Grammar and disciplinary culture : a corpus-based study

The present study provides a usage-based account of how three grammatical structures, declarative content clauses, interrogative content clause and as-predicative constructions, are used in academic research articles. These structures may be used in both knowledge claims and citations, and they often express evaluative meanings. Using the methodology of quantitative corpus linguistics, I investigate how the culture of the academic discipline influences the way in which these constructions are used in research articles. The study compares the rates of occurrence of these grammatical structures and investigates their co-occurrence patterns in articles representing four different disciplines (medicine, physics, law, and literary criticism). The analysis is based on a purpose-built 2-million-word corpus, which has been part-of-speech tagged. The analysis demonstrates that the use of these grammatical structures varies between disciplines, and further shows that the differences observed in the corpus data are linked with differences in the nature of knowledge and the patterns of enquiry. The constructions in focus tend to be more frequently used in the soft disciplines, law and literary criticism, where their co-occurrence patterns are also more varied. This reflects both the greater variety of topics discussed in these disciplines, and the higher frequency of references to statements made by other researchers. Knowledge-building in the soft fields normally requires a careful contextualisation of the arguments, giving rise to statements reporting earlier research employing the constructions in focus. In contrast, knowledgebuilding in the hard fields is typically a cumulative process, based on agreed-upon methods of analysis. This characteristic is reflected in the structure and contents of research reports, which offer fewer opportunities for using these constructions.

Eläinruumis - seuraeläin : Erittävä ruumis englantilaisessa lääketieteessä ja kohteliaisuuskulttuurissa 1700-1780

Tutkielmani tarkastelee käsityksiä ruumiista englantilaisessa kulttuurissa n. 1700-1780. Ruumiin historia on varsin uusi historiantutkimuksen ala ja sitä on leimannut feministinen tutkimusote, joka on keskittynyt diskurssiin naisen ruumiista ja seksuaalisuudesta. Itse kuitenkin lähestyn ruumista sen sivuutetun ulottuvuuden, ruumiineritteiden, kautta ja yhdistän käsitykset myös toiminnan tasolle. Tarkastelen erittävää ruumiista kulttuurihistoriallisesti sijoittamalla sen kahteen 1700-luvulla merkittävään kontekstiin: lääketieteeseen ja kohteliaisuuskulttuuriin. Tällä pyrin paitsi avaamaan ruumiiseen ja ruumiineritteisiin liittyvien käsitysten kirjoa myös osoittamaan näiden kahden kontekstin väliset vuorovaikutussuhteet. Lääketieteen ja kohteliaisuuskulttuurin puitteissa käsittelen erittävää ruumista mahdollisimman kokonaisvaltaisesti niin fysiologian, terveydenhoidon, hajujen, hy­gienian, aineellisen kulttuurin kuin sosiaalisten suhteiden kannalta, käyttäen lähteinäni mm. lääketieteellisiä tekstejä, kaunokirjallisuutta, matkakertomuksia, päiväkirjoja, kirjeitä, lehtiä ja muuta kohteliasta kirjallisuutta. Käsitykset erittävästä ruumiista osoittautuvat moninaisiksi, jopa ristiriitaisiksi. Toi­saalta lääketiede korostaa ruumiin avoimuutta ja eritteiden vapautta, toisaalta kohteliaisuus vaatii ruumiintoimintojen suhteen pidättyväisyyttä. Tämän moninai­suuden pohjalta kritisoin mm. Norbert Eliaksen sivilisaatioteoriaa ja osoitan näiden vastakkaisten käsitysten tilannesidonnaisuuden, joka mahdollistaa nii­den samanaikaisen olemassaolon. Kohteliaat herrasmiehet ja -naiset ovatkin kovin kiinnostuneita erittävistä ruumiistaan: ihmiset tarkkailevat eritteitään saadakseen tietoa ruumiin sisältä sekä toisten eritteitä karsiakseen kaiken eläimellisen ympäriltään ja kaunokirjallisuudessa ne ovat naurun ja huvituksen lähde. Vuorovai­kutus lääketieteellisen ja kohteliaan kulttuurin välillä osoittautuukin varsin tiiviiksi: kohteliaisuuden takana vaikuttavat periaatteet näkyvät myös lääketieteessä ja toisaalta lääketiede tunkeutuu mm. kohteliaaseen kieleen, hämärtäen näin kontekstien välistä rajaa.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Zebrafish as a model of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. Its etiology is unknown and no disease-modifying drugs are available. Thus, more information concerning its pathogenesis is needed. Among other genes, mutated PTEN-induced kinase 1 (PINK1) has been linked to early-onset and sporadic PD, but its mode of action is poorly understood. Most animal models of PD are based on the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to MPP+ by monoamine oxidase B (MAO B) and causes cell death of dopaminergic neurons in the substantia nigra in mammals. Zebrafish has been a widely used model organism in developmental biology, but is now emerging as a model for human diseases due to its ideal combination of properties. Zebrafish are inexpensive and easy to maintain, develop rapidly, breed in large quantities producing transparent embryos, and are readily manipulated by various methods, particularly genetic ones. In addition, zebrafish are vertebrate animals and results derived from zebrafish may be more applicable to mammals than results from invertebrate genetic models such as Drosophila melanogaster and Caenorhabditis elegans. However, the similarity cannot be taken for granted. The aim of this study was to establish and test a PD model using larval zebrafish. The developing monoaminergic neuronal systems of larval zebrafish were investigated. We identified and classified 17 catecholaminergic and 9 serotonergic neuron populations in the zebrafish brain. A 3-dimensional atlas was created to facilitate future research. Only one gene encoding MAO was found in the zebrafish genome. Zebrafish MAO showed MAO A-type substrate specificity, but non-A-non-B inhibitor specificity. Distribution of MAO in larval and adult zebrafish brains was both diffuse and distinctly cellular. Inhibition of MAO during larval development led to markedly elevated 5-hydroxytryptamine (serotonin, 5-HT) levels, which decreased the locomotion of the fish. MPTP exposure caused a transient loss of cells in specific aminergic cell populations and decreased locomotion. MPTP-induced changes could be rescued by the MAO B inhibitor deprenyl, suggesting a role for MAO in MPTP toxicity. MPP+ affected only one catecholaminergic cell population; thus, the action of MPP+ was more selective than that of MPTP. The zebrafish PINK1 gene was cloned in zebrafish, and morpholino oligonucleotides were used to suppress its expression in larval zebrafish. The functional domains and expression pattern of zebrafish PINK1 resembled those of other vertebrates, suggesting that zebrafish is a feasible model for studying PINK1. Translation inhibition resulted in cell loss of the same catecholaminergic cell populations as MPTP and MPP+. Inactivation of PINK1 sensitized larval zebrafish to subefficacious doses of MPTP, causing a decrease in locomotion and cell loss in one dopaminergic cell population. Zebrafish appears to be a feasible model for studying PD, since its aminergic systems, mode of action of MPTP, and functions of PINK1 resemble those of mammalians. However, the functions of zebrafish MAO differ from the two forms of MAO found in mammals. Future studies using zebrafish PD models should utilize the advantages specific to zebrafish, such as the ability to execute large-scale genetic or drug screens.

