Molecular Genetics of Schizophrenia and Related Intermediate Phenotypes in a Founder Population

Schizophrenia, affecting about 1% of population worldwide, is a severe mental disorder characterized by positive and negative symptoms, such as psychosis and anhedonia, as well as cognitive deficits. At present, schizophrenia is considered a complex disorder of neurodevelopmental origin with both genetic and environmental factors contributing to its onset. Although a number of candidate genes for schizophrenia have been highlighted, only very few schizophrenia patients are likely to share identical genetic liability. This study is based on the nation-wide schizophrenia family sample of the National Institute for Health and Welfare, and represents one of the largest and most well-characterized familial series in the world. In the first part of this study, we investigated the roles of the DTNBP1, NRG1, and AKT1 genes in the background of schizophrenia in Finland. Although these genes are associated with schizophrenia liability in several populations, any significant association with clinical diagnostic information of schizophrenia remained absent in our sample of 441 schizophrenia families. In the second part of this study, we first replicated schizophrenia linkage on the long arm of chromosome 7 in 352 schizophrenia families. In the following association analysis, we utilized additional clinical disorder features and intermediate phenotypes – endophenotypes - in addition to diagnostic information from altogether 290 neuropsychologically assessed schizophrenia families. An intragenic short tandem repeat allele of the regional RELN gene, supposed to play a role in the background of several neurodevelopmental disorders, showed significant association with poorer cognitive functioning and more severe schizophrenia symptoms. Additionally, this risk allele was significantly more prevalent among the individuals affected with schizophrenia spectrum disorders. We have previously identified linkage of schizophrenia and its cognitive endophenotypes on the long arms of chromosomes 2, 4, and 5. In the last part of this study, we selected altogether 104 functionally relevant candidate genes from the linked regions. We detected several promising associations, of which especially interesting are the ERBB4 gene, showing association with the severity of schizophrenia symptoms and impairments in traits related to verbal abilities, and the GRIA1 gene, showing association with the severity of schizophrenia symptoms. Our results extend the previous evidence that the genetic risk for schizophrenia is at least partially mediated via the effects of the candidate genes and their combinations on relevant brain systems, resulting in alterations in different disorder domains, such as the cognitive deficits.

Molecular basis of the kidney filtration barrier : Role of the nephrin protein complex

The kidney filtration barrier consists of fenestrated endothelial cell layer, glomerular basement membrane and slit diaphragm (SD), the specialized junction between glomerular viscelar epithelial cells (podocytes). Podocyte injury is associated with the development of proteinuria, and if not reversed the injury will lead to permanent deterioration of the glomerular filter. The early events are characterized by disruption of the integrity of the SD, but the molecular pathways involved are not fully understood. Congenital nephrotic syndrome of the Finnish type (CNF) is caused by mutations in NPHS1, the gene encoding the SD protein nephrin. Lack of nephrin results in loss of the SD and massive proteinuria beginning before birth. Furthermore, nephrin expression is decreased in acquired human kidney diseases including diabetic nephropathy. This highlights the importance of nephrin and consequently SD in regulating the kidney filtration function. However, the precise molecular mechanism of how nephrin is involved in the formation of the SD is unknown. This thesis work aimed at clarifying the role of nephrin and its interaction partners in the formation of the SD. The purpose was to identify novel proteins that associate with nephrin in order to define the essential molecular complex required for the establishment of the SD. The aim was also to decipher the role of novel nephrin interacting proteins in podocytes. Nephrin binds to nephrin-like proteins Neph1 and Neph2, and to adherens junction protein P-cadherin. These interactions have been suggested to play a role in the formation of the SD. In this thesis work, we identified densin as a novel interaction partner for nephrin. Densin was localized to the SD and it was shown to bind to adherens junction protein beta-catenin. Furthermore, densin was shown to behave in a similar fashion as adherens junction proteins in cell-cell contacts. These results indicate that densin may play a role in cell adhesion and, therefore, may contribute to the formation of the SD together with nephrin and adherens junction proteins. Nephrin was also shown to bind to Neph3, which has been previously localized to the SD. Neph3 and Neph1 were shown to induce cell adhesion alone, whereas nephrin needed to trans-interact with Neph1 or Neph3 from the opposite cell surface in order to make cell-cell contacts. This was associated with the decreased tyrosine phosphorylation of nephrin. These data extend the current knowledge of the molecular composition of the nephrin protein complex at the SD and also provide novel insights of how the SD may be formed. This thesis work also showed that densin was up-regulated in the podocytes of CNF patients. Neph3 was up-regulated in nephrin deficient mouse kidneys, which share similar podocyte alterations and lack of the SD as observed in CNF patients podocytes. These data suggest that densin and Neph3 may have a role in the formation of morphological alterations in podocytes detected in CNF patients. Furthermore, this thesis work showed that deletion of beta-catenin specifically from adult mouse podocytes protected the mice from the development of adriamycin-induced podocyte injury and proteinuria compared to wild-type mice. These results show that beta-catenin play a role in the adriamycin induced podocyte injury. Podocyte injury is a hallmark in many kidney diseases and the changes observed in the podocytes of CNF patient share characteristics with injured podocytes observed in chronic kidney diseases. Therefore, the results obtained in this thesis work suggest that densin, Neph3 and beta-catenin participate in the molecular pathways which result in morphological alterations commonly detected in injured podocytes in kidney diseases.

Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Genomic microarrays in chromosomal analysis of leukemia

Chromosomal alterations in leukemia have been shown to have prognostic and predictive significance and are also important minimal residual disease (MRD) markers in the follow-up of leukemia patients. Although specific oncogenes and tumor suppressors have been discovered in some of the chromosomal alterations, the role and target genes of many alterations in leukemia remain unknown. In addition, a number of leukemia patients have a normal karyotype by standard cytogenetics, but have variability in clinical course and are often molecularly heterogeneous. Cytogenetic methods traditionally used in leukemia analysis and diagnostics; G-banding, various fluorescence in situ hybridization (FISH) techniques, and chromosomal comparative genomic hybridization (cCGH), have enormously increased knowledge about the leukemia genome, but have limitations in resolution or in genomic coverage. In the last decade, the development of microarray comparative genomic hybridization (array-CGH, aCGH) for DNA copy number analysis and the SNP microarray (SNP-array) method for simultaneous copy number and loss of heterozygosity (LOH) analysis has enabled investigation of chromosomal and gene alterations genome-wide with high resolution and high throughput. In these studies, genetic alterations were analyzed in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). The aim was to screen and characterize genomic alterations that could play role in leukemia pathogenesis by using aCGH and SNP-arrays. One of the most important goals was to screen cryptic alterations in karyotypically normal leukemia patients. In addition, chromosomal changes were evaluated to narrow the target regions, to find new markers, and to obtain tumor suppressor and oncogene candidates. The work presented here shows the capability of aCGH to detect submicroscopic copy number alterations in leukemia, with information about breakpoints and genes involved in the alterations, and that genome-wide microarray analyses with aCGH and SNP-array are advantageous methods in the research and diagnosis of leukemia. The most important findings were the cryptic changes detected with aCGH in karyotypically normal AML and CLL, characterization of amplified genes in 11q marker chromosomes, detection of deletion-based mechanisms of MLL-ARHGEF12 fusion gene formation, and detection of LOH without copy number alteration in karyotypically normal AML. These alterations harbor candidate oncogenes and tumor suppressors for further studies.

