N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.

Effect of long-wave UV radiation on mouse melanoma: an in vitro and in vivo study

The skin cancer incidence has increased substantially over the past decades and the role of ultraviolet (UV) radiation in the etiology of skin cancer is well established. Ultraviolet B radiation (280-320 nm) is commonly considered as the more harmful part of the UV-spectrum due to its DNA-damaging potential and well-known carcinogenic effects. Ultraviolet A radiation (320-400 nm) is still regarded as a relatively low health hazard. However, UVA radiation is the predominant component in sunlight, constituting more than 90% of the environmentally relevant solar ultraviolet radiation. In the light of the recent scientific evidence, UVA has been shown to have genotoxic and immunologic effects, and it has been proposed that UVA plays a significant role in the development of skin cancer. Due to the popularity of skin tanning lamps, which emit high intensity UVA radiation and because of the prolonged sun tanning periods with the help of effective UVB blockers, the potential deleterious effects of UVA has emerged as a source of concern for public health. The possibility that UV radiation may affect melanoma metastasis has not been addressed before. UVA radiation can modulate various cellular processes, some of which might affect the metastatic potential of melanoma cells. The aim of the present study was to investigate the possible role of UVA irradiation on the metastatic capacity of mouse melanoma both in vitro and in vivo. The in vitro part of the study dealt with the enhancement of the intercellular interactions occurring either between tumor cells or between tumor cells and endothelial cells after UVA irradiation. The use of the mouse melanoma/endothelium in vitro model showed that a single-dose of UVA to melanoma cells causes an increase in melanoma cell adhesiveness to non-irradiated endothelium after 24-h irradiation. Multiple-dose irradiation of melanoma cells already increased adhesion at a 1-h time-point, which suggests the possible cumulative effect of multiple doses of UVA irradiation. This enhancement of adhesiveness might lead to an increase in binding tumor cells to the endothelial lining of vasculature in various internal organs if occurring also in vivo. A further novel observation is that UVA induced both decline in the expression of E-cadherin adhesion molecule and increase in the expression of the N-cadherin adhesion molecule. In addition, a significant decline in homotypic melanoma-melanoma adhesion (clustering) was observed, which might result in the reduction of E-cadherin expression. The aim of the in vivo animal study was to confirm the physiological significance of previously obtained in vitro results and to determine whether UVA radiation might increase melanoma metastasis in vivo. The use of C57BL/6 mice and syngeneic melanoma cell lines B16-F1 and B16-F10 showed that mice, which were i.v. injected with B16-F1 melanoma cells and thereafter exposed to UVA developed significantly more lung metastases when compared with the non-UVA-exposed group. To study the mechanism behind this phenomenon, the direct effect of UVA-induced lung colonization capacity was examined by the in vitro exposure of B16-F1 cells. Alternatively, the UVA-induced immunosuppression, which might be involved in increased melanoma metastasis, was measured by standard contact hypersensitivity assay (CHS). It appears that the UVA-induced increase of metastasis in vivo might be caused by a combination of UVA-induced systemic immunosuppression, and to the lesser extent, it might be caused by the increased adhesiveness of UVA irradiated melanoma cells. Finally, the UVA effect on gene expression in mouse melanoma was determined by a cDNA array, which revealed UVA-induced changes in the 9 differentially expressed genes that are involved in angiogenesis, cell cycle, stress-response, and cell motility. These results suggest that observed genes might be involved in cellular response to UVA and a physiologically relevant UVA dose have previously unknown cellular implications. The novel results presented in this thesis offer evidence that UVA exposure might increase the metastatic potential of the melanoma cells present in blood circulation. Considering the wellknown UVA-induced deleterious effects on cellular level, this study further supports the notion that UVA radiation might have more potential impact on health than previously suggested. The possibility of the pro-metastatic effects of UVA exposure might not be of very high significance for daily exposures. However, UVA effects might gain physiological significance following extensive sunbathing or solaria tanning periods. Whether similar UVA-induced pro-metastatic effects occur in people sunbathing or using solaria remains to be determined. In the light of the results presented in this thesis, the avoidance of solaria use could be well justified.