Experimental and computational studies on lipoprotein lipolysis at acidic pH

Atherosclerosis is a disease of the arteries; its characteristic features include chronic inflammation, extra- and intracellular lipid accumulation, extracellular matrix remodeling, and an increase in extracellular matrix volume. The underlying mechanisms in the pathogenesis of advanced atherosclerotic plaques, that involve local acidity of the extracellular fluid, are still incompletely understood. In this thesis project, my co-workers and I studied the different mechanisms by which local extracellular acidity could promote accumulation of the atherogenic apolipoprotein B-100 (apoB-100)-containing plasma lipoprotein particles in the inner layer of the arterial wall, the intima. We found that lipolysis of atherogenic apoB-100-containing plasma lipoprotein particles (LDL, IDL, and sVLDL) by the secretory phospholipase A2 group V (sPLA2-V) enzyme, was increased at acidic pH. Also, the binding of apoB-100-containing plasma lipoprotein particles to human aortic proteoglycans was dramatically enhanced at acidic pH. Additionally, lipolysis by sPLA2-V enzyme further increased this binding. Using proteoglycan-affinity chromatography, we found that sVLDL lipoprotein particles consist of populations, differing in their affinities toward proteoglycans. These populations also contained different amounts of apolipoprotein E (apoE) and apolipoprotein C-III (apoC-III); the amounts of apoC-III and apoE per particle were highest in the population with the lowest affinity toward proteoglycans. Since PLA2-modification of LDL particles has been shown to change their aggregation behavior, we also studied the effect of acidic pH on the monolayer structure covering lipoprotein particles after PLA2-induced hydrolysis. Using molecular dynamics simulations, we found that, in acidity, the monolayer is more tightly packed laterally; moreover, its spontaneous curvature is negative, suggesting that acidity may promote lipoprotein particles fusion. In addition to extracellular lipid accumulation, the apoB-100-containing plasma lipoprotein particles can be taken up by inflammatory cells, namely macrophages. Using radiolabeled lipoprotein particles and cell cultures, we showed that sPLA2-V-modification of LDL, IDL, and sVLDL lipoproteins particles, at neutral or acidic pH, increased their uptake by human monocyte-derived macrophages.

Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Expression and function of angiotensins in the regulation of intraocular pressure - an experimental study

Glaucoma is a multifactorial long-term ocular neuropathy associated with progressive loss of the visual field, retinal nerve fiber structural abnormalities and optic disc changes. Like arterial hypertension it is usually a symptomless disease, but if left untreated leads to visual disability and eventual blindness. All therapies currently used aim to lower intraocular pressure (IOP) in order to minimize cell death. Drugs with new mechanisms of action could protect glaucomatous eyes against blindness. Renin-angiotensin system (RAS) is known to regulate systemic blood pressure and compounds acting on it are in wide clinical use in the treatment of hypertension and heart failure but not yet in ophthalmological use. There are only few previous studies concerning intraocular RAS, though evidence is accumulating that drugs antagonizing RAS can also lower IOP, the only treatable risk factor in glaucoma. The main aim of this experimental study was to clarify the expression of the renin-angiotensin system in the eye tissues and to test its potential oculohypotensive effects and mechanisms. In addition, the possible relationship between the development of hypertension and IOP was evaluated in animal models. In conclusion, a novel angiotensin receptor type (Mas), as well as ACE2 enzyme- producing agonists for Mas, were described for the first time in the eye structures participating in the regulation of IOP. In addition, a Mas receptor agonist significantly reduced even normal IOP. The effect was abolished by a specific receptor antagonist. Intraocular, local RAS would thus to be involved in the regulation of IOP, probably even more in pathological conditions such as glaucoma though there was no unambiguous relationship between arterial and ocular hypertension. The findings suggest the potential as antiglaucomatous drugs of agents which increase ACE2 activity and the formation of angiotensin (1-7), or activate Mas receptors.

ADAMs, Osteoclastogenesis and Bone Destruction in the Loosening of the Totally Replaced Hip

Total hip replacement is the golden standard treatment for severe osteoarthritis refractory for conservative treatment. Aseptic loosening and osteolysis are the major long-term complications after total hip replacement. Foreign body giant cells and osteoclasts are locally formed around aseptically loosening implants from precursor cells by cell fusion. When the foreign body response is fully developed, it mediates inflammatory and destructive host responses, such as collagen degradation. In the present study, it was hypothesized that the wear debris and foreign body inflammation are the forces driving local osteoclast formation, peri-implant bone resorption and enhanced tissue remodeling. Therefore the object was to characterize the eventual expression and the role of fusion molecules, ADAMs (an abbreviation for A Disintegrin And Metalloproteinase, ADAM9 and ADAM12) in the fusion of progenitor cells into multinuclear giant cells. For generation of such cells, activated macrophages trying to respond to foreign debris play an important role. Matured osteoclasts together with activated macrophages mediate bone destruction by secreting protons and proteinases, including matrix metalloproteinases (MMPs) and cathepsin K. Thus this study also assessed collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. ADAMs were found in the interface tissues of revision total hip replacement patients. Increased expression of ADAMs at both transcriptional and translational levels was found in synovial membrane-like interface tissue of revision total hip replacement (THR) samples compared with that in primary THR samples. These studies also demonstrate that multinucleate cell formation from monocytes by stimulation with macrophage-colony stimiulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) is characterized by time dependent changes of the proportion of ADAMs positive cells. This was observed both in the interface membrane in patients and in two different in vitro models. In addition to an already established MCS-F and RANKL driven model, a new virally (parainfluenza 2) driven model (of human salivary adenocarcinoma (HSY) cells or green monkey kidney (GMK) cells) was developed to study various fusion molecules and their role in cell fusion in general. In interface membranes, collagen was highly degraded and collagen degradation significantly correlated with the number of local cells containing collagenolytic enzymes, particularly cathepsin K. As a conclusion, fusion molecules ADAM9 and ADAM12 seem to be dynamically involved in cell-cell fusion processes and multinucleate cell formation. The highly significant correlation between collagen degradation and collagenolytic enzymes, particularly cathepsin K, indicates that the local acidity of the interface membrane in the pathologic bone and soft tissue destruction. This study provides profound knowledge about cell fusion and mechanism responsible for aseptic loosening as well as increases knowledge helpful for prevention and treatment.