The molecular basis of Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia due to cerebellar cortical atrophy, infantile- or childhood-onset bilateral cataracts, progressive myopathy, and mild to severe mental retardation. Additional features include hypergonadotropic hypogonadism, various skeletal abnormalities, short stature, and strabismus. The neuroradiologic hallmarks are hypoplasia of both the vermis and cerebellar hemispheres. The histopathologic findings include severe cerebellar atrophy and loss of Purkinje and granule cells. The common pathologic findings in muscle biopsy are variation in muscle fiber size, atrophic fibers, fatty replacement, and rimmed vacuole formation. The presence of marked cerebellar atrophy with myopathy distinguishes MSS from another rare syndrome, the congenital cataracts, facial dysmorphism, and neuropathy syndrome (CCFDN). Previously, work by others had resulted in the identification of an MSS locus on chromosome 5q31. A subtype of MSS with myoglobinuria and neuropathy had been linked to the CCFDN locus on chromosome 18qter, at which mutations in the CTDP1 gene had been identified. We confirmed linkage to the previously identified locus on chromosome 5q31 in two Finnish families with eight affected individuals, reduced the critical region by fine-mapping, and identified SIL1 as a gene underlying MSS. We found a common homozygous founder mutation in all Finnish patients. The same mutation was also present in patient samples from Norway and Sweden. Altogether, we identified eight mutations in SIL1, including nonsense, frameshift, splice site alterations, and one missense mutation. SIL1 encodes a nucleotide exchange factor for the endoplasmic reticulum (ER) resident heat-shock protein 70 chaperone GRP78. GRP78 functions in protein synthesis and quality control of the newly synthesized polypeptides. It senses and responds to stressful cellular conditions. We showed that in mice, SIL1 and GRP78 show highly similar spatial and temporal tissue expression in developing and mature brain, eye, and muscle. Studying endogenous proteins in mouse primary hippocampal neurons, we found that SIL1 and GRP78 colocalize and that SIL1 localizes to the ER. We studied the subcellular localization of two mutant proteins, a missense mutant found in two patients and an artificial mutant lacking the ER retrieval signal, and found that both mutant proteins formed aggregates within the ER. Well in line with our findings and the clinical features of MSS, recent work by Zhao et al. showed that a truncation of SIL1 causes ataxia and cerebellar Purkinje cell loss in the naturally occurring woozy mutant mouse. Prior to Purkinje cell degeneration, the unfolded protein response is initiated and abnormal protein accumulations are present. MSS thus joins the group of protein misfolding and accumulation diseases. These findings highlight the importance of SIL1 and the role of the ER in neuronal function and survival. The results presented in this thesis provide tools for the molecular genetic diagnostics of MSS and give a basis for future studies on the molecular pathogenesis of MSS. Understanding the mechanisms behind this pleiotropic syndrome may provide insights into more common forms of ataxia, myopathy, and neurodegeneration.

Genetic basis of hereditary colorectal cancers: Hereditary nonpolyposis colorectal cancer and Familial adenomatous polyposis

Hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP) are characterized by a high risk and early onset of colorectal cancer (CRC). HNPCC is due to a germline mutation in one of the following MMR genes: MLH1, MSH2, MSH6 and PMS2. A majority of FAP and attenuated FAP (AFAP) cases are due to germline mutations of APC, causing the development of multiple colorectal polyps. To date, over 450 MMR gene mutations and over 800 APC mutations have been identified. Most of these mutations lead to a truncated protein, easily detected by conventional mutation detection methods. However, in about 30% of HNPCC and FAP, and about 90% of AFAP families, mutations remain unknown. We aimed to clarify the genetic basis and genotype-phenotype correlation of mutation negative HNPCC and FAP/AFAP families by advanced mutation detection methods designed to detect large genomic rearrangements, mRNA and protein expression alterations, promoter mutations, phenotype linked haplotypes, and tumoral loss of heterozygosity. We also aimed to estimate the frequency of HNPCC in Uruguayan CRC patients. Our expression based analysis of mutation negative HNPCC divided these families into two categories: 1) 42% of families linked to the MMR genes with a phenotype resembling that of mutation positive, and 2) 58% of families likely to be associated with other susceptibility genes. Unbalanced mRNA expression of MLH1 was observed in two families. Further studies revealed that a MLH1 nonsense mutation, R100X was associated with aberrant splicing of exons not related to the mutation and an MLH1 deletion (AGAA) at nucleotide 210 was associated with multiple exon skipping, without an overall increase in the frequency of splice events. APC mutation negative FAP/AFAP families were divided into four groups according to the genetic basis of their predisposition. Four (14%) families displayed a constitutional deletion of APC with profuse polyposis, early age of onset and frequent extracolonic manifestations. Aberrant mRNA expression of one allele was observed in seven (24%) families with later onset and less frequent extracolonic manifestations. In 15 (52%) families the involvement of APC could neither be confirmed nor excluded. In three (10%) of the families a germline mutation was detected in genes other than APC: AXIN2 in one family, and MYH in two families. The families with undefined genetic basis and especially those with AXIN2 or MYH mutations frequently displayed AFAP or atypical polyposis. Of the Uruguayan CRC patients, 2.6% (12/461) fulfilled the diagnostic criteria for HNPCC and 5.6% (26/461) were associated with increased risk of cancer. Unexpectedly low frequency of molecularly defined HNPCC cases may suggest a different genetic profile in the Uruguayan population and the involvement of novel susceptibility genes. Accurate genetic and clinical characterization of families with hereditary colorectal cancers, and the definition of the genetic basis of "mutation negative" families in particular, facilitate proper clinical management of such families.