The role of productivity in the ecological and evolutionary dynamics of predator-prey interaction

Productivity is predicted to drive the ecological and evolutionary dynamics of predator-prey interaction through changes in resource allocation between different traits. However, resources are seldom constantly available and thus temporal variation in productivity could have considerable effect on the species' potential to evolve. To study this, three long-term microbial laboratory experiments were established where Serratia marcescens prey bacteria was exposed to predation of protist Tetrahymena thermophila in different prey resource environments. The consequences of prey resource availability for the ecological properties of the predator-prey system, such as trophic dynamics, stability, and virulence, were determined. The evolutionary changes in species traits and prey genetic diversity were measured. The prey defence evolved stronger in high productivity environment. Increased allocation to defence incurred cost in terms of reduced prey resource use ability, which probably constrained prey evolution by increasing the effect of resource competition. However, the magnitude of this trade-off diminished when measured in high resource concentrations. Predation selected for white, non-pigmented, highly defensive prey clones that produced predation resistant biofilm. The biofilm defence was also potentially accompanied with cytotoxicity for predators and could have been traded off with high motility. Evidence for the evolution of predators was also found in one experiment suggesting that co-evolutionary dynamics could affect the evolution and ecology of predator-prey interaction. Temporal variation in resource availability increased variation in predator densities leading to temporally fluctuating selection for prey defences and resource use ability. Temporal variation in resource availability was also able to constrain prey evolution when the allocation to defence incurred high cost. However, when the magnitude of prey trade-off was small and the resource turnover was periodically high, temporal variation facilitated the formation of predator resistant biofilm. The evolution of prey defence constrained the transfer of energy from basal to higher trophic levels, decreasing the strength of top-down regulation on prey community. Predation and temporal variation in productivity decreased the stability of populations and prey traits in general. However, predation-induced destabilization was less pronounced in the high productivity environment where the evolution of prey defence was stronger. In addition, evolution of prey defence weakened the environmental variation induced destabilization of predator population dynamics. Moreover, protozoan predation decreased the S. marcescens virulence in the insect host moth (Parasemia plantaginis) suggesting that species interactions outside the context of host-pathogen relationship could be important indirect drivers for the evolution of pathogenesis. This thesis demonstrates that rapid evolution can affect various ecological properties of predator-prey interaction. The effect of evolution on the ecological dynamics depended on the productivity of the environment, being most evident in the constant environments with high productivity.

The biological functions of mouse twinfilin isoforms

The actin cytoskeleton is essential for many cellular processes, including motility, morphogenesis, endocytosis and signal transduction. Actin can exist in monomeric (G-actin) or filamentous (F-actin) form. Actin filaments are considered to be the functional form of actin, generating the protrusive forces characteristic for the actin cytoskeleton. The structure and dynamics of the actin filament and monomer pools are regulated by a large number of actin-binding proteins in eukaryotic cells. Twinfilin is an evolutionarily conserved small actin monomer binding protein. Twinfilin is composed of two ADF/cofilin-like domains, separated by a short linker and followed by a C-terminal tail. Twinfilin forms a stable, high affinity complex with ADP-G-actin, inhibits the nucleotide exchange on actin monomers, and prevents their assembly into filament ends. Twinfilin was originally identified from yeast and has since then been found from all organisms studied except plants. Not much was known about the role of twinfilin in the actin dynamics in mammalian cells before this study. We set out to unravel the mysteries still covering twinfilins functions using biochemistry, cell biology, and genetics. We identified and characterized two mouse isoforms for the previously identified mouse twinfilin-1. The new isoforms, twinfilin-2a and -2b, are generated from the same gene through alternative promoter usage. The three isoforms have distinctive expression patterns, but are similar biochemically. Twinfilin-1 is the major isoform during development and is expressed in high levels in almost all tissues examined. Twinfilin-2a is also expressed almost ubiquitously, but at lower levels. Twinfilin-2b turned out to be a muscle-specific isoform, with very high expression in heart and skeletal muscle. It seems all mouse tissues express at least two twinfilin isoforms, indicating that twinfilins are important regulators of actin dynamics in all cell and tissue types. A knockout mouse line was generated for twinfilin-2a. The mice homozygous for this knockout were viable and developed normally, indicating that twinfilin-2a is dispensable for mouse development. However, it is important to note that twinfilin-2a shows similar expression pattern to twinfilin-1, suggesting that these proteins play redundant roles in mice. All mouse isoforms were shown to be able to sequester actin filaments and have higher affinity for ADP-G-actin than ATP-G-actin. They are also able to directly interact with heterodimeric capping protein and PI(4,5)P2 similar to yeast twinfilin. In this study we also uncovered a novel function for mouse twinfilins; capping actin filament barbed ends. All mouse twinfilin isoforms were shown to possess this function, while yeast and Drosophila twinfilin were not able to cap filament barbed ends. Twinfilins localize to the cytoplasm but also to actin-rich regions in mammalian cells. The subcellular localizations of the isoforms are regulated differently, indicating that even though twinfilins biochemical functions in vitro are very similar, in vivo they can play different roles through different regulatory pathways. Together, this study show that twinfilins regulate actin filament assembly both by sequestering actin monomers and by capping filament barbed ends, and that mammals have three biochemically similar twinfilin isoforms with partially overlapping expression patterns.

