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  • Santangeli, Andrea (Helsingin yliopisto, 2013)
    Humans are the main cause of the on-going large-scale biodiversity crisis, mostly through processes like habitat loss and fragmentation, and habitat degradation. The recent recognition of the scale and rate of biodiversity erosion has stimulated strong political and institutional reactions, culminating in the implementation of a large number of conservation initiatives. Such efforts have been largely insufficient to revert or slow down the rapid loss of biodiversity. Commonly, conservation resources have been allocated based on decisions supported by traditional knowledge or expert opinion rather than scientific evidence. Therefore, it is relevant that interventions are evaluated, to ultimately allow learning from past actions and taking better decisions in the future. With this thesis I aim to provide evidence needed to improve the effectiveness of different approaches to conservation of some species affected by anthropogenic activities. In doing so, I considered conservation interventions implemented mostly on private land with different underlying approaches: voluntary and inexpensive (based on self-motivation of landowners); voluntary market-based (landowners are compensated); Compulsory land reservation or legislation (landowners have no choice). I first evaluate the effectiveness of a conservation program aimed at protecting raptor nests in private forests of North Karelia in eastern Finland. I show that here an inexpensive voluntary approach, based on self-motivation of landowners, may represent an effective instrument for achieving conservation with very limited financial resources. This approach was effective not only at eliciting participation of local forest owners, but it also provided ecological benefits to the raptor species considered. Relevant outcomes for practical conservation can also emerge when multiple interventions are compared. This was the case for nest protection of the Montagu s harrier breeding in cropland of Spain and France. In France, protection of nests from harvesting operations has been achieved on a voluntary basis. Here I show that the most effective interventions to enhance nest productivity were those that not only protect from harvesting, but also from predation. This was achieved by erecting a protective fence around the nest. On the other hand, some nest protection measures in Spain were more expensive due to payments to farmers. Here, temporary removal of the chicks during harvest operations or relocation of the nest to a nearby safe place, as well as harvest delay, were the most effective measures to enhance nest productivity. Harvest delay was also the most expensive among all measures, therefore removal or relocation of the nest should be prioritized wherever it is operationally feasible. Interestingly, the most commonly employed measure, the retention of a small buffer of un-harvested crop, was also less effective compared to the other means. Unexpected but positive outcomes for conservation management emerged also from an evaluation of the effects of nest site protection for breeding White-tailed eagles in south-western Finland. The species was breeding as often and successfully in protected and unprotected areas, which suggests that compulsory and expensive protection through land reservation may not be necessary under the studied conditions. The species apparently thrives also in unprotected land subject to some levels of anthropogenic activities. I found opposite results in a study on protection of flying squirrel sites in Finland. Here I provide evidence indicating that the enforced legislation to protect the species habitat in Finnish forests is ineffective. The species occupancy at sites protected according to the law strongly declined following tree harvest. This indicates that the primary objective of the legislation (i.e. prevent deterioration of the sites where the species occurs) are not met. This is due to the fact that conservation of flying squirrel s habitat may conflict with forestry interests, and thus restrictions have been largely set in favour of the latter and at the detriment of the former. The case studies presented here indicate that evaluating the effectiveness of past actions is important. This step allows understanding whether past efforts have reached their initial objectives. Only with the strength of this evidence it is possible to adaptively revise current conservation plans and increase the chances of reaching the desired outcome from any given action. This is particularly relevant in the modern era, where conservation challenges are enormous, and the resources limited. Therefore, it is crucial that any implemented effort produces the best possible outcome for conservation.
