Kuluttaminen ja ylhäisaatelin elämäntapa 1700-luvun Ruotsissa

Consumption and the lifestyle of the high nobility in eighteenth-century Sweden This monograph is an analysis of the lifestyle, consumption and private finances of the Swedish high nobility during the eighteenth century (ca 1730 1795). It describes the lifestyle of one noble house, the House of Fersen. The Fersen family represents the leading political, economic and cultural elite in eighteenth-century Sweden. The analysis concentrates on Count Carl von Fersen (1716 1786) and his brother Count Axel von Fersen (1719 1794), their spouses and children. Carl von Fersen was a courtier whilst Axel von Fersen was an officer and one of the leaders of the Francophile Hat party. His son, Axel von Fersen the younger, was in his time an officer and a favourite of Gustavus III, King of Sweden, as well as a favourite and trusted confidant of Marie-Antoinette, Queen of France. The research is based upon the Fersen family s private archives, the Counts personal account books, probate inventories, letters and diaries. The study discusses the Fersens landed property and investments in ironworks and manufacturing, the indebtedness of the high nobility, high offices in civil administration, the militia and at court, as well as marriages as the foundations of noble wealth and power. It analyses the Count von Fersens revenue and expenditure, their career options and personal expenses, their involvement in the building and decorating of palaces, and the servants in service of the Fersen family as well as the ideal nobleman and his consumption. Central themes are inheritance, children s education, marriages and ladies preparing their trousseaux, the nobility ordering luxury goods from France, the consumption of Counts and Countesses before and after marrying and having children, the pleasures of a noble life as well as the criticism of luxury and sumptuousness. The study contributes to the large body of research on consumption and nobility in the eighteenth century by connecting the lifestyle, consumption and private finances of the Swedish high nobility to their European context. Key words: nobility, Fersen, lifestyle, consumption, private finances, Sweden, eighteenth century

Intergenerational Transfer of Sabbath Observance : How is Religious Tradition Transferred to Children among Finnish Seventh-day Adventists?

This study examined religious home education in educational, psychological, and sociological context. Growing up within a religious denomination is a process of learning the rules, norms, opinions, and attitudes, which serve to make the individual an active member of the group. It is a process of transferring the cultural inheritance between generations. Sabbathkeeping can be regarded as a strong indicator of the Seventh-day Adventist value system, which is also why I have concentrated on this specific issue in my study. The purpose of the study was to find out, how the Sabbath is transferred from parents to children among Finnish Adventists. It was also examined how parents could make the day of rest positively exceptional for children, and how the parental authoritativeness affects the process of transference. According to Bull & Lockhart s (1989) theory, the amount of Adventist generations in family history influences the transfer of religious tradition. This study aimed to find out whether or not this theory would apply to the present-day Finland. The nature of religious development among Adventist young people was also one of the interests of the research. The methods used in the study were in-depth interviews (n = 10) and a survey (n = 106). The majority of the interviewees was young adults (age 15-30) grown up in Adventist families. The interviews were taped and transcribed for the study, and survey answers were analysed with SPSS-data analysis program. The amount of survey questionnaires evaluated was 106, whole population of 15-30 year-old Finnish Adventists being about one thousand. Democratic relationship between parents and children, parents' example, encouragement to own thinking, and positive experiences of Sabbath and the whole religion, including the social dimension of the Adventism, seem to be some of the most significant factors in transference of religious tradition. Both too severe and too permissive education were considered to lead to similar results: unsuccessful transfer of values, or even rebellion and adopting a totally opposite way of life than that of the parents. In this study the amount of Adventist generations in family history does not correlate significantly with the end results of value transference. Keywords: Sabbath, intergenerational, value transference, religious home education Avainsanat: sapatti, arvojen siirtyminen vanhemmilta lapsille, uskonnollinen kotikasvatus

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Novel Insights into the Molecular Genetic Background of Colorectal Tumors