Structural basis of cytoskeletal regulation by twinfilin

The actin cytoskeleton is required, in all eukaryotic organisms, for several key cellular functions such as cell motility, cytokinesis, and endocytosis. In cells, actin exists either in a monomeric state (G-actin) or in a filamentous form (F-actin). F-actin is the functional form, which can assemble into various structures and produce direct pushing forces that are required for different motile processes. The assembly of actin monomers into complicated three-dimensional structures is tightly regulated by a large number of actin regulating proteins. One central actin regulating protein is twinfilin. Twinfilin consists of two actin depolymerizing-factor homology (ADF-H) domains, which are capable of binding actin, and is conserved from yeast to mammals. Previously it has been shown that twinfilin binds to and sequesters G-actin, and interacts with the heterodimeric capping protein. More recently it has been found that twinfilin also binds to the fast growing actin filament ends and prevents their growth. However, the cellular role of twinfilin and the molecular mechanisms of these interactions have remained unclear. In this study we characterized the molecular mechanisms behind the functions of twinfilin. We demonstrated that twinfilin forms a high-affinity complex with ADP-bound actin monomers (ADP-G-actin). Both ADF-H domains are capable of binding G-actin, but the C-terminal domain contains the high-affinity binding site. Our biochemical analyses identified twinfilin s C-terminal tail region as the interaction site for capping protein. Contrary to G-actin binding, both ADF-H domains of twinfilin are required for the actin filament barbed end capping activity. The C-terminal domain is structurally homologous to ADF/cofilin and binds to filament sides in a similar manner, providing the main affinity for F-actin during barbed end capping. The structure of the N-terminal domain is more distant from ADF/cofilin, and thus it can only associate with G-actin or the terminal actin monomer at the filament barbed end, where it regulates twinfilin s affinity for barbed ends. These data suggest that the mechanism of barbed end capping is similar for twinfilin and gelsolin family proteins. Taken together, these studies revealed how twinfilin interacts with G-actin, filament barbed ends, and capping protein, and also provide a model for how these activities evolved through a duplication of an ancient ADF/cofilin-like domain.

