Investigation of molecular mechanisms regulating biomineralization of pearl oyster Pinctada maxima

Biomineralization is a process encompassing all mineral containing tissues produced within an organism. The most dynamic example of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this remarkable architecture. Subsequently, for the past decade considerable research have been undertaken to identify and characterize the protein components involved in biomineralization. Despite these efforts the general understanding of the process remains ambiguous. This study employs a novel molecular approach to further the elucidation of the shell biomineralization. A microarray platform has been custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from the mantle, an organ involved in shell formation. This microarray has been used as the primary tool for three separate investigations in an effort to associate transcriptional gene expression from P. maxima to the process of shell biomineralization. The first investigation analyzes the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and each analyzed for gene expression with PmaxArray 1.0. Over 2000 ESTs were differentially expressed among the tissue sections, identifying five major expression regions. Three of these regions have been proposed to have shell formation functions belonging to nacre, prismatic calcite and periostracum. The spatial gene expression map was confirmed by in situ hybridization, localizing a subset of ESTs from each expression region to the same mantle area. Comparative sequence analysis of ESTs expressed in the proposed shell formation regions with the BLAST tool, revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell formation genes. The second investigation correlates temporal EST expression during P. maxima larval ontogeny with transitions in shell mineralization during the same period. A timeline documenting the morphologicat microstructural and mineralogical shell characteristics of P. maxima throughout larval ontogeny has been established. Three different shell types were noted based on the physical characters and termed, prodissoconch I, prodissoconch 11 and dissoconch. PmaxArray 1.0 analyzed ESTs expression of animals throughout the larval development of P. maxima, noting up-regulation of 359 ESTs in association with the shell transitions from prodissoconch 1 to prodissoconch 11 to dissoconch. Comparative sequence analysis of these ESTs indicates a number of the transcripts are novel as well as showing significant sequence similarities between ESTs and known shell matrix associated genes and proteins. These ESTs are discussed in relation to the shell characters associated with their temporal expression. The third investigation uses PmaxArray 1.0 to analyze gene expression in the mantle tissue of P. maxima specimens exposed to sub-lethal concentrations of a shell-deforming toxin, tributyltin (TBT). The shell specific effects of TBT are used in this investigation to interpret differential expression of ESTs with respect to shell formation functions. A lethal and sublethal TBT concentration range was established for P. maxima, noting a concentration of 50 ng L- 1 TBT as sub-lethal over a 21 day period. Mantle tissue from P. maxima animals treated with 50 ng L- 1 TBT was assessed for differential EST expression with untreated control animals. A total of 102 ESTs were identified as differentially expressed in association with TBT exposure, comparative sequence identities included an up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of transcripts encoding novel peptides were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This thesis has used a microarray platform to analyze gene expression in spatial, temporal and toxicity investigations, revealing the involvement of numerous gene transcripts in specific shell formation functions. Investigation of thousands of transcripts simultaneously has provided a holistic interpretation of the organic components regulating shell biomineralization.

Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer.

Background The purpose of this study was to identify candidate metastasis suppressor genes from a mouse allograft model of prostate cancer (NE-10). This allograft model originally developed metastases by twelve weeks after implantation in male athymic nude mice, but lost the ability to metastasize after a number of in vivo passages. We performed high resolution array comparative genomic hybridization on the metastasizing and non-metastasizing allografts to identify chromosome imbalances that differed between the two groups of tumors. Results This analysis uncovered a deletion on chromosome 2 that differed between the metastasizing and non-metastasizing tumors. Bioinformatics filters were employed to mine this region of the genome for candidate metastasis suppressor genes. Of the 146 known genes that reside within the region of interest on mouse chromosome 2, four candidate metastasis suppressor genes (Slc27a2, Mall, Snrpb, and Rassf2) were identified. Quantitative expression analysis confirmed decreased expression of these genes in the metastasizing compared to non-metastasizing tumors. Conclusion This study presents combined genomics and bioinformatics approaches for identifying potential metastasis suppressor genes. The genes identified here are candidates for further studies to determine their functional role in inhibiting metastases in the NE-10 allograft model and human prostate cancer.

Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

Molecular approaches to diagnosis and management of ovarian cancer

The recent advances in the understanding of the pathogenesis of ovarian cancer have been helpful in addressing issues in diagnosis, prognosis and management. The study of ovarian tumours by novel techniques such as immunohistochemistry, fluorescent in situ hybridisation, comparative genomic hybridisation, polymerase chain reaction and new tumour markers have aided the evaluation and application of new concepts into clinical practice. The correlation of novel surrogate tumour specific features with response to treatment and outcome in patients has defined prognostic factors which may allow the future design of tailored therapy based on a molecular profile of the tumour. These have also been used to design new approaches to therapy such as antibody targeting and gene therapy. The delineation of roles of c-erbB2, c-fms and other novel receptor kinases in the pathogenesis of ovarian cancer has led initially to the development of anti-c-erbB2 monoclonal antibody therapy. The discovery of BRCA1 and BRCA2 genes will have an impact in the diagnosis and the prevention of familial ovarian cancer. The important role played by recessive genes such as p53 in cancer has raised the possibility of restoration of gene function by gene therapy. Although the pathological diagnosis of ovarian cancer is still confirmed principally on morphological features, addition of newer investigations will increasingly be useful in addressing difficult diagnostic problems. The increasingly rapid pace of discovery of genes important in disease, makes it imperative that the evaluation of their contribution in the pathogenesis of ovarian cancer is undertaken swiftly, thus improving the overall management of patients and their outcome.

Molecular biomarkers in non-small-cell lung cancer : a retrospective analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 FLEX study showed that the addition of cetuximab to cisplatin and vinorelbine significantly improved overall survival, compared with cisplatin and vinorelbine alone, in the first-line treatment of EGFR-expressing, advanced non-small-cell lung cancer (NSCLC). We investigated whether candidate biomarkers were predictive for the efficacy of chemotherapy plus cetuximab in this setting. Methods: Genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour tissue of patients enrolled in the FLEX study was screened for KRAS codon 12 and 13 and EGFR kinase domain mutations with PCR-based assays. In FFPE tissue sections, EGFR copy number was assessed by dual-colour fluorescence in-situ hybridisation and PTEN expression by immunohistochemistry. Treatment outcome was investigated according to biomarker status in all available samples from patients in the intention-to-treat population. The primary endpoint in the FLEX study was overall survival. The FLEX study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT00148798. Findings: KRAS mutations were detected in 75 of 395 (19%) tumours and activating EGFR mutations in 64 of 436 (15%). EGFR copy number was scored as increased in 102 of 279 (37%) tumours and PTEN expression as negative in 107 of 303 (35%). Comparisons of treatment outcome between the two groups (chemotherapy plus cetuximab vs chemotherapy alone) according to biomarker status provided no indication that these biomarkers were of predictive value. Activating EGFR mutations were identified as indicators of good prognosis, with patients in both treatment groups whose tumours carried such mutations having improved survival compared with those whose tumours did not (chemotherapy plus cetuximab: median 17·5 months [95% CI 11·7-23·4] vs 8·5 months [7·1-10·8], hazard ratio [HR] 0·52 [0·32-0·84], p=0·0063; chemotherapy alone: 23·8 months [15·2-not reached] vs 10·0 months [8·7-11·0], HR 0·35 [0·21-0·59], p<0·0001). Expression of PTEN seemed to be a potential indicator of good prognosis, with patients whose tumours expressed PTEN having improved survival compared with those whose tumours did not, although this finding was not significant (chemotherapy plus cetuximab: median 11·4 months [8·6-13·6] vs 6·8 months [5·9-12·7], HR 0·80 [0·55-1·16], p=0·24; chemotherapy alone: 11·0 months [9·2-12·6] vs 9·3 months [7·6-11·9], HR 0·77 [0·54-1·10], p=0·16). Interpretation: The efficacy of chemotherapy plus cetuximab in the first-line treatment of advanced NSCLC seems to be independent of each of the biomarkers assessed. Funding: Merck KGaA. © 2011 Elsevier Ltd.

