Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Oncolytic Adenoviruses for Gynecologic Cancer

Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly developing field is as well replication competent agents, which allow improved tumor penetration and local amplification of the anti-tumor effect. Adenoviral cancer gene therapy approaches lack cross-resistance with other treatment options and therefore synergistic effects are possible. This study focused on development of adenoviral vectors suitable for treatment of various gynecologic cancer types, describing the development of the field from non-replicating adenoviral vectors to multiple-modified conditional replicating viruses. Transcriptional targeting of gynecologic cancer cells by the use of the promoter of vascular endothelial growth factor receptor type 1 (flt-1) was evaluated. Flt-1 is not expressed in the liver and thus an ideal promoter for transcriptional targeting of adenoviruses. Our studies implied that the flt-1 promoter is active in teratocarcinomas.and therefore a good candidate for development of oncolytic adenoviruses for treatment of this often problematic disease with then poor outcome. A tropism modified conditionally replicating adenovirus (CRAd), Ad5-Δ24RGD, was studied in gynecologic cancers. Ad5-Δ24RGD is an adenovirus selectively replication competent in cells defective in the p16/Rb pathway, including many or most tumor cells. The fiber of Ad5-Δ24RGD contains an integrin binding arginine-glycine-aspartic acid motif (RGD-4C), allowing coxackie-adenovirus receptor independent infection of cancer cells. This approach is attractive because expression levels of CAR are highly variable and often low on primary gynecological cancer cells. Oncolysis could be shown for a wide variety of ovarian and cervical cancer cell lines as well as primary ovarian cancer cell spheroids, a novel system developed for in vitro analysis of CRAds on primary tumor substrates. Biodistribution was evaluated and preclinical safety data was obtained by demonstrating lack of replication in human peripheral blood mononuclear cells. The efficicacy of Ad5-Δ24RGD was shown in different orthotopic murine models including a highly aggressive intraperitoneal model of disseminated ovarian cancer cells, where Ad5-Δ24RGD resulted in complete eradication of intraperitoneal disease in half of the mice. To further improve the selectivity and specificity of CRAds, triple-targeted oncolytic adenoviruses were cloned, featuring the cyclo-oxygenase-2 (cox-2) promoter, E1A transcomplementation and serotype chimerism. Those viruses were evaluated on ovarian cancer cells for specificity and oncolytic potency with regard to two different cox2 versions and three different variants of E1A (wild type, delta24 and delta2delta24). Ad5/3cox2Ld24 emerged as the best combination due to enhanced selectivity without potency lost in vitro or in an aggressive intraperitoneal orthotopic ovarian tumor model. In summary, the preclinical therapeutic efficacy of the CRAds tested in this study, taken together with promising biodistribution and safety data, suggest that these CRAds are interesting candidates for translation into clinical trials for gynecologic cancer.

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.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

Is prosopis a curse or a blessing? : An ecological economic analysis of an invasive alien tree species in Sudan.

The issue of the usefulness of different prosopis species versus their status as weeds is a matter of hot debate around the world. The tree Prosopis juliflora had until 2000 been proclaimed weedy in its native range in South America and elsewhere in the dry tropics. P. juliflora or mesquite has a 90-year history in Sudan. During the early 1990s a popular opinion in central Sudan and the Sudanese Government had begun to consider prosopis a noxious weed and a problematic tree species due to its aggressive ability to invade farmlands and pastures, especially in and around irrigated agricultural lands. As a consequence prosopis was officially declared an invasive alien species also in Sudan, and in 1995 a presidential decree for its eradication was issued. Using a total economic valuation (TEV) approach, this study analysed the impacts of prosopis on the local livelihoods in two contrasting irrigated agricultural schemes. Primarily a problem-based approach was used in which the derivation of non-market values was captured using ecological economic tools. In the New Halfa Irrigation Scheme in Kassala State, four separate household surveys were conducted due to diversity between the respective population groups. The main aim was here to study the magnitude of environmental economic benefits and costs derived from the invasion of prosopis in a large agricultural irrigation scheme on clay soil. Another study site, the Gandato Irrigation Scheme in River Nile State represented impacts from prosopis that an irrigation scheme was confronted with on sandy soil in the arid and semi-arid ecozones along the main River Nile. The two cases showed distinctly different effects of prosopis but both indicated the benefits to exceed the costs. The valuation on clay soil in New Halfa identified a benefit/cost ratio of 2.1, while this indicator equalled 46 on the sandy soils of Gandato. The valuation results were site-specific and based on local market prices. The most important beneficial impacts of prosopis on local livelihoods were derived from free-grazing forage for livestock, environmental conservation of the native vegetation, wood and non-wood forest products, as well as shelterbelt effects. The main social costs from prosopis were derived from weeding and clearing it from farm lands and from canalsides, from thorn injuries to humans and livestock, as well as from repair expenses vehicle tyre punctures. Of the population groups, the tenants faced most of the detrimental impacts, while the landless population groups (originating from western and eastern Sudan) as well as the nomads were highly dependent on this tree resource. For the Gandato site the monetized benefit-cost ratio of 46 still excluded several additional beneficial impacts of prosopis in the area that were difficult to quantify and monetize credibly. In River Nile State the beneficial impact could thus be seen as completely outweighing the costs of prosopis. The results can contributed to the formulation of national and local forest and agricultural policies related to prosopis in Sudan and also be used in other countries faced with similar impacts caused by this tree.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Radical Enhanced Atomic Layer Deposition of Metals and Oxides

