Studies on the Cell Wall Structure and on the Mechanical Properties of Norway Spruce

The structure and the mechanical properties of wood of Norway spruce (Picea abies [L.] Karst.) were studied using small samples from Finland and Sweden. X-ray diffraction (XRD) was used to determine the orientation of cellulose microfibrils (microfibril angle, MFA), the dimensions of cellulose crystallites and the average shape of the cell cross-section. X-ray attenuation and x-ray fluorescence measurements were used to study the chemical composition and the trace element content. Tensile testing with in situ XRD was used to characterise the mechanical properties of wood and the deformation of crystalline cellulose within the wood cell walls. Cellulose crystallites were found to be 192 284 Å long and 28.9 33.4 Å wide in chemically untreated wood and they were longer and wider in mature wood than in juvenile wood. The MFA distribution of individual Norway spruce tracheids and larger samples was asymmetric. In individual cell walls, the mean MFA was 19 30 degrees, while the mode of the MFA distribution was 7 21 degrees. Both the mean MFA and the mode of the MFA distribution decreased as a function of the annual ring. Tangential cell walls exhibited smaller mean MFA and mode of the MFA distribution than radial cell walls. Maceration of wood material caused narrowing of the MFA distribution and removed contributions observed at around 90 degrees. In wood of both untreated and fertilised trees, the average shape of the cell cross-section changed from circular via ambiguous to rectangular as the cambial age increased. The average shape of the cell cross-section and the MFA distribution did not change as a result of fertilisation. The mass absorption coefficient for x-rays was higher in wood of fertilised trees than in that of untreated trees and wood of fertilised trees contained more of the elements S, Cl, and K, but a smaller amount of Mn. Cellulose crystallites were longer in wood of fertilised trees than in that of untreated trees. Kraft cooking caused widening and shortening of the cellulose crystallites. Tensile tests parallel to the cells showed that if the mean MFA is initially around 10 degrees or smaller, no systematic changes occur in the MFA distribution due to strain. The role of mean MFA in defining the tensile strength or the modulus of elasticity of wood was not as dominant as that reported earlier. Crystalline cellulose elongated much less than the entire samples. The Poisson ratio νca of crystalline cellulose in Norway spruce wood was shown to be largely dependent on the surroundings of crystalline cellulose in the cell wall, varying between -1.2 and 0.8. The Poisson ratio was negative in kraft cooked wood and positive in chemically untreated wood. In chemically untreated wood, νca was larger in mature wood and in latewood compared to juvenile wood and earlywood.

A study of the nanostructure of the cell wall of the tracheids of conifer xylem by x-ray scattering

The purpose of this study was to develop practical and reliable x-ray scattering methods to study the nanostructure of the wood cell wall and to use these methods to systematically study the nanostructure of Norway spruce and Scots pine grown in Finland and Sweden. Methods to determine the microfibril angle (MFA) distribution, the crystallinity of wood, and the average size of cellulose crystallites using wide-angle x-ray scattering were developed and these parameters were determined as a function of the number of the year ring. The mean MFA in Norway spruce decreases rapidly as a function of the number of the year ring and after the 7th year ring it varies between 6° and 10°. The mean MFA of Scots pine behaves the same way as the mean MFA of Norway spruce. The thickness of cellulose crystallites for Norway spruce and Scots pine appears to be constant as a function of the number of the year ring. The obtained mean values are 32 Å for Norway spruce and 31 Å for Scots pine. The length of the cellulose crystallites was also quite constant as a function of the year ring. The mean length of the crystallites for Norway spruce was 364 Å, while the standard deviation was 27 Å. The mass fraction of crystalline cellulose in wood is the crystallinity of wood and the intrinsic crystallinity of cellulose is the crystallinity of cellulose. The crystallinity of wood increases from the 2nd year ring to the 10th year ring from the pith and is constant after the 10th year ring. The crystallinity of cellulose obtained using nuclear magnetic resonance spectroscopy was 52% for both species. The crystallinity of wood and the crystallinity of cellulose behave the same way in Norway spruce and Scots pine. The methods were also applied to studies on thermally modified Scots pine wood grown in Finland. Wood is modified thermally by heating and steaming in order to improve its properties such as biological resistance and dimensional stability. Modification temperatures varied from 100 °C to 240 °C. The thermal modification increases the crystallinity of wood and the thickness of cellulose crystallites but does not influence the MFA distribution. When the modification temperature was 230 °C and time 4 h, the thickness of the cellulose crystallites increased from 31 Å to 34 Å.

Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line

Background: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging. Methods: We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations. Results: This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. qRT-PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P = 0.006). Conclusion: We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers.

Morphogenesis of Mammalian Endoplasmic Reticulum and Golgi Apparatus throughout the Cell Cycle

The endoplasmic reticulum (ER) and the Golgi apparatus are organelles that produce, modify and transport proteins and lipids and regulate Ca2+ environment within cells. Structurally they are composed of sheets and tubules. Sheets may take various forms: intact, fenestrated, single or stacked. The ER, including the nuclear envelope, is a single continuous network, while the Golgi shows only some level of connectivity. It is often unclear, how different morphologies correspond to particular functions. Previous studies indicate that the structures of the ER and Golgi are dynamic and regulated by fusion and fission events, cytoskeleton, rate of protein synthesis and secretion, and specific structural proteins. For example, many structural proteins shaping tubular ER have been identified, but sheet formation is much more unclear. In this study, we used light and electron microscopy to study morphological changes of the ER and Golgi in mammalian cells. The proportion, type, location and dynamics of ER sheets and tubules were found to vary in a cell type or cell cycle stage dependent manner. During interphase, ER and Golgi structures were demonstrated to be regulated by p37, a cofactor of the fusion factor p97, and microtubules, which also affected the localization of the organelles. Like previously shown for the Golgi, the ER displayed a tendency for fenestration and tubulation during mitosis. However, this shape change did not result in ER fragmentation as happens to Golgi, but a continuous network was retained. The activity of p97/p37 was found to be important for the reassembly of both organelles after mitosis. In EM images, ER sheet membranes appear rough, since they contain attached ribosomes, whereas tubular membranes appear smooth. Our studies revealed that structural changes of the ER towards fenestrated and tubular direction correlate with loss of ER-bound ribosomes and vice versa. High and low curvature ER membranes have a low and high density of ribosomes, respectively. To conclude, both ER and Golgi architecture depend on fusion activity of p97/p37. ER morphogenesis, particularly of the sheet shape, is intimately linked to the density of membrane bound ribosomes.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Merkel cell carcinoma : Epidemiological study with special reference to polyomavirus and vascular factors in pathogenesis

Merkel cell carcinoma (MCC) is a rare cutaneous malignancy that occurs predominantly on sun exposed skin areas. A new polyomavirus (MCPyV) was identified in MCC tumor tissues in 2008 suggesting that a viral infection might be an etiological factor. A typical MCC is a rapidly growing painless purple nodule. In its early stage it can be misjudged by its appearance as a cyst or abscess. Recurrences are common and approximately half of the patients will develop lymph node metastases and third of the patents will have distant metastases. It affects mostly elderly persons at an average age of 70 at the time of diagnosis. MCC was first described in 1972 and the first MCC patient in Finland was identified in 1983. MCC has been poorly recognized, but increased awareness and better diagnostic accuracy has increased the incidence since the early years. In this study, all cases with a notation of MCC during 1979 2008 were obtained from the Finnish Cancer Registry. Based on this data, the incidence is 0.11 for men and 0.12 for women. It is similar than that of other Nordic countries, but lower than in the USA. For clinical series, the files of patients diagnosed with MCC during 1983 2004 were reviewed, and the tissue samples were re-evaluated, if available (n=181). Third of the patients were men, and the most common site of the primary tumor was the head and neck (53%). The majority of the patients (86%) presented with a clinically node-negative (Stage I or II) disease, but the disease recurred in 38% of them. The treatment schemes were heterogeneous. No additional benefit from a wide margin (≥2 cm) was found compared to a margin of 0.1-1.9 cm, but intralesional excision was more often associated with local recurrence. None of the patients with Stage I-II disease who had received postoperative radiotherapy had local recurrence during the follow-up period. The 5-year relative survival ratio for Stage I disease was 68%, for Stage II 67%, for Stage III 16%, and for Stage IV 0%. The relative excess risk of death was significantly lower among women than among men. Some of these tissue samples were further analyzed for vascular invasion (n=126) by immunohistochemistry using vascular endothelial markers CD-31 and D2-40. Vascular invasion was seen in 93% of the samples and it was observed already in very small, <5mm tumors. The tissue samples were also analyzed for the presence of MCPyV by using a polymerase chain reaction (PCR) and quantitative PCR. MCPyV DNA was present in 80% of 114 samples studied. The patients with virus-positive tumors had better overall survival than patients with virus-negative tumors. Immunohistochemical analyses were performed for the expression of VEGFR-2 (n=21) and endostatin (n=19), but they had no prognostic value. Our results support the concept of treating MCC with margin-negative excision and radiotherapy to the tumor bed to reduce local recurrence. The finding of a high frequency of lymphovascular invasion reduces its value as a prognostic factor, but emphasizes the role of sentinel node biopsy even in very small primary MCC.