Molecular characterization of "Candidatus Similichlamydia latridicola" gen. nov., sp. nov. (Chlamydiales: "Candidatus Parilichlamydiaceae"), a novel Chlamydia-like epitheliocystis agent in the striped trumpeter, Latris lineata (Forster)

Histological analysis of gill samples taken from individuals of Latris lineata reared in aquaculture in Tasmania, Australia, and those sampled from the wild revealed the presence of epitheliocystis-like basophilic inclusions. Subsequent morphological, in situ hybridization, and molecular analyses were performed to confirm the presence of this disease and discovered a Chlamydia-like organism associated with this condition, and the criteria set by Fredericks and Relman's postulates were used to establish disease causation. Three distinct 16S rRNA genotypes were sequenced from 16 fish, and phylogenetic analyses of the nearly full-length 16S rRNA sequences generated for this bacterial agent indicated that they were nearly identical novel members of the order Chlamydiales. This new taxon formed a well-supported clade with "Candidatus Parilichlamydia carangidicola" from the yellowtail kingfish (Seriola lalandi). On the basis of sequence divergence over the 16S rRNA region relative to all other members of the order Chlamydiales, a new genus and species are proposed here for the Chlamydia-like bacterium from L. lineata, i.e., "Candidatus Similichlamydia latridicola" gen. nov., sp. nov.

Genetic evidence for natural hybridization between Red Snow Crab (Chionoecetes japonicus) and Snow Crab (Chionoecetes opilio) in Korea

Both red snow crab (Chionoecetes japonicus Rathbun, 1932) and snow crab (Chionoecetes opilio Fabricius, 1788) are commercially important species in Korea. The geographical ranges of the two species overlap in the East Sea, where both species are fished commercially. Morphological identification of the two species and putative hybrids can be difficult because of their overlapping morphological characteristics. The presence of putative hybrids can affect the total allowable catch (TAC) of C. japonicus and C. opilio, and causes problems managing C. japonicus and C. opilio wild resources. To date, however, no natural hybridization has been reported between C. japonicus and C. opilio, despite their overlapping distributions along the coast of the East Sea. In this study, the internal transcribed spacer (ITS) region of major ribosomal RNA genes from the nuclear genome and the cytochrome oxidase I (CO I) gene from the mitochondrial genome were sequenced to determine whether natural hybridization occurs between the two species. Our results revealed that all putative hybrids identified using morphological traits had two distinct types of ITS sequences corresponding to those of both parental species. Mitochondrial CO I gene sequencing showed that all putative hybrids had sequences identical to C. japonicus. A genotyping assay based on single nucleotide polymorphisms in the ITS1 region and the CO I gene produced the most efficient and accurate identification of all hybrid individuals. Molecular data clearly demonstrate that natural hybridization does occur between C. japonicus and C. opilio, but only with C. japonicus as the maternal parent.

Ecology of uncultivated oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Array Comparative Genomic Hybridization in Sarcomas

Over the past years, much research on sarcomas based on low-resolution cytogenetic and molecular cytogenetic methods has been published, leading to the identification of genetic abnormalities partially underlying the tumourigenesis. Continued progress in the identification of genetic events such as copy number aberrations relies upon adapting the rapidly evolving high-resolution microarray technology, which will eventually provide novel insights into sarcoma biology, and targets for both diagnostics and drug development. The aim of this Thesis was to characterize DNA copy number changes that are involved in the pathogenesis of soft tissue leiomyosarcoma (LMS), dermatofibrosarcoma protuberans (DFSP), osteosarcoma (OS), malignant fibrous histiocytoma (MFH), and uterine leiomyosarcoma (ULMS) by applying fine resolution array comparative genomic hybridization (aCGH) technology. Both low- and high-grade LMS tumours showed distinct copy number patterns, in addition to sharing two minimal common regions of gains and losses. Small aberrations were detected by aCGH, which were beyond the resolution of chromosomal comparative genomic hybridization (cCGH). DFSP tumours analysed by aCGH showed gains in 17q, 22q, and 21 additional gained regions, but only one region (22q) with copy number loss. Recurrent amplicons identified in OS by aCGH were 12q11-q15, 8q, 6p12-p21, and 17p. Amplicons 12q and 17p were further characterized in detail. The amplicon at 17p was characterized by aCGH in low- and high-grade LMS, OS, and MFH. In all but one case this amplicon, with minimal common regions of gains at 17p11-p12, started with the distal loss of 17p13-pter. OS and high-grade LMS were grouped together as they showed a complex pattern of copy number gains and amplifications at 17p, whereas MFH and low-grade LMS showed a continuous pattern of copy number gains and amplification at 17p. In addition to the commonly gained and lost regions identified in ULMS by aCGH, various biological processes affected by these copy number changes were also indicated by pathway analysis. The three novel findings obtained in this work were: characterization of amplicon 17p in low- and high-grade LMS and MFH, profiles of DNA copy number changes in LMS, and detection of various pathways affected by copy number changes in ULMS. These studies have not been undertaken previously by aCGH technology, thus this Thesis adds new information regarding DNA copy number changes in sarcomas. In conclusion, the aCGH technique used in this Thesis has provided new insights into the genetics of sarcomas by detecting the precise regions affected by copy number changes and some potential candidate target genes within those regions, which had not been uncovered by previously applied low resolution techniques.