The Role of Genes on Chromosome Locus 4q12 in Human Central Nervous System Tumors

Human central nervous system (CNS) tumors are a heterogeneous group of tumors occurring in brain, brainstem and spinal cord. Malignant gliomas (astrocytic and oligodendroglial tumors), which arise from the neuroepithelial cells are the most common CNS neoplasms in human. Malignant gliomas are highly aggressive and invasive tumors, and have a very poor prognosis. The development and progression of gliomas involve a stepwise accumulation of genetic alterations that generally affect either signal transduction pathways activated by receptor tyrosine kinases (RTKs), or cell cycle arrest pathways. Constitutive activation or deregulated signaling by RTKs is caused by gene amplification, overexpression or mutations. The aberrant RTK signaling results in turn in the activation of several downstream pathways, which ultimately lead to malignant transformation and tumor proliferation. Many genetic abnormalities implicated in nervous system tumors involve the genes located at the chromosomal region 4q12. This locus harbors the receptor tyrosine kinases KIT, PDGFRA and VEGFR2, and other genes (REST, LNX1) with neural function. Gene amplification and protein expression of KIT, PDGFRA, and VEGFR2 was studied using clinical tumor material. REST and LNX1, as well as NUMBL, the interaction partner of LNX1, were studied for their gene mutations and amplifications. In our studies, amplification of LNX1 was associated with KIT and PDGFRA amplification in glioblastomas, and coamplification of KIT, PDGFRA and VEGFR2 was detected in medulloblastomas and CNS primitive neuroectodermal tumors. PDGFRA amplification was also correlated with poor overall survival. Coamplification of KIT, PDGFRA and VEGFR2 was observed in a subset of human astrocytic and oligodendroglial tumors. We suggest that genes at 4q12 could be a part of a larger amplified region, which is deregulated in gliomas, and could be used as a prognostic marker of tumorigenic process. The signaling pathways activated due to gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment. This study also includes the characterization of KIT overexpressing astrocytes, analyzed by various in vitro functional assays. Our results show that overexpression of KIT in mouse astrocytes promotes cell proliferation and anchorage-independent growth, as well as phenotypic changes in the cells. Furthermore, the increased proliferation is partly inhibited by imatinib, a small molecule inhibitor of KIT. These results suggest that KIT may play a role in astrocyte growth regulation, and might have an oncogenic role in brain tumorigenesis. Elucidation of the altered signaling pathways due to specific gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment.

Molecular classification of familial colorectal and endometrial carcinoma

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common known clearly hereditary cause of colorectal and endometrial cancer (CRC and EC). Dominantly inherited mutations in one of the known mismatch repair (MMR) genes predispose to HNPCC. Defective MMR leads to an accumulation of mutations especially in repeat tracts, presenting microsatellite instability. HNPCC is clinically a very heterogeneous disease. The age at onset varies and the target tissue may vary. In addition, families that fulfill the diagnostic criteria for HNPCC but fail to show any predisposing mutation in MMR genes exist. Our aim was to evaluate the genetic background of familial CRC and EC. We performed comprehensive molecular and DNA copy number analyses of CRCs fulfilling the diagnostic criteria for HNPCC. We studied the role of five pathways (MMR, Wnt, p53, CIN, PI3K/AKT) and divided the tumors into two groups, one with MMR gene germline mutations and the other without. We observed that MMR proficient familial CRC consist of two molecularly distinct groups that differ from MMR deficient tumors. Group A shows paucity of common molecular and chromosomal alterations characteristic of colorectal carcinogenesis. Group B shows molecular features similar to classical microsatellite stable tumors with gross chromosomal alterations. Our finding of a unique tumor profile in group A suggests the involvement of novel predisposing genes and pathways in colorectal cancer cohorts not linked to MMR gene defects. We investigated the genetic background of familial ECs. Among 22 families with clustering of EC, two (9%) were due to MMR gene germline mutations. The remaining familial site-specific ECs are largely comparable with HNPCC associated ECs, the main difference between these groups being MMR proficiency vs. deficiency. We studied the role of PI3K/AKT pathway in familial ECs as well and observed that PIK3CA amplifications are characteristic of familial site-specific EC without MMR gene germline mutations. Most of the high-level amplifications occurred in tumors with stable microsatellites, suggesting that these tumors are more likely associated with chromosomal rather than microsatellite instability and MMR defect. The existence of site-specific endometrial carcinoma as a separate entity remains equivocal until predisposing genes are identified. It is possible that no single highly penetrant gene for this proposed syndrome exists, it may, for example be due to a combination of multiple low penetrance genes. Despite advances in deciphering the molecular genetic background of HNPCC, it is poorly understood why certain organs are more susceptible than others to cancer development. We found that important determinants of the HNPCC tumor spectrum are, in addition to different predisposing germline mutations, organ specific target genes and different instability profiles, loss of heterozygosity at MLH1 locus, and MLH1 promoter methylation. This study provided more precise molecular classification of families with CRC and EC. Our observations on familial CRC and EC are likely to have broader significance that extends to sporadic CRC and EC as well.