On the ecology of cold-water phytoplankton in the Baltic Sea

Increased anthropogenic loading of nitrogen (N) and phosphorus (P) has led to an eutrophication problem in the Baltic Sea, and the spring bloom is a key component in the biological uptake of increased nutrient concentrations. The spring bloom in the Baltic Sea is dominated by both diatoms and dinoflagellates. However, the sedimentation of these groups is different: diatoms tend to sink to the sea floor at the end of the bloom, while dinoflagellates to a large degree are been remineralized in the euphotic zone. Understanding phytoplankton competition and species specific ecological strategies is thus of importance for assessing indirect effects of phytoplankton community composition on eutrophication problems. The main objective of this thesis was to describe some basic physiological and ecological characteristics of the main cold-water diatoms and dinoflagellates in the Baltic Sea. This was achieved by specific studies of: (1) seasonal vertical positioning, (2) dinoflagellate life cycle, (3) mixotrophy, (4) primary production, respiration and growth and (5) diatom silicate uptake, using cultures of common cold-water diatoms: Chaetoceros wighamii, C. gracilis, Pauliella taeniata, Thalassiosira baltica, T. levanderi, Melosira arctica, Diatoma tenuis, Nitzschia frigida, and dinoflagellates: Peridiniella catenata, Woloszynskia halophila and Scrippsiella hangoei. The diatoms had higher primary production capacity and lower respiration rate compared with the dinoflagellates. This difference was reflected in the maximum growth rate, which for the examined diatoms range from 0.6 to 1.2 divisions d-1, compared with 0.2 to 0.3 divisions d-1 for the dinoflagellates. Among diatoms there were species specific differences in light utilization and uptake of silicate, and C. wighamii had the highest carbon assimilation capacity and maximum silicate uptake. The physiological properties of diatoms and dinoflagellates were used in a model of the onset of the spring bloom: for the diatoms the model could predict the initiation of the spring bloom; S. hangoei, on the other hand, could not compete successfully and did not obtain positive growth in the model. The other dinoflagellates did not have higher growth rates or carbon assimilation rates and would thus probably not perform better than S. hangoei in the model. The dinoflagellates do, however, have competitive advantages that were not included in the model: motility and mixotrophy. Previous investigations has revealed that the chain-forming P. catenata performs diurnal vertical migration (DVM), and the results presented here suggest that active positioning in the water column, in addition to DVM, is a key element in this species' life strategy. There was indication of mixotrophy in S. hangoei, as it produced and excreted the enzyme leucine aminopeptidase (LAP). Moreover, there was indirect evidence that W. halophila obtains carbon from other sources than photosynthesis when comparing increase in cell numbers with in situ carbon assimilation rates. The results indicate that mixotrophy is a part of the strategy of vernal dinoflagellates in the Baltic Sea. There were also indications that the seeding of the spring bloom is very important for the dinoflagellates to succeed. In mesocosm experiments dinoflagellates could not compete with diatoms when their initial numbers were low. In conclusion, this thesis has provided new information about the basic physiological and ecological properties of the main cold-water phytoplankton in the Baltic Sea. The main phytoplankton groups, diatoms and dinoflagellates, have different physiological properties, which clearly separate their life strategies. The information presented here could serve as further steps towards better prognostic models of the effects of eutrophication in the Baltic Sea.

Micromolar concentration of pentoxifylline improves development in vitro of hamster 8-cell embryos: conrmation of biological viability by embryo transfer

The inßuence of the sperm motility stimulant pentoxifylline (PF) on preimplantation embryo development in hamsters was evaluated. Eight-cell embryos were cultured in hamster embryo culture medium (HECM)-2, with or without PF (0· 0233·6 mM). There was 90%, 37% and 29% inhibition of blastocyst development by 3·6 (used for human sperm), 0·9 and 0 ·45 mM PF, respectively. However, 23 µM PF (exposed to hamster oocytes during IVF) signicantly (P < 0·05) improved blastocyst development (63· 6% v. 51· 8%); morulae development was, however, not curtailed by 0·45 mM or 0·9 mM PF (51·8%±6·0 or 50·5%±11·3, respectively). Post-implantation viability of PF-treated embryos was assessed by embryo transfer; 43% of 80 PF-treated embryos implanted compared with 40% of 79 control embryos. Of the 9 recipients, 6 females delivered pups (19, i.e. 16% of transferred embryos or 53% of implanted embryos). These data show that in hamsters, continuous presence of PF at 0·45-3·6 mM is detrimental to 8-cell embryo development whereas 23 µM PF improves the development of embryos to viable blastocysts which produce live offspring.