Many of the genes predisposing to highly penetrant colorectal cancer (CRC) syndromes, including hereditary non-polyposis colorectal cancer (MLH1, MSH2, MSH6, PMS2), familial adenomatous polyposis (APC), Peutz-Jeghers syndrome (LKB1), juvenile polyposis (SMAD4, BMPR1A), MYH-associated polyposis (MYH), and Cowden syndrome (PTEN) have already been discovered. Identification of these genes has allowed a more precise classification of the hereditary CRC syndromes and provided a means for predictive genetic testing and surveillance. Some of the genes are also involved in sporadic cancer forms, and therefore the investigation of the rare CRC syndromes has been a breakthrough for general cancer research. Despite the accumulating knowledge on hereditary cancer syndromes, a significant number of familial CRCs remain molecularly unexplained after genetic testing, reflecting the possibility of other predisposing genes or existence of novel syndromes. Moreover, genetic variants conferring low-penetrance risk are still largely unknown. In this study, we examined the role of some new high- and low-penetrance alleles on CRC predisposition. We identified disease causing MYH mutations in a subset (9%) of patients with APC and AXIN2 mutation negative adenomatous polyposis. Due to differences in the pattern of inheritance and clinical manifestation, screening for mutations in MYH is beneficial in view of genetic counselling and surveillance. A novel functionally deficient MYH founder mutation A459D was identified in the Finnish population, and this finding had immediate clinical implications for genetic counselling of at risk families. Many patients with hamartomatous polyposis remain without molecular diagnosis due to atypical phenotypes. We therefore sought to classify 49 patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analyses of PTEN, LKB1, BMPR1A, SMAD4, ENG, BRAF, MYH, and BHD along with revision of polyp histology. Mutations were identified in 11/49 (22%) of the patients. In 6 cases the molecular diagnosis was re-classified guiding surveillance and decisions for prophylactic surgery. Re-evaluation of polyp histology with subsequent more accurate selection of candidate gene analyses is beneficial and can be recommended for patients with unexplained polyposis. Furthermore, germline mutations in ENG underlying juvenile polyposis were described for the first time, characterizing a possible novel genetically defined form of hereditary CRC. Association analyses on two putative low-penetrance alleles, NOD2 3020insC and MDM2 SNP309 were performed in a population-based series of 1042 Finnish CRC patients and in cancer-free controls. In contrast to previous results, NOD2 3020insC did not associate with CRC or age at disease onset in the Finnish population. These data suggest that NOD2 3020insC alone might not be sufficient for CRC predisposition. MDM2 SNP309 was as common in the CRC cohort as in the healthy controls. Interesting trends, however, were observed, which after correction for multiple testing did not reach statistical significance. SNP309 was more common in female CRC patients and a trend towards an earlier age at disease onset was observed in women with SNP309. Subsequent studies have supported this observation and SNP309 could affect gender- or hormone-related tumorigenesis. Finally, a large-scale unbiased effort was designed to characterize the complete mutatome of CRC with microsatellite instability (MSI). Using an approach combining expression microarray and genome database searches, we were able to identify putative MSI target genes. Further characterization of one of the genes suggested that it might play a role also in microsatellite stable CRC and Peutz-Jeghers syndrome pathogenesis.

Zinc, cadmium and lead resistance mechanisms in bacteria and their contribution to biosensing

In bacteria resistance to heavy metals is mainly achieved through active efflux, but also sequestration with proteins or as insoluble compounds is used. Although numerous studies have dealt with zinc, cadmium and lead resistance mechanisms in bacteria, it has still remained unclear how different transporters are integrated into an effective homeostasis/resistance network and whether specific mechanisms for lead sequestration exist. Furthermore, since metals are toxic not only to bacteria but to higher organisms as well, it is important to be able to estimate possible biological effects of heavy metals in the environment. This could be done by determining the bioavailable amount of the metals in the environment with bacterial bioreporters. That is, one can employ bacteria that respond to metal contamination by a measurable signal to assess the property of metals to cross biological membranes and to cause harmful effects in a possibly polluted environment. In this thesis a new lead resistance mechanism is described, interplay between CBA transporters and P-type ATPases in zinc and cadmium resistance is presented and finally the acquired knowledge is used to construct bacterial bioreporters for heavy metals with increased sensitivity and specificity. The new lead resistance model employs a P-type ATPase that removes Pb2+ ions from the cytoplasm and a phosphatase that produces inorganic phosphate for lead sequestration in the periplasm. This was the first study where the molecular mechanism of lead sequestration has been described. Characterization of two P-type ATPases and two CBA transporters showed that resistance mechanisms for Zn2+ and Cd2+ are somewhat different than for Pb2+ as these metals cannot be sequestered as insoluble compounds as easily. Resistance to Zn2+ was conferred merely by the CBA transporter that could export both cytoplasmic and periplasmic ions; whereas, full resistance to Cd2+ required interplay of a P-type ATPase that exported cytoplasmic ions to periplasm and a CBA transporter that further exported periplasmic ions to the outside. The knowledge on functionality of the transporters and metal-inducible promoters was exploited in bioreporter technology. A transporter-deficient bioreporter strain that lacked exporters for Zn2+/Cd2+/Pb2+ could detect up to 45-fold lower metal concentrations than its wild type counterpart due to the accumulation of metals in the cell. The broad specificity issue of bioreporters was overcome by using Zn-specific promoter as a sensor element, thus achieving Zn-specific bioreporter.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