Genetic Mechanisms of Tumourigenesis in von Hippel-Lindau-Associated Tumours with the Emphasis on Capillary Hemangioblastoma

The von Hippel-lindau (VHL) disease is a dominantly inherited neoplastic disorder which predisposes patients to multiple tumours including capillary haemangioblastomas (CHBs), pheochromocytomas (PCCs), renal cell carcinomas (RCCs). CHBs are the most common manifestations of VHL disease, occurring sporadically or as a manifestation of VHL disease. Inactivation of the VHL gene at 3p25-26 is believed to cause both familial and sporadic VHL-associated tumours and germ-line mutation of the VHL gene have been detected in 100% of the CHBs studied. However, a limited number of sporadic CHBs, PCCs display VHL inactivation. Other molecular alterations involved in tumourigenesis of sporadic CHBs, PCCs remain largely unknown. The purpose of the present work was to search for genetic alterations, or other mechanisms of inactivation, in addition to the VHL gene, that may be important in the development of VHL-associated tumours. Though less satisfactory than cure, prevention and early detection are the most promising and feasible means reducing cancer morbidity and mortality. This work is based on the view that increasing knowledge about the molecular events underlying tumour development will eventually aid in early detection and lead to improved treatment. We evaluated a large set of VHL-associated patients, searched for a clinical and radiologic signs of the disease. We succesfully performed a germ-line mutation analysis and characterised three patient groups, VHL, suspect VHL and sporadic, a germ-line mutation analysis revealed a 50% mutation rate only in the VHL groups, no sporadic or suspect cases displayed any mutation. We also utilized comparative genomic hybridization (CGH) to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed (27%) DNA copy number losses. The most common finding was loss of chromosomal arm 6q, seen in (23%) cases, No differences were noted between VHL-associated and sporadic tumours. Furthermore a loss of heterozygosity (LOH) study on chromosome 3p and 6q was done with the purpose to determine allele losses not observable by CGH, and to uncover the location of putative tumour suppressor genes important in CHB and PCC tumourigenesis. We identified loss of chromosome 6q and a minimal deleted area at 6q23-24 in CHBs. We also showed LOH at 6q23-24 in PCCs and identified the ZAC1 (6q24-25) as a candidate gene, ZAC1 is a maternally imprinted tumour suppressor gene with anti proliferative properties. To study further the role of ZAC inactivation in CHBs, we investigated LOH, promoter hypermethylation and expression status of the ZAC1 gene in mainly sporadic CHBs. Our LOH analysis revealed that the majority of the tumours with allele loss. The gene promoter methylation analysis similarly detected predominance of the methylated ZAC sequence in almost all tumours. Immunohistochemistry exhibited a strongly reduced expression of ZAC in stromal cells of all CHBs studied. Our current results indicate that the absence of the unmethylated, ZAC1 promoter sequence was highly concurrent with LOH for the ZAC1 region or 6q loss. This observation together with lack of ZAC expression, points to preferential loss of the non imprinted, expressed ZAC allele in CHB, in summary, our series of studies reveal a new chromosomal region 6q, emphasizes the importance of ZAC1 gene in the development of CHB and PCC, particularly in non-VHL associated cases.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja sydänlihaksen supistumisyksikkö, sarkomeeri, koostuu tarkoin järjestyneistä aktiini- ja myosiinisäikeistä. Rakenne eroaa muista solutyypeistä, joissa aktiinisäikeistö muovautuu jatkuvasti ja sen järjestyminen säätelee solun muotoa, solujakautumista, soluliikettä ja solunsisäisten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteenä ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyvät aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementteinä ja säätelijöinä. Myotilinia ja myopalladinia ilmennetään poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeissä kuten hermostossa, ja eri muodoilla saattaa olla solutyypistä riippuvia tehtäviä. Poikkijuovaisessa lihaksessa kaikki perheen jäsenet sijaitsevat aktiinisäikeitä yhdistävässä Z-levyssä ja ne sitovat Z-levyn rakenneproteiinia, -aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyvän periytyvään haimasyöpään ja lisääntyneeseen sydäninfarktin riskiin. Tässä tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa löydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niistä on aktiinidynamiikkaa säätelevä profilin. Profilinilla on kahdenlaisia tehtäviä; se edesauttaa aktiinisäikeiden muodostumista, mutta se voi myös eristää yksittäisiä aktiinimolekyylejä ja edistää säikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenevä runsaasti aktiinia sisältävillä solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin tehtävään aktiinisäideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinisäikeitä yhteensitova -aktiniini. -Aktiniini liittää solutukirangan solukalvon proteiineihin ja ankkuroi solunsisäisiä viestintämolekyylejä. Sitoutumista välittävä alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttejä sisältäviä tiivistymiä ja lopulta uusia sarkomeereja. Löydöstemme perusteella myotilinin uudelleenjärjestyminen noudattaa aktiinin muutoksia. Tämä viittaa siihen, että myotilin liittää yhteen uudismuodostuvia aktiinisäikeitä ja vakauttaa niitä. Myotilin saattaa myös ankkuroida viesti- tai rakennemolekyylejä, joiden tehtävänä on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, että myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinisäikeiden säätelijänä. Palladinin puute johtaa sikiöaikaiseen kuolemaan hiirillä, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyvän ja kehittyvän normaalisti eikä niillä esiintynyt rakenteellisia tai toiminnallisia häiriöitä. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, nähdään samankaltaisia kuin sairailla ihmisillä. Näin ollen muuntunut myotilin näyttä olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvittääksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilmentää puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilmentävien hiirten sydänlihaksessa todettiin vähäisiä hienorakenteen muutoksia, mutta risteytetyillä hiirillä tavattiin rakenteellisia ja toiminnallisia muutoksia myös luurankolihaksessa. Tulosten perusteella voidaan todeta, että palladinin 200 kDa muoto säätelee sydänlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla näyttäisi olevan päällekkäisiä tehtäviä.