Atomic Layer Deposition (ALD) is a chemical, gas-phase thin film deposition method. It is known for its ability for accurate and precise thickness control, and uniform and conformal film growth. One area where ALD has not yet excelled is film deposition at low temperatures. Also deposition of metals, besides the noble metals, has proven to be quite challenging. To alleviate these limitations, more aggressive reactants are required. One such group of reactants are radicals, which may be formed by dissociating gases. Dissociation is most conveniently done with a plasma source. For example, dissociating molecular oxygen or hydrogen, oxygen or hydrogen radicals are generated. The use of radicals in ALD may surmount some of the above limitations: oxide film deposition at low temperatures may become feasible if oxygen radicals are used as they are highly reactive. Also, as hydrogen radicals are very effective reducing agents, they may be used to deposit metals. In this work, a plasma source was incorporated in an existing ALD reactor for radical generation, and the reactor was used to study five different Radical Enhanced ALD processes. The modifications to the existing reactor and the different possibilities during the modification process are discussed. The studied materials include two metals, copper and silver, and three oxides, aluminium oxide, titanium dioxide and tantalum oxide. The materials were characterized and their properties were compared to other variations of the same process, utilizing the same metal precursor, to understand what kind of effect the non-metal precursor has on the film properties and growth characteristics. Both metals were deposited successfully, and silver for the first time by ALD. The films had low resistivity and grew conformally in the ALD mode, demonstrating that the REALD of metals is true ALD. The oxide films had exceptionally high growth rates, and aluminium oxide grew at room temperature with low cycle times and resulted in good quality films. Both aluminium oxide and titanium dioxide were deposited on natural fibres without damaging the fibre. Tantalum oxide was also deposited successfully, with good electrical properties, but at slightly higher temperature than the other two oxides, due to the evaporation temperature required by the metal precursor. Overall, the ability of REALD to deposit metallic and oxide films with high quality at low temperatures was demonstrated.