LFA-1 Integrin beta2 Chain Phosphorylation Regulates Protein Interactions and Mediates Signals in T Cells

Integrins are heterodimeric transmembrane adhesion receptors composed of alpha- and beta-subunits and they are vital for the function of multicellular organisms. Integrin-mediated adhesion is a complex process involving both affinity regulation and coupling to the actin cytoskeleton. Integrins also function as bidirectional signaling devices, regulating cell adhesion and migration after inside-out signaling, but also signal into the cell to regulate growth, differentiation and apoptosis after ligand binding. The LFA-1 integrin is exclusively expressed in leukocytes and is of fundamental importance for the function of the immune system. The LFA-1 integrins have short intracellular tails, which are devoid of catalytic activity. These cytoplasmic domains are important for integrin regulation and both the alpha and beta chain become phosphorylated. The alpha chain is constitutively phosphorylated, but the beta chain becomes phosphorylated on serine and functionally important threonine residues only after cell activation. The cytoplasmic tails of LFA-1 bind to many cytoskeletal and signaling proteins regulating numerous cell functions. However, the molecular mechanisms behind these interactions have been poorly understood. Phosphorylation of the cytoplasmic tails of the LFA-1 integrin could provide a mechanism to regulate integrin-mediated cytoskeletal interactions and take part in T cell signaling. In this study, the effects of phosphorylation of LFA-1 integrin cytoplasmic tails on different cellular functions were examined. Site-specific phosphorylation of both the alpha- and beta-chains of the LFA-1 was shown to have a role in the regulation of the LFA-1 integrin.Alpha-chain Ser1140 is needed for integrin conformational changes after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1, whereas beta-chain binds to 14-3-3 proteins through the phosphorylated Thr758 and mediates cytoskeletal reorganization. Thr758 phosphorylation also acts as a molecular switch to inhibit filamin binding and allows 14-3-3 protein binding to integrin cytoplasmic domain, and it was also shown to lead to T cell adhesion, Rac-1/Cdc42 activation and expression of the T cell activation marker CD69, indicating a signaling function for Thr758 phosphorylation in T cells. Thus, phosphorylation of the cytoplasmic tails of LFA-1 plays an important role in different functions of the LFA-1 integrin in T cells. It is of vital importance to study the mechanisms and components of integrin regulation since leukocyte adhesion is involved in many functions of the immune system and defects in the regulation of LFA-1 contributes to auto-immune diseases and fundamental defects in the immune system.

Electron and proton transfer in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

Cells of every living organism on our planet − bacterium, plant or animal − are organized in such a way that despite differences in structure and function they utilize the same metabolic energy represented by electrochemical proton gradient across a membrane. This gradient of protons is generated by the series of membrane bound multisubunit proteins, Complex I, II, III and IV, organized in so-called respiratory or electron transport chain. In the eukaryotic cell it locates in the inner mitochondrial membrane while in the bacterial cell it locates in the cytoplasmic membrane. The function of the respiratory chain is to accept electrons from NADH and ubiquinol and transfer them to oxygen resulting in the formation of water. The free energy released upon these redox reactions is converted by respiratory enzymes into an electrochemical proton gradient, which is used for synthesis of ATP as well as for many other energy dependent processes. This thesis is focused on studies of the first member of the respiratory chain − NADH:ubiquinone oxidoreductase or Complex I. This enzyme has a boot-shape structure with hydrophilic and hydrophobic domains, the former of which has all redox groups of the protein, the flavin and eight to nine iron-sulfur clusters. Complex I serves as a proton pump coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. So far the mechanism of energy transduction by Complex I is unknown. In the present study we applied a set of different methods to study the electron and proton transfer reactions in Complex I from Escherichia coli. The main achievement was the experiment that showed that the electron transfer through the hydrophilic domain of Complex I is unlikely to be coupled to proton transfer directly or to conformational changes in the protein. In this work for the first time properties of all redox centers of Complex I were characterized in the intact purified bacterial enzyme. We also probed the role of several conserved amino acid residues in the electron transfer of Complex I. Finally, we found that highly conserved amino acid residues in several membrane subunits form a common pattern with a very prominent feature – the presence of a few lysines within the membrane. Based on the experimental data, we suggested a tentative principle which may govern the redox-coupled proton pumping in Complex I.