Phospholipids of lipid-containing bacteriophages and their transbilayer distribution

In this study we used electro-spray ionization mass-spectrometry to determine phospholipid class and molecular species compositions in bacteriophages PM2, PRD1, Bam35 and phi6 as well as their hosts. To obtain compositional data of the individual leaflets, phospholipid transbilayer distribution in the viral membranes was studied. We found that 1) the membranes of all studied bacteriophage are enriched in PG as compared to the host membranes, 2) molecular species compositions in the phage and host membranes are similar, and 3) phospholipids in the viral membranes are distributed asymmetrically with phosphatidylglycerol enriched in the outer leaflet and phosphatidylethanolamine in the inner one (except Bam35). Alternative models for selective incorporation of phospholipids to phages and for the origins of the asymmetric phospholipid transbilayer distribution are discussed. Notably, the present data are also useful when constructing high resolution structural models of bacteriophages, since diffraction methods cannot provide a detailed structure of the membrane due to high motility of the lipids and lack of symmetric organization of membrane proteins.

Long-term outcomes of esophageal atresia

Esophageal atresia (EA), a common congenital anomaly comprising interrupted esophagus with or without a tracheoesophageal fistula (TEF), affects one in 2840 newborns. Over half have associated anomalies. After EA repair in infancy, gastroesophageal reflux (GER) and esophageal dysmotility and respiratory problems are common. As there exist no previous population-based long-term follow-up-studies on EA, its long-term sequelae are unclear. The aims of this study were to assess the cancer incidence (I), esophageal morbidity and function (II), respiratory morbidity (III), and the spinal defects (IV) in adults with repaired EA. All patients treated for EA at the Hospital for Children and Adolescents, University of Helsinki, from 1947 to 1985 were identified, and those alive with their native esophagus were contacted, and the first hundred who replied made up the study group. The patients were interviewed, they filled in symptom questionnaires, and they underwent esophageal endoscopy and manometry, pulmonary function tests, and a full orthopedic evaluation was performed with radiographs of the spine. The questionnaire was also sent by mail to adults with repaired EA not attending the clinical study, and to 287 general population-derived controls matched for age, gender, and municipality of residence. Incidence of cancer among the study population was evaluated from the population-based countrywide cancer registry. 169 (72%) adults with repaired EA replied; 101 (42%) (58 male) participated in the clinical studies at a median age of 36 years (range, 22-56). Symptomatic GER occurred in 34% and dysphagia in 85% of the patients and in 8% and 2% of the controls (P<0.001 for both). The main endoscopic findings included hiatal hernia (28%), Barrett´s esophagus (11%), esophagitis (8%), and stenotic anastomosis (8%). Histology revealed esophagitis in 25 individuals, and epithelial metaplasia in another 21. At immunohistochemistry, CDX2-positive columnar epithelial metaplasia was present in all 21 individuals, and 6 of these also demonstrated goblet cells and MUC2 positivity. In all histological groups, GER and dysphagia were equally common (P=ns). Esophageal manometry demonstrated non-propagating peristalsis in most of the patients, and low ineffective pressure