The Catholic Internet discussion on cloning : A study in cognitive rhetoric

This work combines the cognitive theory of folk-theoretical thought with the classical Aristotelian theory of artistic proof in rhetoric. The first half of the work discusses the common ground shared by the elements of artistic proof (logos, pathos, ethos) and the elements of folk-theoretical thought (naïve physics, folk biology, folk psychology, naïve sociology). Combining rhetoric with the cognitive theory of folk-theoretical thought creates a new point of view for argumentation analysis. The logos of an argument can be understood as the inferential relations established between the different parts of an argument. Consequently, within this study the analysis of logos is to be viewed as the analysis of the inferential folk-theoretical elements that make the suggested factual states-of-things appear plausible within given argumentative structures. The pathos of an argumentative structure can be understood as determining the quality of the argumentation in question in the sense that emotive elements play a great part in what can be called a distinction between good and deceptive rhetoric. In the context of this study the analysis of pathos is to be viewed as the analysis of the emotive content of argumentative structures and of whether they aim at facilitating surface- or deep cognitive elaboration of the suggested matters. The ethos of an argumentative structure means both the speaker-presentation and audience-construct that can be discerned within a body of argumentation. In the context of this study, the analysis of ethos is to be understood as the analysis of mutually manifest cognitive environments in the context of argumentation. The theory is used to analyse Catholic Internet discussion concerning cloning. The discussion is divided into six themes: Human Dignity, Sacred Family, Exploitation / Dehumanisation, Playing God, Monsters and Horror Scenarios and Ensoulment. Each theme is analysed for both the rhetorical and the cognitive elements that can be seen creating persuasive force within the argumentative structures presented. It is apparent that the Catholic voices on the Internet extensively oppose cloning. The voices utilise rhetoric that is aggressive and pejorative more often than not. Furthermore, deceptive rhetoric (in the sense presented above) plays a great part in argumentative structures of the Catholic voices. The theory of folk-theoretical thought can be seen as a useful tool for analysing the possible reasons why the Catholic speakers think about cloning and choose to present cloning in their argumentation as they do. The logos utilized in the argumentative structures presented can usually be viewed as based on folk-theoretical inference concerning biology and psychology. The structures of pathos utilized generally appear to aim at generating fear appeal in the assumed audiences, often incorporating counter-intuitive elements. The ethos utilised in the arguments generally revolves around Christian mythology and issues of social responsibility. These structures can also be viewed from the point of view of folk psychology and naïve sociological assumptions.

The Population Consequences of Sex and Conflict

Natural selection generally operates at the level of the individual, or more specifically at the level of the gene. As a result, individual selection does not always favour traits which benefit the population or species as a whole. The spread of an individual gene may even act to the detriment of the organism in which it finds. Thus selection at the level of the individual can affect processes at the level of the organism, group or even at the level of the species. As most behaviours ultimately affect births, deaths and the distribution of individuals, it seems inevitable that behavioural decisions will have an impact on population dynamics and population densities. Behavioural decisions can often involve costs through allocation of energy into behavioural strategies, such as the investment into armaments involved in fighting over resources or increased mortality due to injury or increased predation risk. Similarly, behaviour may act o to benefit the population, in terms of higher survival and increased fecundity. Examples include increased investment through parental care, choosing a mate based on the nuptial gifts they may supply and choosing territories in the face of competition. Investigating the impact of behaviour on population ecology may seem like a trivial task, but it is likely to have important consequences at different levels. For example, antagonistic behaviour may occasionally become so extreme that it increases the risk of extinction, and such extinction risk may have important implications for conservation. As a corollary, any such behaviour may also act as a macroevolutionary force, weeding out populations with traits which, whilst beneficial to the individuals in the short term, ultimately result in population extinction. In this thesis, I examine how behaviours, specifically conflict and competition over a resource and aspects of behaviour involved in sexual selection, can affect population densities, and what the implications are for the evolution and ecology of the populations in question. It is found that both behaviours related to individual conflict and mating strategies can have an effect at the level of the population, but that various factors, such as a feedback between selection and population densities or macroevolution caused by species extinctions, may act to limit the intensity of conflicts that we observe in nature.

Effects of 17α-ethinyl estradiol on the mating system of the sand goby (Pomatoschistus minutus)

In aquatic environments, endocrine disrupting chemicals (EDCs) that interfere with the endocrinology of males and females form a threat to the maintenance of populations. EDCs are a diverse group of natural and manmade chemicals that already at very low concentrations (at nanogram levels) can have severe effects on reproduction by individuals, e.g. complete sex reversal, feminisation of males, impaired reproduction even resulting in near extinction of populations. With regard to fish, despite the extensive literature on physiological effects of EDCs, very little is known about potential population-level effects. In this thesis, I examined how 17α-ethinyl estradiol (EE2), a synthetic estrogen used in oral contraceptive pills, affects the reproductive behaviour of a marine fish, the sand goby (Pomatoschistus minutus). The aims were fourfold. First, I investigated how exposure to EE2 affects courtship and parental care of sand goby males. Secondly, I looked at effects on the mating system and sexual selection. In the third study, I observed the effects of exposure in a social context where exposed males had to compete with non-exposed males for resources and mates. Finally, I studied the effects of exposure on male-male competition and male aggressive behaviour. This work revealed that EE2 exposure impairs the ability of males to acquire and defend a nest, as well as diminishes the attractiveness of males to females by decreasing their courtship and aggressive behaviour. These effects are harmful for a male whose reproductive success is determined by the ability to compete for limited resources and to attract mates. Furthermore, this thesis showed that selection on male size was relaxed after EE2 exposure and male size had a smaller effect on mating success. These effects can be of a profound nature as they interfere with sexual selection, and may in the long run lead to the loss of traits maintained through sexual selection. The thesis shows that an exposure to environmentally relevant levels of EE2 clearly reduces the chances of individuals to reproduce successfully. Furthermore, it strongly suggests that several types of biomarkers should be used to detect and assess the effects of EDC exposure because severe behavioural effects can sometimes be seen before effects are detectable at the molecular or morphometric level. Behavioural assays should be considered an important complementary tool for the standard ecotoxicological assays because observed behavioural changes have direct and negative effects on fitness, while the connection between changes in molecular expression and fitness may be less obvious.