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.

Non-small cell lung cancer : studies on pathogenesis, tumour targeting and treatment outcomes

Lung cancer accounts for more cancer-related deaths than any other cancer. In Finland, five-year survival ranges from 8% to 13%. The main risk factor for lung cancer is long-term cigarette smoking, but its carcinogenesis requires several other factors. The aim of the present study was to 1) evaluate post-operative quality of life, 2) compare clinical outcomes between minimally invasive and conventional open surgery, 3) evaluate the role of oxidative stress in the carcinogenesis of non-small lung cancer (NSCLC), and 4) to identify and characterise targeted agents for therapeutic and diagnostic use in surgery. For study I, pneumonectomy patients replied to 15D quality of life and baseline dyspnea questionnaires. Study III involved a prospective quality of life assessment using the 15D questionnaire after lobectomy or bi-lobectomy. Study IV was a retrospective comparison of clinical outcomes between 212 patients treated with open thoracotomy and 116 patients who underwent a minimally invasive technique. Study II measured parameters of oxidative metabolism (myeloperoxidase activity, glutathione content and NADPH oxidase activity) and DNA adducts. Study V employed the phage display method and identified a core motif for homing peptides. This method served in cell-binding, cell-localisation, and biodistribution studies. Following both pneumonectomy and lobectomy, NSCLC patients showed significantly decreased long-term quality of life. No significant correlation was noted between post-operative quality of life and pre-operative pulmonary function tests. Women suffered more from increased dyspnea after pneumonectomy which was absent after lobectomy or bi-lobectomy. Patients treated with video-assisted thoracoscopy showed significantly decreased morbidity and shorter periods of hospitalization than did open surgery patients. This improvement was achieved even though the VATS patients were older and suffered more comorbid conditions and poorer pulmonary function. No significant differences in survival were noted between these two groups. An increase in NADPH oxidase activity was noted in tumour samples of both adenocarcinoma and squamous cell carcinoma. This increase was independent from myeloperoxidase activity. Elevated glutathione content was noted in tumour tissue, especially in adenocarcinoma. After panning the clinical tumour samples with the phage display method, an amino acid sequence of ARRPKLD, the Thx, was chosen for further analysis. This method proved selective of tumour tissue in both in vitro and in vivo cell-binding assay, and biodistribution showed tumour accumulation. Because of the significantly reduced quality of life following pneumonectomy, other operative strategies should be implemented as an alternative (e.g. sleeve-lobectomy). To treat this disease, implementation of a minimally invasive surgical technique is safe, and the results showed decreased morbidity and a shorter period of hospitalisation than with thoracotomy. This technique may facilitate operative treatment of elderly patients with comorbid conditions who might otherwise be considered inoperable. Simultaneous exposure to oxidative stress and altered redox states indicates the important role of oxidative stress in the pathogenesis and malignant transformation of NSCLC. The studies showed with great specificity and with favourable biodistribution that Thx peptide is specific to NSCLC tumours. Thx thus shows promise in imaging, targeted therapy, and monitoring of treatment response.