Candidate gene studies on cardiovascular traits

Cardiovascular diseases (CVD) are a major cause of death and disability in Western countries and a growing health problem in the developing world. The genetic component of both coronary heart disease (CHD) and ischemic stroke events has been established in twin studies, and the traits predisposing to CVD, such as hypertension, dyslipidemias, obesity, diabetes, and smoking behavior, are all partly hereditary. Better understanding of the pathophysiology of CVD-related traits could help to target disease prevention and clinical treatment to individuals at an especially high disease risk and provide novel pharmaceutical interventions. This thesis aimed to clarify the genetic background of CVD at a population level using large Nordic population cohorts and a candidate gene approach. The first study concentrated on the allelic diversity of the thrombomodulin (THBD) gene in two Finnish cohorts, FINRISK-92 and FINRISK-97. The results from this study implied that THBD variants do not substantially contribute to CVD risk. In the second study, three other candidate genes were added to the analyses. The study investigated the epistatic effects of coagulation factor V (F5), intercellular adhesion molecule -1 (ICAM1), protein C (PROC), and THBD in the same FINRISK cohorts. The results were encouraging; we were able to identify several single SNPs and SNP combinations associating with CVD and mortality. Interestingly, THBD variants appeared in the associating SNP combinations despite the negative results from Study I, suggesting that THBD contributes to CVD through gene-gene interactions. In the third study, upstream transcription factor -1 (USF1) was analyzed in a cohort of Swedish men. USF1 was associated with metabolic syndrome, characterized by accumulation of different CVD risk factors. A putative protective and a putative risk variant were identified. A direct association with CVD was not observed. The longitudinal nature of the study also clarified the effect of USF1 variants on CVD risk factors followed in four examinations throughout adulthood. The three studies provided valuable information on the study of complex traits, highlighting the use of large study samples, the importance of replication, and the full coverage of the major allelic variants of the target genes to assure reliable findings. Although the genetic basis of coronary heart disease and ischemic stroke remains unknown, single genetic findings may facilitate the recognition of high-risk subgroups.

Stem Cell Markers in Cancer: Do They Have Clinical Significance?

In cancer, a subpopulation of malignant cells expresses markers of normal stem cells. These cells have the potential of initiating tumor growth and therefore also tumor recurrence. Thus, these cells are called cancer stem cells. A myriad of markers have been applied to identify these cells, but no single marker can be found exclusively in cancer stem cells. In many types of cancer, clinical recurrence and tumor progression are the main causes of mortality, despite intense oncological treatment. It has been proposed that the presence of cancer stem cells causes this resistance to therapy. The scope of this thesis is to investigate the role of stem cell markers and genes in the clinical setting. Especially, the aim was to elucidate the clinical significance of stem cell markers as novel prognostic and diagnostic tools in cancer. Tumor biopsy material from central nervous system tumors (oligodendroglioma, astrocytoma and glioblatoma), neural crest derived tumors (pheochromocytomas) and oral carcinoma was screened for stem cell markers. Initially, 15 stem cell markers were screened in a test series of gliomas. The markers applied for expanded tumor analyses (in 305 cases of glioma, 42 cases of pheochromocytoma, and 73 cases of oral carcinoma) were BMI-1, Snail, p16, mdm2, and c-Myc. Data on marker expression was compared with clinical and pathological parameters. In gliomas, BMI-1 expression was found in nearly all tumors analyzed, but the frequency of BMI-1 expressing cells was highly variable, ranging from 1 to 100%. In oligodendroglioma, BMI-1 expression was identified as a prognostic marker independent of tumor grade and clinical parameters. In pheochromocytoma, Snail expression was shown to distinguish between the metastatic and non-metastatic forms of the tumor. Snail expression was seen only in metastatic tumors, whereas non-metastatic tumors did not commonly express Snail. Finally, in oral carcinoma, BMI-1 expression was seen in roughly 80% of tumors, and Snail expression was high or very high in all cases. The lack of BMI-1 expression was associated with early relapse in oral carcinoma.

The Interplay Between KSHV-encoded Viral Cyclin and CDK-inhibitors p27Kip1 and p21Cip1 -implications for Viral Lymphomagenesis

Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells.

Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

Blood pressure lowering effects of Lactobacillus helveticus fermented milk containing bioactive peptides Ile-Pro-Pro and Val-Pro-Pro; mechanistic, kinetic and clinical studies

The purpose of the present study was to evaluate the effects of Lactobacillus helveticus fermented milk (peptide milk) containing the casein-derived tripeptides Isoleucyl-prolyl-proline (Ile-Pro-Pro) and Valyl-prolyl-proline (Val-Pro-Pro) on blood pressure and vascular function in hypertensive subjects. The peptide milk lowered systolic and diastolic blood pressure in long-term use in hypertensive subjects when blood pressure was measured by using 24-hour ambulatory blood pressure measurement (ABPM). The blood pressure lowering effect was seen with the dose of 50 mg of tripeptides, and a tendency for lowering blood pressure was also observed when the dose was 5 mg. No adverse effects compared to the placebo group were reported or detected in laboratory analysis. The effect of the peptide milk on arterial stiffness was shown using two different methods, the ambulatory arterial stiffness index (AASI) and pulse wave analysis (PWA). According to the AASI, arterial stiffness was significantly reduced in the peptide milk group compared to the baseline level, but the difference was not significant compared to the placebo group. PWA showed that the peptide milk reduced arterial stiffness significantly compared to the placebo group. Endothelium-independent relaxation (nitroglycerin) and endothelium-dependent relaxation (salbutamol) did not differ between the groups. The blood pressure lowering mechanisms of the tripeptides and the kinetics of Ile-Pro-Pro were investigated using spontaneously hypertensive rats (SHR) and Sprague-Dawley rats. Previous studies have suggested that the blood pressure lowering effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro is based on angiotensin-converting enzyme inhibition, but the present findings did not agree with these previous studies. It was shown in SHR that calcium, potassium and magnesium may also have an important role in attenuating the development of hypertension as part of the peptide milk effect. In addition, the present study suggests indirectly that improved endothelial nitric oxide release capacity is not the mechanism by which peptide milk mediates its favourable circulatory effects. The kinetics of Ile-Pro-Pro were studied using adult Sprague-Dawley rats. The results showed that orally administered Ile-Pro-Pro is absorbed at least partly intact from the gastrointestinal tract. Radiolabelled Ile-Pro-Pro was distributed in different tissues and considerable radioactivity levels were found in tissues related to the renin-angiotensin system (RAS), adrenals, aorta and kidneys. Ile-Pro-Pro does not bind to plasma proteins, and therefore it is possible that its blood pressure lowering effect is mediated by free Ile-Pro-Pro. In conclusion, consumption of the peptide milk lowers blood pressure and reduces arterial stiffness in hypertensive subjects. Ile-Pro-Pro can be absorbed partly intact from the gastrointestinal tract and might accumulate in tissues related to the RAS. The precise blood pressure lowering mechanism of peptide milk remains to be studied.

Dietary elecrolytes and cyclosporine toxicity. Experimental studies in spontaneuosly hypertensive rats

Cyclosporine-A (CsA) is widely used after organ transplantation to prevent rejection and in the treatment of autoimmune diseases. Hypertension and nephrotoxicity are common side-effects of CsA. Studies in patients on the prevention of the side-effects of CsA are difficult to conduct because the patients often receive a combination of different drugs thus making study of the side-effects of a single drug impossible. A challenge in experimental studies has been the lack of an animal model in which the side-effects concomitantly occur. Epidemiological data show an association between sodium (Na) intake and blood pressure. There is also evidence on low dietary intake of magnesium (Mg) and potassium (K) and high blood pressure. Our study was designed to develop an experimental model to study the side-effects of CsA in spontaneously hypertensive rats (SHR). On high dietary sodium, CsA caused hypertension, left ventricular hypertrophy (LVH), narrowing of the coronary arteries, small myocardial infarctions, and proteinuria, reduced creatinine clearance and histopathological renal injury in SHR. Loss of Mg into the urine caused by CsA resulted in Mg depletion in the tissues. Renal excretion of dopamine was reduced and the renin-angiotensin-aldosterone system was activated. We investigated the effects of dietary Mg and/or K and the calcium antagonist drug, isradipine, on the prevention of CsA toxicity. Dietary supplementation of Mg alone or in combination with K prevented from the deleterious pathophysiological and histopathological changes in the kidneys and the heart. K alone had little effect. Isradipine protected better than Mg from LVH, but the combination of isradipine and Mg was the most effective. Isradipine did not, however, protect against Mg loss. In our animal model, the combination of high dietary Na and treatment with CsA accelerated the development of the cardiovascular and renal changes clinically known as the side-effects of CsA. Dietary supplementation of Mg and K and reduction of Na intake and the calcium antagonist drug isradipine prevent from the deleterious effects of CsA.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.