Extraction methods in soil phosphorus characterisation : Limitations and applications

The quantification and characterisation of soil phosphorus (P) is of agricultural and environmental importance and different extraction methods are widely used to asses the bioavailability of P and to characterize soil P reserves. However, the large variety of extractants, pre-treatments and sample preparation procedures complicate the comparison of published results. In order to improve our understanding of the behaviour and cycling of P in soil, it is crucial to know the scientific relevance of the methods used for various purposes. The knowledge of the factors affecting the analytical outcome is a prerequisite for justified interpretation of the results. The aim of this thesis was to study the effects of sample preparation procedures on soil P and to determine the dependence of the recovered P pool on the chemical nature of extractants. Sampling is a critical step in soil testing and sampling strategy is dependent on the land-use history and the purpose of sampling. This study revealed that pre-treatments changed soil properties and air-drying was found to affect soil P, particularly extractable organic P, by disrupting organic matter. This was evidenced by an increase in the water-extractable small-sized (<0.2 µm) P that, at least partly, took place at the expense of the large-sized (>0.2 µm) P. However, freezing induced only insignificant changes and thus, freezing can be taken to be a suitable method for storing soils from the boreal zone that naturally undergo periodic freezing. The results demonstrated that chemical nature of the extractant affects its sensitivity to detect changes in soil P solubility. Buffered extractants obscured the alterations in P solubility induced by pH changes; however, water extraction, though sensitive to physicochemical changes, can be used to reveal short term changes in soil P solubility. As for the organic P, the analysis was found to be sensitive to the sample preparation procedures: filtering may leave a large proportion of extractable organic P undetected, whereas the outcome of centrifugation was found to be affected by the ionic strength of the extractant. Widely used sequential fractionation procedures proved to be able to detect land-use -derived differences in the distribution of P among fractions of different solubilities. However, interpretation of the results from extraction experiments requires better understanding of the biogeochemical function of the recovered P fraction in the P cycle in differently managed soils under dissimilar climatic conditions.

Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.

Time to rest : Signals in shoot apex developmental transitions underlying dormancy

Vuodenajat rytmittävät monivuotisten kasvien elämää pohjoisella pallonpuoliskolla, jolla varmin merkki lähestyvästä talvikaudesta on asteittain lyhenevä päivänpituus. Kun päivänpituus on lyhentynyt tiettyyn raja-arvoon saakka, kasvu hiipuu ja kasvin kehityksessä tapahtuu suuria muutoksia. Väitöskirjatyössäni tutkittiin mekanismeja, jotka liittyvät pituuskasvun päättymiseen, silmujen lepotilan kehittymiseen ja kärkisilmun muodostumiseen hybridihaavan ja koivuntaimilla lyhyen päivänpituuden seurauksena kasvihuoneolosuhteissa. Vain lepotilaiset silmut selviytyvät luonnossa ankaran talvikauden yli, joten etenkin lepotilan kehittymisen tutkiminen on keskeistä pyrittäessä selvittämään monivuotisille kasveille tyypillisen kasvutavan mekanismeja. Jo pitkään on tiedetty, että täysikasvuiset lehdet vastaanottavat tiedon päivänpituudesta ja lähettävät signaaleja varren johtojänteissä ylöspäin kohti kasvin kärkiosaa. Sen sijaan varren kärjen ja kärkikasvupisteen roolia lepotilan kehittymisessä on selvitetty vain vähän. Kuitenkin juuri kärkikasvupisteen selviytyminen vuodesta toiseen on tärkeää, koska sen jakautumiskykyiset solukot tuottavat kasvin maanpäälliset osat. Tässä työssä tehdyissä varttamiskokeissa osoitettiin, että varren kärki ei ainoastaan vastaanota signaaleja lehdistä ja ajoita toimintaansa niiden mukaan, vaan myös kärjellä itsellään on aktiivinen rooli lepotilan kehittymisessä. Erityisesti kiinnitettiin huomiota kärkikasvupisteen eri alueiden, ns. apikaalimeristeemin ja rib-meristeemin erilaisiin tehtäviin ja pääteltiin, että molemmat vaikuttavat lepotilan kehittymiseen. Kokeissa käytettiin normaalien hybridihaapojen lisäksi siirtogeenisiä hybridihaapoja, jotka eivät lopeta kasvuaan lyhyt päivä –olosuhteissa. Siirtogeeniset hybridihaavat ilmensivät voimakkaasti fytokromi A -nimistä valon vastaanottajamolekyyliä rib-meristeemin alueella, mikä saattoi osaltaan vaikuttaa poikkeavaan pituuskasvukäyttäytymiseen. Myös useiden lepotilan kehittymiseen liittyvien geenien ilmenemisessä havaittiin poikkeavuuksia verrattuna ei-siirtogeenisiin kontrolleihin, joiden silmuissa kehittyi lepotila lyhyt päivä –altistuksen seurauksena. Väitöskirjatyössäni havaittiin, että myös kaasumainen kasvihormoni etyleeni toimii viestinvälittäjänä silmujen lepotilan kehittymisessä ja vaikuttaa etenkin lepotilan oikeaan ajoittumiseen. Etyleenillä huomattiin olevan määräävä rooli päätesilmun muodostumisessa: siirtogeeniset koivut, jotka eivät aisti etyleeniä, eivät muodostaneet päätesilmua. Silti siirtogeeniset koivut vaipuivat lepotilaan, joskin myöhemmin kuin ei-siirtogeeniset kontrollit. Tämän perusteella todettiin, että lepotilan ja päätesilmun kehittyminen ovat erillisiä kehitystapahtumia, vaikka ne saattavatkin ajoittua osaksi päällekkäin.