of the distal esophageal body in all. The changes were significantly worse in those with epithelial metaplasia (P≤0.022). Anastomotic complications (OR 8.6-24, 95%CI 1.7-260, P=0.011-0.008), age (OR 20, 95%CI 1.3-310, P=0.034), low distal esophageal body pressure (OR 2.6, 95%CI 0.7-10, P=0.002), and defective esophageal peristalsis (OR 2.2, 95%CI 0.4-11, P=0.014) all predicted development of epithelial metaplasia. Despite the high incidence of esophageal metaplasia, none of the EA patients had suffered esophageal cancer, according to the Finnish Cancer Registry. Although three had had cancer (SIR, 1.0; 95% CI, 0.20-2.8). The overall cancer incidence among adults with repaired EA did not differ from that of the general Finnish population. Current respiratory symptoms occurred in 11% of the patients and 2% of the controls (P<0.001). Of the patients, 16%, and 6% of the controls had doctor-diagnosed asthma (P<0.001). A total of 56% and 70% of the patients and 20% and 50% of the controls had a history of pneumonia and of bronchitis (P<0.001 for both). Respiratory-related impaired quality of life was observable in 11% of the patients in contrast to 6% of the controls (P<0.001). PFT revealed obstruction in 21 of the patients, restriction in 21, and both in 36. A total of 41 had bronchial hyper-responsiveness (BHR) in HCT, and 15 others had an asthma-like response. Thoracotomy-induced rib fusion (OR 3.4, 95%CI 1.3-8.7, P=0.01) and GER-associated epithelial metaplasia in adulthood (OR 3.0, 95%CI 1.0-8.9, P=0.05) were the most significant risk factors for restrictive ventilatory defect. Vertebral anomalies were evident in 45 patients, predominating in the cervical spine in 38. The most significant risk factor for the occurrence of vertebral anomalies was any additional anomaly (OR 27, 95%C I8-100). Scoliosis (over 10 degrees) was observable in 56 patients, over 20 degrees in 11, and over 45 degrees in one. In the EA patients, risk for scoliosis over 10 degrees was 13-fold (OR 13, 95%CI 8.3-21) and over 20 degrees, 38-fold (OR 38, 95%CI 14-106) when compared to that of the general population. Thoracotomy-induced rib fusion (OR 3.6, 95%CI 0.7-19) and other associated anomalies (OR 2.1, 95%CI 0.9-2.9) were the strongest predictive factors for scoliosis. Significant esophageal morbidity associated with EA extends into adulthood. No association existed between the esophageal symptoms and histological findings. Surgical complications, increasing age, and impaired esophageal motility predicted development of epithelial metaplasia after repair of EA. According to our data, the risk for esophageal cancer is less than 500-fold that of the general population. However, the overall cancer incidence among adults with repaired EA did not differ from that of the general population. Adults with repaired EA have had significantly more respiratory symptoms and infections, as well as more asthma, and allergies than does the general population. Thoracotomy-induced rib fusion and GER-associated columnar epithelial metaplasia were the most significant risk factors for the restrictive ventilatory defect that occurred in over half the patients. Over half the patients with repaired EA are likely to develop scoliosis. Risk for scoliosis is 13-fold after repair of EA in relation to that of the general population. Nearly half the patients had vertebral anomalies. Most of these deformities were diagnosed neither in infancy nor during growth. The natural history of spinal deformities seems, however, rather benign, with spinal surgery rarely indicated.

