the subject of this thesis. In this thesis, to study the effects of estrogen, progestin, androgens and selective estrogen receptor modulators (SERMs), a modified tissue explant culture system was used. The different types of human breast tissues (HBTs) used in this study were normal HBTs, obtained from reduction mammoplasties of premenopausal women (prem-HBTs) or postmenopausal (postm-HBTs) women and peritumoral HBTs (peritum-HBTs) which were obtained from surgeries on postmenopausal BC patients. The explants were cultured up to three weeks in the presence or absence of estradiol (E2), medroxyprogesterone acetate (MPA), testosterone (T), dihydrotestosterone (DHT) and SERMs - ospemifene (OSP), raloxifene (RAL) and tamoxifen (TAM). The cultured HBTs maintained morphological integrity and responded to hormonal treatment in vitro. E2, MPA or E2/MPA increased proliferative activity and was associated with increased cyclin-D1 and caused changes in the cell cycle inhibitors p21 and p27, whereas the androgens T and DHT inhibited proliferation and increased apoptosis in HBT epithelia and opposed E2-stimulated proliferation and cell survival. The postm-HBTs were more sensitive to E2 than prem-HBTs. The effects of OSP, RAL and TAM on HBT epithelium were antiproliferative. E2, androgens and SERMs were associated with marked changes in the proportions of epithelial cells expressing steroid hormone receptors: E2 increased ERα expressing cells and decreased androgen receptor (AR) positive cells, whereas T and DHT had opposite effects. The OSP, RAL and TAM, also decreased a proportion of ERα positive cells in HBT epithelium. At 100 nM, these compounds maintained the relative number of AR positive cells, present at control level, which may partly explain proliferative inhibition. In conclusion, the proliferative activity of E2, in the epithelium of postm-HBTs, is opposed by T and DHT, which suggests that the inclusion of androgens in MHT may decrease the risk for developing BC.

Rasvasuodatus UV-valon avulla

Tämä kandidaatintyö käsittelee rasva-aerosolien vähentämistä liesituulettimissa ultraviolettivalon (UV) ja titaanioksidin avulla (TiO2). Työssä käsitellään rasva-aerosolien aiheuttamia haittoja julkisissa keittiöissä ja liesituulettimen merkitystä rasva-aerosolipitoisuuksiin. Työn tavoitteena on selvittää, miten rasva-aerosolit käyttäytyvät kun niitä suodatetaan UV-valon ja TiO2-katalyytin avulla. Työssä myös pohditaan onko UV/TiO2-menetelmä tehokas liesituulettimen rasvan vähentäjä.

Characterisation of Functional Brown Adipose Tissue In Adult Humans

The white adipose tissue mainly serves the purpose of energy storage, while brown adipose tissue (BAT) has the capacity to generate heat under cold conditions in mammals and in human infants. BAT is controlled by the central nervous system, and BAT function is accompanied by increased energy expenditure. However, it was not previously certain whether adult humans also have functional BAT. The aim of this doctoral work was to identify functional BAT in adult humans and to characterise its glucose uptake and blood flow under cold and insulin stimulation conditions in lean and in obese humans, by using positron emission tomography. Further, the impact of weight loss on BAT glucose uptake was assessed. Cerebral glucose uptake was also studied in relation to BAT function and cold exposure. The results showed that healthy adult humans have functional BAT, as assessed by the intense cold-induced glucose uptake and by biopsies. BAT was also found to be a highly insulinsensitive tissue in lean humans, but the effects of insulin and cold exposure were attenuated in obese humans, although the glucose uptake capacity of cold-activated BAT might be increased by weight loss. Blood flow in the BAT of lean humans was associated with whole-body energy expenditure. The presence of cold-activated BAT was related to lower body mass index and higher insulin sensitivity. Finally, BAT activation was linked to the activity of the cerebellum, the thalamus and certain neocortical regions. The cold-induced cerebral glucose uptake was also lower in obese than in lean adult humans.

Phenomena and mechanisms in purification of animal fat

Työn tarkoitus oli tutkia eläinrasvan puhdistusta biodieselin valmistusta varten. Eläinrasvaa syntyy elintarviketeollisuuden sivutuotteena ja sitä saadaan myös myymättä jääneistä elintarvikkeista. Rasva sisältää epäpuhtauksia, jotka on poistettava ennen biodieselprosessia. Tässä työssä tutkittavat epäpuhtaudet ovat typpi, fosfori, rauta, natrium, kalsium ja magnesium. Puhdistusmenetelminä käytettiin saostamista sitruunahapolla sekä adsorbointia kahdella eri adsorbentilla. Tavoitteena oli selvittää riittävä määrä happoa ja adsorbenttia sekä tutkia puhdistuksen mekanismia. Lisäksi tarkasteltiin lämpötilan vaikutusta adsorption aikana.