The neuronal cell adhesion molecule ICAM-5

The neuronal cell adhesion molecule ICAM-5 ICAM-5 (telencephalin) belongs to the intercellular adhesion molecule (ICAM)-subgroup of the immunoglobulin superfamily (IgSF). ICAMs participate in leukocyte adhesion and adhesion-dependent functions in the central nervous system (CNS) through interacting with the leukocyte-specific b2 integrins. ICAM-5 is found in the mammalian forebrain, appears at the time of birth, and is located at the cell soma and neuronal dendrites. Recent studies also show that it is important for the regulation of immune functions in the brain and for the development and maturation of neuronal synapses. The clinical importance of ICAM-5 is still under investigation; it may have a role in the development of Alzheimer s disease (AD). In this study, the role of ICAM-5 in neuronal differentiation and its associations with a-actinin and N-methyl-D-aspartic acid (NMDA) receptors were examined. NMDA receptors (NMDARs) are known to be involved in many neuronal functions, including the passage of information from one neuron to another one, and thus it was thought important to study their role related to ICAM-5. The results suggested that ICAM-5 was able to induce dendritic outgrowth through homophilic adhesion (ICAM-5 monomer binds to another ICAM-5 monomer in the same or neighbouring cell), and the homophilic binding activity appeared to be regulated by monomer/multimer transition. Moreover, ICAM-5 binding to a-actinin was shown to be important for neuritic outgrowth. It was examined whether matrix metalloproteinases (MMPs) are the main enzymes involved in ICAM-5 ectodomain cleavage. The results showed that stimulation of NMDARs leads to MMP activation, cleavage of ICAM-5 and it is accompanied by dendritic spine maturation. These findings also indicated that ICAM-5 and NMDA receptor subunit 1 (NR1) compete for binding to a-actinin, and ICAM-5 may regulate the NR1 association with the actin cytoskeleton. Thus, it is concluded that ICAM-5 is a crucial cell adhesion molecule involved in the development of neuronal synapses, especially in the regulation of dendritic spine development, and its functions may also be involved with memory formation and learning.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

Integration of miRNA and mRNA expression with DNA copy number of Ewing sarcoma cell lines

Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.

Relative Contribution of Cell Proliferation and Microvascularity to Metastasis in Malignant Uveal Melanoma