Kallikrein-related peptidase 4 induces tumour microenvironment alterations that support tumour growth

Kallikrein-related peptidase 4 (KLK4) is a protease with elevated production in prostate cancer versus benign tissue. KLK4 expression is associated with prostate cancer risk, and its activity favours tumour progression through increasing cell motility and growth. Importantly, over-production of KLK4 in prostate glandular cells precedes tumour formation, positioning the enzyme to play a role in early remodelling of the tumour microenvironment, a process essential for tumour growth. We sought to identify the proteins and downstream signalling pathways targeted by KLK4 activity, to define its role in tumour microenvironment remodelling and evaluate the efficacy of KLK4 inhibition as a cancer therapy.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

Adhesion,Presence and Antifouling of Deinococcus geothermalis in Paper Machine Environment

This thesis has two items: biofouling and antifouling in paper industry. Biofouling means unwanted microbial accumulation on surfaces causing e.g. disturbances in industrial processes, contamination of medical devices or of water distribution networks. Antifouling focuses on preventing accumulation of the biofilms in undesired places. Deinococcus geothermalis is a pink-pigmented, thermophilic bacterium, and extremely resistant towards radiation, UV-light and desiccation and known as a biofouler of paper machines forming firm and biocide resistant biofilms on the stainless steel surfaces. The compact structure of biofilm microcolonies of D. geothermalis E50051 and the adhesion into abiotic surfaces were investigated by confocal laser scanning microscope combined with carbohydrate specific fluorescently labelled lectins. The extracellular polymeric substance in D. geothermalis microcolonies was found to be a composite of at least five different glycoconjugates contributing to adhesion, functioning as structural elements, putative storages for water, gliding motility and likely also to protection. The adhesion threads that D. geothermalis seems to use to adhere on an abiotic surface and to anchor itself to the neighbouring cells were shown to be protein. Four protein components of type IV pilin were identified. In addition, the lectin staining showed that the adhesion threads were covered with galactose containing glycoconjugates. The threads were not exposed on planktic cells indicating their primary role in adhesion and in biofilm formation. I investigated by quantitative real-time PCR the presence of D. geothermalis in biofilms, deposits, process waters and paper end products from 24 paper and board mills. The primers designed for doing this were targeted to the 16S rRNA gene of D. geothermalis. We found D. geothermalis DNA from 9 machines, in total 16 samples of the 120 mill samples searched for. The total bacterial content varied in those samples between 107 to 3 ×1010 16S rRNA gene copies g-1. The proportion of D. geothermalis in those same samples was minor, 0.03 1.3 % of the total bacterial content. Nevertheless D. geothermalis may endanger paper quality as its DNA was shown in an end product. As an antifouling method towards biofilms we studied the electrochemical polarization. Two novel instruments were designed for this work. The double biofilm analyzer was designed for search for a polarization program that would eradicate D. geothermalis biofilm or from stainless steel under conditions simulating paper mill environment. The Radbox instrument was designed to study the generation of reactive oxygen species during the polarization that was effective in antifouling of D. geothermalis. We found that cathodic character and a pulsed mode of polarization were required to achieve detaching D. geothermalis biofilm from stainless steel. We also found that the efficiency of polarization was good on submerged, and poor on splash area biofilms. By adding oxidative biocides, bromochloro-5,5-dimethylhydantoin, 2,2-dibromo-2-cyanodiacetamide or peracetic acid gave additive value with polarization, being active on splash area biofilms. We showed that the cathodically weighted pulsed polarization that was active in removing D. geothermalis was also effective in generation of reactive oxygen species. It is possible that the antifouling effect relied on the generation of ROS on the polarized steel surfaces. Antifouling method successful towards D. geothermalis that is a tenacious biofouler and possesses a high tolerance to oxidative stressors could be functional also towards other biofoulers and applicable in wet industrial processes elsewhere.

Role and function of nonmuscle alpha-actinin-1 and -4 in regulating distinct subcategories of actin stress fibers in mammalian cells

Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration.

Components of fractionated stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the accessory glands before and during ejaculation. The stallion s ejaculate is a series of jets that differ in sperm concentration, semen volume and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the total number of sperm. The semen volume and sperm concentration in each of the jets decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with a low sperm concentration. The aims of these studies were to examine the effects of the different SP fractions, and the presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five experiments. The levels of selected enzymes, electrolytes and proteins in different SP fractions were determined. These studies also aimed at assessing the individual variation in the levels of the selected SP components and in the effects of SP on spermatozoa. The association between the components of SP and semen quality, sperm longevity, and fertility was examined with a stepwise linear regression analysis. Compared to samples containing SP during storage, centrifugation and the subsequent removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw motility parameters were also higher in samples containing SP compared to samples stored without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the samples stored with SP than those without SP, and this effect was seen in both sperm-rich and sperm-poor fractions. There were no differences in DNA integrity between fractions stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor fraction after SP removal. The differences between fractions in sperm motility after cooled storage were non-significant. The levels of alkaline phosphatase, acid phosphatase and β-glucuronidase were higher in the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions. The concentrations of sodium and chloride were highest in pre-sperm fluid. In the sperm-poor fraction, the level of potassium was associated with the maintenance of sperm motility during storage. The levels of alkaline and acid phosphatase were associated with sperm concentration and the total number of spermatozoa in the ejaculates. None of the measured SP components were correlated to the first cycle pregnancy rate. In summary, the removal of SP improved DNA integrity after cooled storage compared with samples containing SP. There were no differences in the maintenance of sperm motility between the sperm-rich and sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was found in the sperm-rich fractions stored without SP. In practice, the results presented in this thesis support the use of individual modifications of semen processing techniques for cooled transported semen from subfertile stallions.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.