  • Rintala, Janne-Markus (Helsingin yliopisto, 2009)
    The seasonal occurrence of sea ice that annually covers almost half the Baltic Sea area provides a unique habitat for halo- and cold temperature-tolerant extremophiles. Baltic Sea ice biology has more than 100 years of tradition that began with the floristic observation of species by the early pioneers using light microscopic techniques that were the only thing available at the time. Since the discovery of life within sea ice, more technologies have become available for taxonomy. Electron microscopy and genetic evidence have been used to identify sea ice biota revealing increased numbers of taxa. Meanwhile ecologists have used light microscopic cell enumeration in addition to the chemical and physical properties of sea ice in attempts to explain the food web structure of sea ice and its functions. Thus, during the Baltic winter, the sea ice hosts more abundant and diverse microbial communities than the water column beneath it. These communities are typically dominated by autotrophic diatoms together with a diverse assortment of dinoflagellates, auto- and heterotrophic flagellates, ciliates, metazoan rotifers and bacteria, which are mostly responsible for the recycling of nutrients. This thesis comprises ecological and systematic studies. In addition to the results of the previous studies carried out on landfast ice, the data presented here provide new insight into the spatial distribution of pelagial sea ice, which has remained largely unexplored. The studies reveal spatial heterogeneity in the pelagial sea ice of the Gulf of Bothnia. There were mismatches in chlorophyll-a concentrations and in photosynthetic efficiencies of the communities studied. The temporal succession was followed and experimental studies performed investigating the community responses towards increased or decreased light in landfast ice in the Gulf of Finland. The systematic studies carried out with established dinoflagellate cultures revealed a new resting cyst belonging to common sea ice dinoflagellate, Scrippsiella hangoei (Schiller) Larsen 1995. The cyst can be used to explain the overwintering of this species during prolonged periods of darkness. The dissimilarities and similarities in the material isolated from the sea ice called for description of a new subspecies Heterocapsa arctica ssp. frigida. The cells obtained in the cultured material were unlike those of the previously described species, necessitating description of ssp. frigida. As a result of its own unique habitus, the subspecies had been noted by Finnish taxonomists during the past three decades and thus its annual occurrence and geographical distribution in the Baltic Sea. This illustrates how combining ecology and systematics increases our understanding of organisms.
  • Lehikoinen, Annukka (Helsingin yliopisto, 2014)
    Environmental risk assessment (ERA) is a process of estimating the probability and consequences of an adverse event due to pressures or changes in environmental conditions resulting from human activities. Its purpose is to search the optimal courses of action under uncertainty when striving for the sustainable use of environment through minimizing the potential losses. As environmental issues are typically multidisciplinary, addressing large amount of eco-societal inter-linkages, an optimal tool for the ERA should enable the efficient integration and meta-analysis of multidisciplinary knowledge. By describing the causalities and studying the interactions among its components, this kind of integrative analysis provides us better understanding about the environmental system in focus. In addition, the functional ERA application should allow exploring, explaining and forecasting the responses of an environmental system to changes in natural and human induced stressors, serving as a decision support model that enables the search of optimal management strategy, also in the presence of imperfect knowledge. Bayesian Network (BN) is a graphical model that enables the integration of both quantitative and qualitative data and knowledge to a causal chain of inference. It is a powerful tool for synthesising knowledge, logic and rules, providing aid for thinking about complex systems that are too demanding to be analysed by human brains alone. In a BN, all the knowledge is handled in the form of probability distributions, thus the result represents the prevailing state of knowledge. The method facilitates analysing the location and amount of uncertainty explicitly, as well as enables studying its significance when it comes to the decision making. The main contribution of this thesis is to share experiences and ideas about the development and use of the ERA applications executed by using the BN as method. The perspective of the work is dichotomic. The objective in the separate studies presented in the articles have been on one hand to develop tools for integrating available knowledge and materials to enable the quantitative assessment of the environmental risks. On the other hand, the ultimate aim has been to learn more about the environmental risks and their potential management in the case study area of the Gulf of Finland. In this thesis, both of these perspectives are considered. Eutrophication and oil transportations at the Gulf of Finland are used as the case issues. The thesis concludes that Bayesian networks have plenty of properties that are useful for ERA and the method can be used for solving problems typical for that field analytically. By planting the developed graphical BNs in the commonly used Drivers-Pressures-States-Impacts-Responses -problem structuring framework, it is also demonstrated that combining these two approaches can be helpful in conceptual modeling, enabling the better framing of the research problem at hand and thinking about it systematically. The greatest challenges concerning the BN-ERA modeling are found to be related to the computational limitations of the current BN software, when it comes to the joint use of the discretised and continuous variables, as well as the restricted capacity to include the spatial resolution to the models. Producing the prior probability distributions by using deterministic models is also noted to be relatively tedious and time-consuming. The issues of end use of the applications, problems related to the scientific publishing of them, as well as the advantages and challenges of working in the multidisciplinary research teams are discussed.