Uveal melanoma (UM) is the second most common primary intraocular cancer worldwide. It is a relatively rare cancer, but still the second most common type of primary malignant melanoma in humans. UM is a slowly growing tumor, and gives rise to distant metastasis mainly to the liver via the bloodstream. About 40% of patients with UM die of metastatic disease within 10 years of diagnosis, irrespective of the type of treatment. During the last decade, two main lines of research have aimed to achieve enhanced understanding of the metastasis process and accurate prognosis of patients with UM. One emphasizes the characteristics of tumor cells, particularly their nucleoli, and markers of proliferation, and the other the characteristics of tumor blood vessels. Of several morphometric measurements, the mean diameter of the ten largest nucleoli (MLN) has become the most widely applied. A large MLN has consistently been associated with high likelihood of dying from UM. Blood vessels are of paramount importance in metastasis of UM. Different extravascular matrix patterns can be seen in UM, like loops and networks. This presence is associated with death from metastatic melanoma. However, the density of microvessels is also of prognostic importance. This study was undertaken to help understanding some histopathological factors which might contribute to developing metastasis in UM patients. Factors which could be related to tumor progression to metastasis disease, namely nucleolar size, MLN, microvascular density (MVD), cell proliferation, and The Insulin-like Growth Factor 1 Receptor(IGF-1R), were investigated. The primary aim of this thesis was to study the relationship between prognostic factors such as tumor cell nucleolar size, proliferation, extravascular matrix patterns, and dissemination of UM, and to assess to what extent there is a relationship to metastasis. The secondary goal was to develop a multivariate model which includes MLN and cell proliferation in addition to MVD, and which would fit better with population-based, melanoma-related survival data than previous models. I studied 167 patients with UM, who developed metastasis even after a very long time following removal of the eye, metastatic disease was the main cause of death, as documented in the Finnish Cancer Registry and on death certificates. Using an independent population-based data set, it was confirmed that MLN and extravascular matrix loops and networks were unrelated, independent predictors of survival in UM. Also, it has been found that multivariate models including MVD in addition to MLN fitted significantly better with survival data than models which excluded MVD. This supports the idea that both the characteristics of the blood vessels and the cells are important, and the future direction would be to look for the gene expression profile, whether it is associated more with MVD or MLN. The former relates to the host response to the tumor and may not be as tightly associated with the gene expression profile, yet most likely involved in the process of hematogenous metastasis. Because fresh tumor material is needed for reliable genetic analysis, such analysis could not be performed Although noninvasive detection of certain extravascular matrix patterns is now technically possible,in managing patients with UM, this study and tumor genetics suggest that such noninvasive methods will not fully capture the process of clinical metastasis. Progress in resection and biopsy techniques is likely in the near future to result in fresh material for the ophthalmic pathologist to correlate angiographic data, histopathological characteristics such as MLN, and genetic data. This study supported the theory that tumors containing epithelioid cells grow faster and have poorer prognosis when studied by cell proliferation in UM based on Ki-67 immunoreactivity. Cell proliferation index fitted best with the survival data when combined with MVD, MLN, and presence of epithelioid cells. Analogous with the finding that high MVD in primary UM is associated with shorter time to metastasis than low MVD, high MVD in hepatic metastasis tends to be associated with shorter survival after diagnosis of metastasis. Because the liver is the main organ for metastasis from UM, growth factors largely produced in the liver hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-1 (IGF-1) together with their receptors may have a role in the homing and survival of metastatic cells. Therefore the association between immunoreactivity for IGF-1R in primary UM and metastatic death was studied. It was found that immunoreactivity for IGF-IR did not independently predict metastasis from primary UM in my series.

Patents, Ethics and Stem Cell Research : The Case of hESC Innovation in Australia, Europe and the United States