Molecular Genetic Background of Tumours in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a hereditary tumour predisposition syndrome. Its phenotype includes benign cutaneous and uterine leiomyomas (CLM, ULM) with high penetrance and rarer renal cell cancer (RCC), most commonly of papillary type 2 subtype. Over 130 HLRCC families have been identified world-wide but the RCC phenotype seems to concentrate in families from Finland and North America for unknown reasons. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. FH encodes the enzyme fumarase from mitochondrial citric acid cycle. Fumarase enzyme activity or type or site of the FH mutation are unassociated with disease phenotype. The strongest evidence for tumourigenesis mechanism in HLRCC supports a hypoxia inducible factor driven process called pseudohypoxia resulting from accumulation of the fumarase substrate fumarate. In this study, to assess the importance of gene- or exon-level deletions or amplifications of FH in patients with HLRCC-associated phenotypes, multiplex ligation-dependent probe amplification (MLPA) method was used. One novel FH mutation, deletion of exon 1, was found in a Swedish male patient with an evident HLRCC phenotype with CLM, RCC, and a family history of ULM and RCC. Six other patients with CLM and 12 patients with only RCC or uterine leiomyosarcoma (ULMS) remained FH mutation-negative. These results suggest that copy number aberrations of FH or its exons are an infrequent cause of HLRCC and that only co-occurrence of benign tumour types justifies FH-mutation screening in RCC or ULMS patients. Determination of the genomic profile of 11 HLRCC-associated RCCs from Finnish patients was performed by array comparative genomic hybridization. The most common copy number aberrations were gains of 2, 7, and 17 and losses of 13q12.3-q21.1, 14, 18, and X. When compared to aberrations of sporadic papillary RCCs, HLRCC-associated RCCs harboured a distinct DNA copy number profile and lacked many of the changes characterizing the sporadic RCCs. The findings suggest a divergent molecular pathway for tumourigenesis of papillary RCCs in HLRCC. In order to find a genetic modifier of RCC risk in HLRCC, genome-wide linkage and identical by descent (IBD) analysis studies were performed in Finnish HLRCC families with microsatellite marker mapping and SNP-array platforms. The linkage analysis identified only one locus of interest, the FH gene locus in 1q43, but no mutations were found in the genes of the region. IBD analysis yielded no convincing haplotypes shared by RCC patients. Although these results do not exclude the existence of a genetic modifier for RCC risk in HLRCC, they emphasize the role of FH mutations in the malignant tumourigenesis of HLRCC. To study the benign tumours in HLRCC, genome-wide DNA copy number and gene expression profiles of sporadic and HLRCC ULMs were defined with modern SNP- and gene-expression array platforms. The gene expression array suggests novel genes involved in FH-deficient ULM tumourigenesis and novel genes with putative roles in propagation of sporadic ULM. Both the gene expression and copy number profiles of HLRCC ULMs differed from those of sporadic ULMs indicating distinct molecular basis of the FH-deficient HLRCC tumours.