  • Laakkonen, Mika (Helsingin yliopisto, 2007)
    Predation forms one of the main selective forces in nature and in a vast number of prey species the behavioural responses form the main way to avoid predation. World wide numerous captive breeding programs are used to produce fish and other animal species for conservational reintroductions. However, rearing animals in the absence of predators in captivity has been shown to weaken their predator avoidance skills and lead to behavioural divergence between wild and captive-bred populations. In my thesis I studied the effects of predator odour exposures on antipredator behavioural and physiological responses of captive reared Saimaa Arctic charr. This charr population is the most endangered fish population in Finland and a sample of the remaining population has been taken to captive breeding and used for an extensive reintroduction program. Lowered responsiveness to predators is probably one of the major reasons for the poor survival probability of the charr after release into the wild. The main aims of my thesis were to explore the reasons for behavioural phenotypic variation in this charr population and whether naïve charr young could be trained to recognise their natural predators. The predator species in my thesis were burbot (Lota lota) and pikeperch (Sander lucioperca). In my thesis I showed that the captive-bred charr responded to chemical cues from burbot and pikeperch, but the magnitude of responses was linked to the predator species. The burbot odour increased the spatial odour avoidance of the charr young. On the other hand, in the pikeperch treatment charr reduced their relative swimming activity and tended to show more freezing behaviour relative to the burbot treatment. It seems evident that these different responses are related to the different hunting tactics of predator species. Furthermore, I detected wide between-family differences in antipredator responsiveness (i.e. inherited variation in antipredator behaviours) in this captive stock. Detected differences were greater in the response towards burbot than towards pikeperch. These results, in addition to predator-specific antipredator responses, suggest that there is a clear inherited component in antipredator responsiveness in Saimaa charr population and that the detected inherited differences could explain a part of the behavioural phenotypic variation in this population. In my thesis I also found out that both social learning and direct exposure to live predators enhance the antipredator responsiveness of charr young. In addition, I obtained indications that predator odour exposures (i.e. life-skills training) in alevin and fry stages can fine-tune the innate antipredator responsiveness of charr. Thus, all these methods have the potential to enhance the innate antipredator responsiveness of naïve charr young, possibly also improving the post-release survival of these trained individuals in the wild. However, the next logical phase would be to carry out large scale survival studies in the wild to test this hypothesis. Finally, the results of my thesis emphasize that possible long-term life-skills training methods should take into account not only the behavioural but also the physiological effects of training.
  • Sarhan, Alia (Helsingin yliopisto, 2006)
    In my thesis I have been studying the effects of population fragmentation and extinction-recolonization dynamics on genetic and evolutionary processes in the Glanville fritillary butterfly (Melitaea cinxia). By conducting crosses within and among newly-colonized populations and using several fitness measures, I found a strong decrease in fitness following colonization by a few related individuals, and a strong negative relationship between parental relatedness and offspring fitness. Thereafter, I was interested in determining the number and relatedness of individuals colonizing new populations, which I did using a set of microsatellites I had previously developed for this species. Additionally, I am interested in the evolution of key life-history traits. By following the lifetime reproductive success of males emerging at different times in a semi-natural setup, I demonstrated that protandry is adaptive in males, and I was able to rule out, for M. cinxia, alternative incidental hypotheses evoked to explain the evolution of protandry in insects. Finally, in work I did together with Prof. Hanna Kokko, I am proposing bet-hedging as a new mechanism that could explain the evolution of polyandry in M. cinxia.