Embryonic stem cells offer potentially a ground-breaking insight into health and diseases and are said to offer hope in discovering cures for many ailments unimaginable few years ago. Human embryonic stem cells are undifferentiated, immature cells that possess an amazing ability to develop into almost any body cell such as heart muscle, bone, nerve and blood cells and possibly even organs in due course. This remarkable feature, enabling embryonic stem cells to proliferate indefinitely in vitro (in a test tube), has branded them as a so-called miracle cure . Their potential use in clinical applications provides hope to many sufferers of debilitating and fatal medical conditions. However, the emergence of stem cell research has resulted in intense debates about its promises and dangers. On the one hand, advocates hail its potential, ranging from alleviating and even curing fatal and debilitating diseases such as Parkinson s, diabetes, heart ailments and so forth. On the other hand, opponents decry its dangers, drawing attention to the inherent risks of human embryo destruction, cloning for research purposes and reproductive cloning eventually. Lately, however, the policy battles surrounding human embryonic stem cell innovation have shifted from being a controversial research to scuffles within intellectual property rights. In fact, the ability to obtain patents represents a pivotal factor in the economic success or failure of this new biotechnology. Although, stem cell patents tend to more or less satisfy the standard patentability requirements, they also raise serious ethical and moral questions about the meaning of the exclusions on ethical or moral grounds as found in European and to an extent American and Australian patent laws. At present there is a sort of a calamity over human embryonic stem cell patents in Europe and to an extent in Australia and the United States. This in turn has created a sense of urgency to engage all relevant parties in the discourse on how best to approach patenting of this new form of scientific innovation. In essence, this should become a highly favoured patenting priority. To the contrary, stem cell innovation and its reliance on patent protection risk turmoil, uncertainty, confusion and even a halt on not only stem cell research but also further emerging biotechnology research and development. The patent system is premised upon the fundamental principle of balance which ought to ensure that the temporary monopoly awarded to the inventor equals that of the social benefit provided by the disclosure of the invention. Ensuring and maintaining this balance within the patent system when patenting human embryonic stem cells is of crucial contemporary relevance. Yet, the patenting of human embryonic stem cells raises some fundamental moral, social and legal questions. Overall, the present approach of patenting human embryonic stem cell related inventions is unsatisfactory and ineffective. This draws attention to a specific question which provides for a conceptual framework for this work. That question is the following: how can the investigated patent offices successfully deal with patentability of human embryonic stem cells? This in turn points at the thorny issue of application of the morality clause in this field. In particular, the interpretation of the exclusions on ethical or moral grounds as found in Australian, American and European legislative and judicial precedents. The Thesis seeks to compare laws and legal practices surrounding patentability of human embryonic stem cells in Australia and the United States with that of Europe. By using Europe as the primary case study for lessons and guidance, the central goal of the Thesis then becomes the determination of the type of solutions available to Europe with prospects to apply such to Australia and the United States. The Dissertation purports to define the ethical implications that arise with patenting human embryonic stem cells and intends to offer resolutions to the key ethical dilemmas surrounding patentability of human embryonic stem cells and other morally controversial biotechnology inventions. In particular, the Thesis goal is to propose a functional framework that may be used as a benchmark for an informed discussion on the solution to resolving ethical and legal tensions that come with patentability of human embryonic stem cells in Australian, American and European patent worlds. Key research questions that arise from these objectives and which continuously thread throughout the monograph are: 1. How do common law countries such as Australia and the United States approach and deal with patentability of human embryonic stem cells in their jurisdictions? These practices are then compared to the situation in Europe as represented by the United Kingdom (first two chapters), the Court of Justice of the European Union and the European Patent Office decisions (Chapter 3 onwards) in order to obtain a full picture of the present patenting procedures on the European soil. 2. How are ethical and moral considerations taken into account at patent offices investigated when assessing patentability of human embryonic stem cell related inventions? In order to assess this part, the Thesis evaluates how ethical issues that arise with patent applications are dealt with by: a) Legislative history of the modern patent system from its inception in 15th Century England to present day patent laws. b) Australian, American and European patent offices presently and in the past, including other relevant legal precedents on the subject matter. c) Normative ethical theories. d) The notion of human dignity used as the lowest common denominator for the interpretation of the European morality clause. 3. Given the existence of the morality clause in form of Article 6(1) of the Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions which corresponds to Article 53(a) European Patent Convention, a special emphasis is put on Europe as a guiding principle for Australia and the United States. Any room for improvement of the European morality clause and Europe s current manner of evaluating ethical tensions surrounding human embryonic stem cell inventions is examined. 4. A summary of options (as represented by Australia, the United States and Europe) available as a basis for the optimal examination procedure of human embryonic stem cell inventions is depicted, whereas the best of such alternatives is deduced in order to create a benchmark framework. This framework is then utilised on and promoted as a tool to assist Europe (as represented by the European Patent Office) in examining human embryonic stem cell patent applications. This method suggests a possibility of implementing an institution solution. 5. Ultimately, a question of whether such reformed European patent system can be used as a founding stone for a potential patent reform in Australia and the United States when examining human embryonic stem cells or other morally controversial inventions is surveyed. The author wishes to emphasise that the guiding thought while carrying out this work is to convey the significance of identifying, analysing and clarifying the ethical tensions surrounding patenting human embryonic stem cells and ultimately present a solution that adequately assesses patentability of human embryonic stem cell inventions and related biotechnologies. In answering the key questions above, the Thesis strives to contribute to the broader stem cell debate about how and to which extent ethical and social positions should be integrated into the patenting procedure in pluralistic and morally divided democracies of Europe and subsequently Australia and the United States.