Living on voles - plastic life of the Ural owl

Individuals face variable environmental conditions during their life. This may be due to migration, dispersion, environmental changes or, for example, annual variation in weather conditions. Genetic adaptation to a novel environment happens through natural selection. Phenotypic plasticity allows, however, a quick individual response to a new environment. Phenotypic plasticity may also be beneficial for individual if the environment is highly variable. For example, eggs are costly to produce. If the food conditions vary significantly between breeding seasons it is useful to be able to adjust the clutch and egg size according to the food abundance. In this thesis I use Ural owl vole system to study phenotypic plasticity and natural selection using a number of reproduction related traits. The Ural owl (Strix uralensis) is a long-lived and sedentary species. The reproduction and survival of the Ural owl, in fact their whole life, is tied to the dramatically fluctuating vole densities. Ural owls do not cause vole cycles but they have to adjust their behaviour to the rather predictable population fluctuations of these small mammals. Earlier work with this system has shown that Ural owl laying date and clutch size are plastic in relation to vole abundance. Further, individual laying date clutch size reaction norms have been shown to vary in the amount of plasticity. My work extends the knowledge of natural selection and phenotypic plasticity in traits related to reproduction. I show that egg size, timing of the onset of incubation and nest defense aggressiveness are plastic traits with fitness consequences for the Ural owl. Although egg size is in general thought to be a fixed characteristic of an individual, this highly heritable trait in the Ural owl is also remarkably plastic in relation to the changes in vole numbers, Ural owls are laying the largest eggs when their prey is most abundant. Timing of the onset of incubation is an individual-specific property and plastic in relation to clutch size. Timing of incubation is an important underlying cause for asynchronous hatching in birds. Asynchronous hatching is beneficial to offspring survival in Ural owl. Hence, timing of the onset of incubation may also be under natural selection. Ural owl females also adjust their nest defense aggressiveness according to the vole dynamics, being most aggressive in years when they produce the largest broods. Individual females show different levels of nest defense aggressiveness. Aggressiveness is positively correlated with the phenotypic plasticity of aggressiveness. As elevated nest defense aggressiveness is selected for, it may promote the plasticity of aggressive nest defense behaviour. All the studied traits are repeatable or heritable on individual level, and their expression is either directly or indirectly sensitive to changes in vole numbers. My work considers a number of important fitness-related traits showing phenotypic plasticity in all of them. Further, in two chapters I show that there is individual variation in the amount of plasticity exhibited. These findings on plasticity in reproduction related traits suggest that variable environments indeed promote plasticity.

The effects of eutrophication on alternative reproductive tactics in threespine sticklebacks