  • Tuomainen, Ulla (Helsingin yliopisto, 2012)
    When a habitat undergoes change, the first response of an individual is often behavioural adjustment. This immediate response can determine whether the population will survive or not, as behavioural flexibility gives time for genetic changes to arise later on. Habitat changes that alter reproductive behaviours can have long-lasting effects on populations. If the selective regime has changed under the new conditions, mate choice cues may no longer reliably reflect an individual s quality. Thus, animals have to be able to adjust their reproductive behaviours to the local conditions. The aim of my thesis was to discuss if and how animals are able to respond to rapid anthropogenic environmental change, and to study the mechanisms of the responses and the evolutionary consequences. The main focus was on the effects of human-induced eutrophication on the reproductive behaviour of fishes. Eutrophication is the result of increased nutrient input and can cause dense underwater vegetation and algal blooms. I used fishes from two very different ecosystems as model species, the Baltic Sea threespine stickleback (Gasterosteus aculeatus) and the desert goby (Chlamydogobius eremius), an endemic species of the Lake Eyre region in Central Australia. I investigated the effects of increased habitat complexity on courtship behaviour and the possibility of local differentiation in courtship and nest building behaviour depending on the level eutrophication in the habitat of origin. Furthermore, I observed the effect of turbidity on stickleback nest building behaviour. The results show that threespine stickleback males, which were born in areas that have been eutrophied for decades, court females at a higher intensity than males from clear water areas. Similarly, male desert gobies increased their courtship effort in dense vegetation. Intense courtship could be an adjustment to reduced visibility and lowered predation risk in the densely vegetated sites. However, there were no clear differences in nest building between males from clear and eutrophied areas under standardized conditions. This was expected as Baltic Sea sticklebacks prefer to nest under vegetation cover and are fairly rigid in adjusting their nest characteristics. Nest building was affected by increased turbidity: males built smaller nests with a larger nest entrance in turbid water. The large variation in the magnitude of phytoplankton blooms may require a rapid adjustment of the optimal nest structure to the current conditions. This thesis highlights the complex interactions that are set- off by human-induced changes in habitats and are followed by the immediate behavioural responses. It also encourages more research to tease apart the phenotypic and genetic components of the observed behavioural differences.
  • Hellstedt, Paavo (Helsingin yliopisto, 2005)
  • Kanerva, Teri (Helsingin yliopisto, 2006)
    This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.
  • Saarikangas, Juha (Helsingin yliopisto, 2010)
    Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.
  • Viitasalo, Satu (Helsingin yliopisto, 2007)
    Benthic-pelagic coupling describes processes that operate across and between the seafloor and open-water ecosystems. In soft-sediment communities, bioturbation by sediment-dwelling and epibenthic organisms may strongly shape habitat characteristics and influence processes, e.g. biogeochemical cycling, which supplies bioavailable nutrients to pelagic primary producers. In addition, benthic fauna may mediate benthic-pelagic coupling by affecting the survival and hatching of zooplankton dormant eggs in the sediment. In the shallow waters and seasonally fluctuating environment of the Baltic Sea, emergence from the seafloor essentially contributes to the dynamics of zooplankton pelagic populations. In this thesis, I examine how benthic organisms with different functional traits affect the link between the benthic and pelagic systems in the northern Baltic Sea. By means of experimental laboratory studies, the effects of sediment-dwelling (Monoporeia affinis, Macoma balthica and Marenzelleria spp.) and nectobenthic (Mysis spp.) taxa on the survival and hatching of zooplankton benthic eggs and on benthic nutrient fluxes and sediment structure were investigated. In the predation studies, the nectobenthic mysids Mysis spp. preyed upon benthic eggs of the cladoceran Bosmina longispina maritima (syn. B. coregoni maritima), both in pelagic and benthic environments. Of the sediment-dwelling species, the amphipod M. affinis and the bivalve M. balthica reduced the number of cladoceran eggs in the sediment, whereas the polychaetes Marenzelleria spp. had no effects on cladoceran eggs. Both M. balthica and M. affinis also increased the mortality rates of benthic eggs of copepods and rotifers. It was estimated that zooplankton eggs provide an additional carbon source for food-limited benthic communities. The results indicate that predation pressure on zooplankton benthic eggs may be strong, but varies widely depending on the season and the functional characteristics of the macrofauna. Macoma balthica buried cladoceran eggs and a fluorescent tracer from the sediment surface to a depth of 3 4 cm, indicating efficient sediment mixing. In contrast, the other taxa had fewer effects on particle distributions. In addition to organic matter mineralization, particle mixing is crucial to the success of benthic recruitment of zooplankton, since only eggs close to the sediment surface may hatch. Macoma balthica and M. affinis altered the patterns of zooplankton emergence from the sediment. In general, the highest emergence rates were observed in the absence of macroscopic fauna, and M. balthica exerted a stronger suppressive effect than M. affinis. Moreover, copepods were less severely affected than cladocerans, while only one species (Temora longicornis) clearly benefited from the presence of the macrofauna. These differences probably result from species-specific differences in the resistance of eggs to disturbances. The results show that benthic fauna may considerably alter the patterns of zooplankton emergence from the seafloor, thereby shaping zooplankton pelagic populations. The semi-motile M. balthica and Marenzelleria spp. increased the fluxes of phosphate and ammonium from the sediment to the water, whereas the motile M. affinis and Mysis mixta had a contrasting effect. In the eutrophied Baltic Sea, efficient internal cycling of bioavailable nutrients forms a strong feedback inhibiting the recovery of the ecosystem. Based on the results, a change in species dominance from the two motile taxa, susceptible to oxygen deficiency, to the more tolerant semi-motile taxa provides additional feedback, strengthening internal nutrient cycling and accelerating eutrophication, with deteriorating near-bottom oxygen conditions and changes in the benthic communities. In shallow-water ecosystems, benthic nutrient regeneration plays a key role in determining the overall productivity of the ecosystem. In addition, the results of this study show that the communities in the benthos may essentially contribute to the structure of those in the plankton.