Molecular genetics and evolution of Puumala hantavirus

Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome. Finland has the highest documented incidence of NE with around 1000 cases diagnosed annually. PUUV is also found in other Scandinavian countries, Central Europe and the European part of Russia. PUUV belongs to the genus Hantavirus in the family Bunyaviridae. Hantaviruses are rodent-borne viruses each carried by a specific host that is persistently and asymptomatically infected by the virus. PUUV is carried by the bank voles (Myodes glareolus, previously known as Clethrionomys glareolus). Hantaviruses have co-evolved with their carrier rodents for millions of years and these host animals are the evolutionary scene of hantaviruses. In this study, PUUV sequences were recovered from bank voles captured in Denmark and Russian Karelia to study the evolution of PUUV in Scandinavia. Phylogenetic analysis of these strains showed a geographical clustering of genetic variants following the presumable migration pattern of bank voles during the recolonization of Scandinavia after the last ice age approximately 10 000 years ago. The currently known PUUV genome sequences were subjected to in-depth phylogenetic analyses and the results showed that genetic drift seems to be the major mechanism of PUUV evolution. In general, PUUV seems to evolve quite slowly following a molecular clock. We also found evidence for recombination in the evolution of some genetic lineages of PUUV. Viral microevolution was studied in controlled virus transmission in colonized bank voles and changes in quasispecies dynamics were recorded as the virus was transmitted from one animal to another. We witnessed PUUV evolution in vivo, as one synonymous mutation became repeatedly fixed in the viral genome during the experiment. The detailed knowledge on the PUUV diversity was used to establish new sensitive and specific detection methods for this virus. Direct viral invasion of the hypophysis was demonstrated for the first time in a lethal case of NE. PUUV detection was done by immunohistochemistry, in situ hybridization and RT-nested-PCR of the autopsy tissue samples.

Structural studies of analgesics and their interactions. III. The crystal and molecular structure of amidopyrine

Amidopyrine (1-phenyl-2,3-dimethyl-4-dimethylaminopyrazolone), C13HzvN30, a dimethylamino derivative of antipyrine and an important analgesic and antipyretic agent, crystallizes in the triclinic space group P1 with four molecules in a unit cell of dimensions a= 7.458 (5), b = 10.744 (5), c= 17.486 (15)/~,, e=98.6 (2),/~= 85.6 (3), y= 108-6 (2) . The structure was solved by direct methods and refined to an R value of 0.055 for 3706 photographically observed reflexions. The dimensions of the two crystallographically independent molecules are very nearly the same. The pyrazolone moiety in the molecule has dimensions comparable to those in antipyrine. Unlike antipyrine, the molecular dimensions of amidopyrine in the free state (the present structure) are close to those found in some of its hydrogenbonded complexes. Thus it appears that the presence of the dimethylamino group makes the molecule more resistant to changes in its dimensions resulting from molecular association. An attempt has also been made to correlate the polar nature of the pyrazolone moiety and the hybridization state of the hetero nitrogen atoms in antipyrine, amidopyrine and their complexes.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Molecular Alterations in Asbestos-Related Lung Cancer

Background: Asbestos is a well known cancer-causing mineral fibre, which has a synergistic effect on lung cancer risk in combination with tobacco smoking. Several in vitro and in vivo experiments have demonstrated that asbestos can evoke chromosomal damage and cause alterations as well as gene expression changes. Lung tumours, in general, have very complex karyotypes with several recurrently gained and lost chromosomal regions and this has made it difficult to identify specific molecular changes related primarily to asbestos exposure. The main aim of these studies has been to characterize asbestos-related lung cancer at a molecular level. Methods: Samples from asbestos-exposed and non-exposed lung cancer patients were studied using array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to detect copy number alterations (CNA) as well as microsatellite analysis to detect allelic imbalance (AI). In addition, asbestos-exposed cell lines were studied using gene expression microarrays. Results: Eighteen chromosomal regions showing differential copy number in the lung tumours of asbestos-exposed patients compared to those of non-exposed patients were identified. The most significant differences were detected at 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 and 19p13.1-p13.3 (p<0.005). The alterations at 2p and 9q were validated and characterized in detail using AI and FISH analysis in a larger study population. Furthermore, in vitro studies were performed to examine the early gene expression changes induced by asbestos in three different lung cell lines. The results revealed specific asbestos-associated gene expression profiles and biological processes as well as chromosomal regions enriched with genes believed to contribute to the common asbestos-related responses in the cell lines. Interestingly, the most significant region enriched with asbestos-response genes was identified at 2p22, close to the previously identified region showing asbestos-related CNA in lung tumours. Additionally, in this thesis, the dysregulated biological processes (Gene Ontology terms) detected in the cell line experiment were compared to dysregulated processes identified in patient samples in a later study (Ruosaari et al., 2008a). Commonly affected processes such as those related to protein ubiquitination, ion transport and surprisingly sensory perception of smell were identified. Conclusions: The identification of specific CNA and dysregulated biological processes shed some light on the underlying genes acting as mediators in asbestos-related lung carcinogenesis. It is postulated that the combination of several asbestos-specific molecular alterations could be used to develop a diagnostic method for the identification of asbestos-related lung cancer.