When organisms compete for mates and fertilisations, the process of sexual selection drives the evolution of traits that increase reproductive success. The traits targeted by selection, and the extent to which they change, are constrained by the local environment. Sexual selection due to female mate choice can be undermined by alternative reproductive tactics (ARTs), which refers to discontinuous variation in traits or behaviours used in reproduction. As human activities are rapidly changing our planet, this raises the question how ARTs will be affected. Fish show a bewildering diversity of ARTs, which make them good model organisms to answer these questions. One example of human-induced environmental change, which is affecting aquatic ecosystems around the world, is eutrophication, the over-enrichment of water bodies with nutrients. One of its effects is decreased underwater visibility due to increases in both turbidity and vegetation density. The aims of this thesis were to investigate the effects increased turbidity and vegetation density have on an ART in sticklebacks, a fish common to marine and fresh water bodies of the Northern hemisphere. I furthermore investigated how this affected sexual selection for male size, a trait commonly under selection. I used a combination of behavioural observations in microcosms, where I manipulated underwater visibility, with collection of genetic material to reconstruct parentage of broods, and thus identify sneak fertilisations. The results show that turbidity might have weak negative effects on the frequency of sneaking behaviour, although this behaviour was rather infrequent in these experiments, which complicates firm conclusions. In dense vegetation the number of sneak fertilisations decreased slightly, as fewer nesting males sneaked, while the number of non-nesting males sneaking remained constant. The paternity analyses revealed that a significantly smaller fraction of eggs was sneak fertilised under dense vegetation. Furthermore, amongst the nesting males that sneaked, the amount of eggs sneak fertilised correlated positively with courtship success. A reduction in sneaking by these males under dense vegetation equalised the distribution of fertilisation success, in turn contributing to a decrease in the opportunity for selection. Under dense vegetation significantly more males built nests, which has also been observed in previous field studies. In a separate experiment we addressed if such changes in the proportion of nesters and non-nesters, without changes in visibility, affected the incidence of sneak fertilisation. My results show this was not the case, likely because sneaking is an opportunistic tactic shown by both nesters and non-nesters. Non-nesters did sneak proportionately more when there were many of them, which could be due to changes in the cost-benefit ratio of sneaking. As nesters can only attack one intruder at a time, the costs and risks per sneaker will decrease as the number of sneakers increases. The defensive behaviours shown by the nesters before spawning shifted to a more aggressive form of nest defence. This could be because less aggressive behaviours lose their effectiveness when the number of intruders increases. It could also indicate that the risks associated with aggressive behaviours decrease when there are fewer fellow nesters, as other studies indicate nesters are competitive and aggressive individuals. Under turbid conditions I did not detect changes in the opportunity for selection, based on fertilisation success, nor was male size under significant selection under clear or turbid conditions. More thorough analyses under densely vegetated conditions across the nesting, courtship and fertilisation stages revealed a decrease in the opportunity for selection across all stages. A reduction in sneaking by nesters contributed to this. During the nesting stage, but not during later stages, body size was under significant directional selection under sparse, but not dense vegetation. This illustrates the importance of considering all selection stages to get a complete picture of how environmental changes affect sexual selection. Leaving out certain stages or subgroups can result in incomplete or misleading results.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Juvenile neuronal ceroid lipofuscinosis; psychiatric symptoms, GAD-autoantibodies, and response to medication

Juvenile neuronal ceroid lipofuscinosis (JNCL) is one of the most common neurodegenerative diseases in childhood. Its clinical onset, with visual failure as the first sign, is between the ages of 4 to 8 years. During the disease progress, epilepsy, motor symptoms, cognitive decline, and psychiatric symptoms become apparent. It leads to premature death between ages 15 and 30. Treatment consists of symptomatic drug administration and various forms of rehabilitation, but to date, no curative treatment exists. To gain a more comprehensive picture of psychiatric problems, symptoms were evaluated by the Child Behavior Checklist, the Teacher Report Form, and the Children s Depression Inventory. The JNCL patients had a great number of severe psychiatric symptoms, with wide inter-individual variability. The most common symptoms were social, thought, attention, and sleep problems, somatic complaints, and aggressive behaviour. Patients with psychotropic treatment had more problems than did those without psychotropic treatment, and female patients had more problems than did males. Between 10 and 20% of the patients reported depressive symptoms. In a 5-year follow-up, [123I]β-CIT SPECT and MRI revealed a tendency of decreasing serotonin transporter (SERT) availability and progressive brain atrophy. The correlation between changes in midbrain SERT and total brain volume was positive; no correlation appeared between SERT or brain atrophy and depressive symptoms. Thus, it seems likely that the low SERT availability is associated with progressive brain atrophy; it may also predispose towards depression, however. An open survey of psychotropic drugs and their efficacy was performed on JNCL patients in Finland. The most commonly used psychotropic drugs were the antidepressant citalopram and the antipsychotic risperidone. Their efficacy was good or satisfactory in the majority of cases and they seemed well tolerated. Quetiapine had a marked effect on one patient with a history of severe psychotic symptoms. Glutamate decarboxylase 65 autoantibodies (GAD65ab), found in JNCL patients, indicate that an immunomediated reaction against GAD or GABAergic neurons may play a part in the underlying pathogenetic mechanism. GAD65ab s also appeared in the serum of all eight JNCL patients included and intermittent corticosteroid therapy was initiated in all cases. After one year, the GAD65ab s had disappeared in the two oldest patients, who experienced an improvement in motor symptoms and alertness associated with their prednisolone therapy. Two younger patients experienced a significant IQ increase, but no change in GADab s. A randomized study with longer follow-up time is needed, however, to clarify the effect of prednisolone on disease progression.