  • Fagerholm, Susanna (Helsingin yliopisto, 2002)
  • Penttinen, Sari (Helsingin yliopisto, 2010)
    Physicochemical characterization of freshwater samples from Finland, Sweden, the Netherlands, and Spain revealed that water hardness and pH decreased and the quantity and quality of humic substances changed considerably in this geographical series from south to north. Since the ambient water chemistry may affect the availability of chemicals, the total aqueous concentration of a chemical may be insufficient to predict the bioconcentration, subsequent biological response, and thus risk. In addition, organisms could be affected directly by water quality characteristics. In this context the main objective of this thesis was to investigate the bioavailability of selected ecotoxicologically relevant chemicals (cadmium, benzo(a)pyrene, and pyrene) in various European surface waters and to show the importance of certain water chemistry characteristics in interpreting the bioavailability and toxicity results. The bioavailability of cadmium to Daphnia magna was examined in very soft humic lake water. Humic substances as natural ligands decreased the free and bioavailable proportion of cadmium in soft lake water. As a consequence the uptake rate and the acute toxicity decreased compared with the humic-free reference. When the hardness of humic lake water was artificially elevated, the acute toxicity of cadmium decreased, although the proportion of free cadmium increased. The decreased bioavailability of cadmium in hard water was a result of effective competition for uptake by the hardness cations, especially calcium ions. The protective role of humic substances and water hardness against cadmium toxicity was also observed in Lumbriculus variegatus, although D. magna was more sensitive to cadmium. The bioavailability of two polycyclic aromatic hydrocarbons (PAHs), pyrene and benzo(a)pyrene, was studied in European surface waters of varying water chemistry. Humic substances acted as complexing ligands with both PAHs, but the bioavailability of the more lipophilic benzo(a)pyrene to D. magna was affected more by humic substances than that of pyrene. In addition, not only the quantity of humic substances, but also their quality affected the bioavailability of benzo(a)pyrene. Nevertheless, the humic substances played a protective role in the photo-enhanced toxicity of pyrene under UV-B radiation. Water hardness had no effect on pyrene toxicity. Results indicate that the typical physicochemical characteristics of boreal freshwaters should be considered carefully in local and regional risk assessment of chemicals concerning the Fennoscandian region.