Molecular genetics of tooth agenesis

Congenital missing of teeth, tooth agenesis or hypodontia, is one of the most common developmental anomalies in man. The common forms in which one or a few teeth are absent, may cause occlusal or cosmetic harm, while severe forms which are relatively rare always require clinical attention to support and maintain the dental function. Observation of tooth agenesis is also important for diagnosis of malformation syndromes. Some external factors may cause developmental defects and agenesis in dentition. However, the role of inheritance in the etiology of tooth agenesis is well established by twin and family studies. Studies on familial tooth agenesis as well as mouse null mutants have also identified several genetic factors. However, these explain syndromic or rare dominant forms of tooth agenesis, whereas the genes and defects responsible for the majority of cases of tooth agenesis, especially the common and less severe forms, are largely unknown. In this study it was shown, that a dominant nonsense mutation in PAX9 was responsible for severe tooth agenesis (oligodontia) in a Finnish family. In a study of tooth agenesis associated with Wolf-Hirschhorn syndrome, it was shown that severe tooth agenesis was present if the causative deletion in 4p spanned the MSX1 locus. It was concluded that severe tooth agenesis was caused by haploinsufficiency of these transcription factors. A summary of the phenotypes associated with known defects in MSX1 and PAX9 showed that, despite similarities, they were significantly different, suggesting that the genes, in addition to known interactions, also have independent roles during the development of human dentition. The original aim of this work was to identify gene defects that underlie the common incisor and premolar hypodontia. After excluding several candidate genes, a genome-wide search was conducted in seven Finnish families in which this phenotype was inherited in an autosomal dominant manner. A promising locus for second premolar agenesis was identified in chromosome 18 in one family and this finding was supported by results from other families. The results also implied the existence of other loci both for second premolar agenesis and for incisor agenesis. On the other hand the results did not lend support for comprehensive involvement of the most obvious candidate genes in the etiology of incisor and premolar hypodontia. Rather, they suggest remarkable genetic heterogeneity of tooth agenesis. The available evidence suggests that quantitative defects during tooth development predispose to a failure to overcome a developmental threshold and to agenesis. The results of the study increase the understanding of the etiology and heredity of tooth agenesis. Further studies may lead to identification of novel genes that affect the development of teeth.

The role of cyclase-associated protein (CAP) in actin dynamics during cell motility and morphogenesis

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.

ORP2 is a sterol receptor that regulates cellular lipid metabolism

ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.

Entry of the membrane-containing bacteriophages into their hosts

Viruses are biological entities able to replicate only within their host cells. Accordingly, entry into the host is a crucial step of the virus life-cycle. The focus of this study was the entry of bacterial membrane-containing viruses into their host cells. In order to reach the site of replication, the cytoplasm of the host, bacterial viruses have to traverse the host cell envelope, which consists of several distinct layers. Lipid membrane is a common feature among animal viruses but not so frequently observed in bacteriophages. There are three families of icosahedral bacteriophages that contain lipid membranes. These viruses belong to families Cystoviridae, Tectiviridae, and Corticoviridae. During the course of this study the entry mechanisms of phages representing the three viral families were investigated. We employed a range of microbiological, biochemical, molecular biology and microscopy techniques that allowed us to dissect phage entry into discrete steps: receptor binding, penetration through the outer membrane, crossing the peptidoglycan layer and interaction with the cytoplasmic membrane. We determined that bacteriophages belonging to the Cystoviridae, Tectiviridae, and Corticoviridae viral families use completely different strategies to penetrate into their host cells.