Health-Related Quality of Life, Costs and Treatment of Inflammatory Rheumatic Diseases in Routine Practice : With special emphasis on biological drugs

Rheumatoid arthritis (RA) and other chronic inflammatory joint diseases already begin to affect patients health-related quality of life (HRQoL) in the earliest phases of these diseases. In treatment of inflammatory joint diseases, the last two decades have seen new strategies and treatment options introduced. Treatment is started at an earlier phase; combinations of disease-modifying anti-rheumatic drugs (DMARDs) and corticosteroids are used; and in refractory cases new drugs such as tumour necrosis factor (TNF) inhibitors or other biologicals can be started. In patients with new referrals to the Department of Rheumatology of the Helsinki University Central Hospital, we evaluated the 15D and the Stanford Health Assessment Questionnaire (HAQ) results at baseline and approximately 8 months after their first visit. Altogether the analysis included 295 patients with various rheumatic diseases. The mean baseline 15D score (0.822, SD 0.114) was significantly lower than for the age-matched general population (0.903, SD 0.098). Patients with osteoarthritis (OA) and spondyloarthropathies (SPA) reported the poorest HRQoL. In patients with RA and reactive arthritis (ReA) the HRQoL improved in a statistically significant manner during the 8-month follow-up. In addition, a clinically important change appeared in patients with systemic rheumatic diseases. HAQ score improved significantly in patients with RA, arthralgia and fibromyalgia, and ReA. In a study of 97 RA patients treated either with etanercept or adalimumab, we assessed their HRQoL with the RAND 36-Item Health Survey 1.0 (RAND-36) questionnaire. We also analysed changes in clinical parameters and the HAQ. With etanercept and adalimumab, the values of all domains in the RAND-36 questionnaire increased during the first 3 months. The efficacy of each in improving HRQoL was statistically significant, and the drug effects were comparable. Compared to Finnish age- and sex-matched general population values, the HRQoL of the RA patients was significantly lower at baseline and, despite the improvement, remained lower also at follow-up. Our RA patients had long-standing and severe disease that can explain the low HRQoL also at follow-up. In a pharmacoeconomic study of patients treated with infliximab we evaluated medical and work disability costs for patients with chronic inflammatory joint disease during one year before and one year after institution of infliximab treatment. Clinical and economic data for 96 patients with different arthritis diagnoses showed, in all patients, significantly improved clinical and laboratory variables. However, the medical costs increased significantly during the second period by 12 015 (95% confidence interval, 6 496 to 18 076). Only a minimal decrease in work disability costs occurred mean decrease 130 (-1 268 to 1 072). In a study involving a switch from infliximab to etanercept, we investigated the clinical outcome in 49 patients with RA. Reasons for switching were in 42% failure to respond by American College of Rheumatology (ACR) 50% criteria; in 12% adverse event; and in 46% non-medical reasons although the patients had responded to infliximab. The Disease Activity Score with 28 joints examined (DAS28) allowed us to measure patients disease activity and compare outcome between groups based on the reason for switching. In the patients in whom infliximab was switched to etanercept for nonmedical reasons, etanercept continued to suppress disease activity effectively, and 1-year drug survival for etanercept was 77% (95% CI, 62 to 97). In patients in the infliximab failure and adverse event groups, DAS28 values improved significantly during etanercept therapy. However, the 1-year drug survival of etanercept was only 43% (95% CI, 26 to 70) and 50% (95% CI, 33 to 100), respectively. Although the HRQoL of patients with inflammatory joint diseases is significantly lower than that of the general population, use of early and aggressive treatment strategies including TNF-inhibitors can improve patients HRQoL effectively. Further research is needed in finding new treatment strategies for those patients who fail to respond or lose their response to TNF-inhibitors.