  • Dermadi Bebek, Denis (Helsingin yliopisto, 2014)
    Colorectal cancer (CRC) is one of the leading causes of death in developed countries. Although, a small fraction of cancers are caused by inherited genetic predisposition most of the CRCs are sporadic. In CRC, cancer risk is associated with lifestyle factors and aging. Even in dominantly inherited CRC predisposition such as in Lynch syndrome (LS), which is linked to germline mu- tations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2, cancer develops as a result of accumulation of genetic and epigenetic changes. After diagnosing an LS family, to be able to offer contiguous pre-symptomatic surveillance and predictive gene counseling to mutation carriers in a family, the pathogenicity assessment of a mutation is needed. Dependent on the type and the site of a germline mutation, inherited cancer risk may vary from high to low and especially in the latter case cancer risk may be strongly affected by lifestyle factors such as diet. Epidemiological studies on humans and previous studies on mice have shown that especially a Western-style diet (WD) may predispose colon mucosa to CRC. However, the mechanisms, which mediate the effects of diet on tumorigenesis are largely unknown. Since both genetic and lifestyle factors have been shown to predispose to cancer, this the- sis analyzed biochemical defects caused by inherited MMR gene mutations and Western diet exposure. Different MMR gene mutations may compromise MMR function through various biochemical defects. Here, we studied 18 inherited non-truncating mutations in MSH2, the second most frequently mutated gene among Lynch syndrome patients. We assessed protein stability, DNA binding, and ATP mediated DNA release abilities of the MSH2 variants. The majority of variants in the amino terminal region including the connector and lever domains p.V161D, p.G162R, p.G164R, p.L173P, p.L187P, p.C333Y, p.D603N) affected protein stabil- ity. Variations in the ATPase domain (p.A636P, p.G674A, p.C697F, p.I745-I746del, p.E749K) totally abolished either mismatch binding or release. Four protein variants (p.T33P, p.A272 V, p.G322D, p.V923E) expressed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) MSH2 protein, while two variants (p.N127S, p.A834T) were in- distinguishable from WT. To define the effects of Western-style diet, we analyzed protein expression changes in histolog- ically normal colon mucosa of wild type (Mlh1+/+) and CRC predisposed mice (Mlh1+/-) after a long-term feeding experiment with WD and AIN-93G control diet. Using network analysis and data mining we also determined which of the affected proteins might be putative play- ers in early CRC development. Our results pinpoint changes in a complex protein interaction network involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, which are important in the generation of reactive oxygen species (ROS) and cellular protection against ROS toxicity. Additionally, we detected SELENBP1 and LGALS4, which are implied in neoplastic processes. Our studies show that mutations in the MMR gene affect the biochemistry of MMR, can have an effect on the phenotype of the mutation carriers and in the latest study suggest that the high sensitivity to Western diet may be linked to haplo-insufficiency caused by a loss of function mutation in the Mlh1+/- mice.
  • Qian, Kui (Helsingin yliopisto, 2013)
    Human papillomaviruses (HPVs) form a large family among double stranded DNA (dsDNA) viruses, some types of which are the major causes of cervical cancer. HPV 16 is widely distributed and the most common high-risk HPV type and approximately half of the cervical cancers are associated with HPV type 16. Of the three HPV 16 encoded oncogenes, the function of E5 in regulating viral replication and pathogenesis is less well understood than E6 and E7. The microRNAs (miRNAs) are important small noncoding RNA molecules that regulate wide range of cellular functions. Some dsDNA viruses, such as SV40 and human polyomaviruses, have functional viral miRNAs. The functional and molecular similarities among dsDNA viruses suggest that HPV could encode viral miRNAs, which have not been validated thus far. The aim of this thesis was to study the functions of the host miRNAs in HPV 16 oncogene induction and identify novel HPV encoded viral miRNAs. We utilized microarray technology to investigate the effect of E5 on host miRNAs and mRNAs expression in 0 96 hours after E5 induction in a cell line model. Among the differentially expressed cellular miRNAs, we further validated the expression of hsa-mir-146a, hsa-mir-203, and hsa-mir-324-5p and some of their target genes in a time series of 96 hours of E5 induction. Our results indicate that HPV E5 expression has an impact through complex regulatory patterns of gene expression in the host cells, and part of those genes is regulated by the E5 protein. Second, high throughput sequencing was used to identify virus-encoded miRNAs. We prepared small RNA sequencing libraries from ten HPV-associated cervical lesions, including cancer and two HPV-harboring cell lines. For more flexible analysis of the sequencing data we developed miRSeqNovel, an R based workflow for miRNA sequencing data analysis, and applied it to the sequencing data to predict putative viral miRNAs and discovered nine putative papillomavirus encoded miRNAs. Viral miRNA validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38, and one by HPV 68. The expression of two HPV 16 miRNAs was further supported by in situ hybridization, and colocalization with p16INK4A staining, a marker of cervical neoplasia. Prediction of cellular target genes of HPV 16 encoded miRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development and cancer, which were also among the functions targeted by the E5 regulated host cell mRNA and miRNAs. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene, and one in the long control region (LCR).