Natural born weight gainers: The mechanisms of obesity in transgenic mice overexpressing neuropeptide Y

Neuropeptide Y (NPY) is a neurotransmitter promoting energy storage by activating Y-receptors and thus affecting food intake, thermogenesis and adipose tissue metabolism. NPY is expressed both in the central and sympathetic nervous system. Hypothalamic NPY is known to stimulate feeding, but the effects of noradrenergic neuron NPY are more ambiguous. Chronic stress stimulates fat accumulation via NPY release from noradrenergic neurons. Furthermore, polymorphism in the human Npy gene has been associated with metabolic disturbances and increased NPY secretion after sympathetic stimulation. The main objective of this study was to clarify the mechanisms of noradrenergic neuron NPY in the development of obesity. The metabolic phenotype of a homozygous mouse overexpressing NPY in the brain noradrenergic neurons and sympathetic nervous system (OE-NPYDβH mouse) was characterized. OE-NPYDβH mice had an increased fat mass and body weight, which caused impairments of glucose metabolism and hyperinsulinaemia with age. There were no differences in energy intake or expenditure, but the sympathetic tone was down-regulated and the endocannabinoid system activated. Furthermore, peripheral Y2-receptors in energy-rich conditions played an important role in mediating the fat-accumulating effect of NPY. These results indicate that noradrenergic neuron NPY promotes obesity via direct effects in the periphery and by modulating the sympatho-adrenal and endocannabinoid systems. Additionally, NPY in the central noradrenergic neurons is believed to possess many important roles. The phenotype of the OE-NPYDβH mouse resembles the situations of chronic stress and Npy gene polymorphism and thus these mice may be exploited in testing novel drug candidates for the treatment of obesity.

Antosyaniinit ja aterianjälkeinen aineenvaihdunta

Marjojen ja hedelmien luontaisilla sinisillä ja punaisilla antosyaniiniväriaineilla on aterianjälkeiselle aineenvaihdunnalle edullisia ominaisuuksia. Antosyaniinit liittyvät esimerkiksi aterianjälkeiseen plasman glukoosi-, insuliini-, rasva- ja tulehdusmerkkiainepitoisuuksien alenemiseen, glukoosinsietokyvyn kasvamiseen ja insuliiniresistenssin vähenemiseen. Vaikutusmekanismeiksi on ehdotettu useita biokemiallisia reaktioita. Antosyaniinit ovat antioksidatiivisia, ja ne lisäävät elimistön antioksidatiivisten entsyymien aktiivisuutta. Ne vaikuttavat hiilihydraattien ja rasvojen aineenvaihduntaan liittyvien entsyymien toimintaan sekä inhiboivat monosakkaridien imeytymistä ja AMP-kinaaseja. Kokeellisessa osassa tutkittiin tummansinisestä Synkeä Sakari -perunalajikkeesta valmistetun höyrykypsennetyn survoksen vaikutusta aterianjälkeiseen glykemiaan ja insulinemiaan verrattuna keltaisesta perunasta valmistettuun survokseen ja mustikkakiisseliin. Tutkimushenkilöt (n=13) nauttivat yksöissokotetussa vaihtovuorotutkimuksessa antosyaniini- ja tärkkelyspitoisuuksien suhteen vakioidun aterian satunnaistetussa järjestyksessä. Aterian jälkeen heiltä kerättiin plasma- ja virtsanäytteitä tietyin väliajoin. Tutkimusaterioiden antosyaniinien, fenolisten happojen, flavonoliglykosidien, vapaiden sokereiden ja orgaanisten happojen tunnistamisessa käytettiin kromatografisia, massaspektrometrisiä ja spektrofotometrisiä menetelmiä. Antosyaniinien ja niiden aineenvaihduntatuotteiden aterianjälkeistä kertymistä virtsaan aloitettiin tutkia kehittämällä näytteiden esikäsittely- ja UHPLC-ESI-MS/MS -menetelmiä. Sininen perunasurvos aiheutti pienemmän glykemiakuorman kuin mustikkakiisseli ja pienemmän insuliinikuorman kuin keltainen perunasurvos tai mustikkakiisseli. Glukoosi- ja insuliinipitoisuuksissa havaittiin merkitseviä eroja 20 ja 40 minuuttia aterian jälkeen. Tutkimus antaa viitteitä siitä, että antosyaniinipitoiset perunalajikkeet ovat glykeemisiltä ominaisuuksiltaan keltaisia perunoita edullisempia. Tämä on merkittävää, koska perunat ovat tärkeä osa suomalaista ruokavaliota ja toistuvat korkeat aterianjälkeiset veren glukoosi- ja insuliinipitoisuudet ovat yhteydessä elintasosairauksiin.

Common dyslipidemias, high lipoprotein(a) concentrations and endothelial function in the children of the STRIP study families

Extreme lipid values predisposing on illnesses are dyslipidemias. Dyslipidemias evolve in early childhood, but their significance or persistency is not well known. Common dyslipidemias may aggregate in the same families. This thesis is a part of the longitudinal randomized Special Turku coronary Risk factor Intervention Project STRIP, in which 1054 families with six months old children were randomized to a control or to an intervention group. The family lipid data from the first 11 years was used. Fasting samples at the age of five years defined the lipid phenotypes. The dyslipidemias coexisting in the parent and the child were studied. At the age of 11 years 402 children participated artery ultrasound studies. The significance of the childhood dyslipidemias and lipoprotein(a) concentration on endothelial function was evaluated with the flow mediated arterial dilatation test. Frequently elevated non-HDL cholesterol concentration from one to seven-year-old children associated to similar parental dyslipidemia that improved the predictive value of the childhood sample. The familial combinations were hypercholesterolemia (2.3%), hypertriglyceridemia (2.0%), familial combined hyperlipidemia (1.8%), and isolated low HDL-cholesterol concentration (1.4%). Combined hyperlipidemia in a parent predicted most frequently the child’s hyperlipidemia. High lipoprotein(a) concentration aggregated in some families and associated to childhood attenuated brachial artery dilatation. Hypercholesterolemia and high lipoprotein(a) concentration at five years of age predicted attenuated dilatation. This study demonstrated that parental dyslipidemias and high lipoprotein(a) concentration help to find early childhood dyslipidemias. The association of hypercholesterolemia and lipoprotein(a) concentration with endothelial function emphasizes the importance of the early recognition of the dyslipidemias.