Ion mobility spectrometry in liquid analysis

Ion mobility spectrometry (IMS) is a straightforward, low cost method for fast and sensitive determination of organic and inorganic analytes. Originally this portable technique was applied to the determination of gas phase compounds in security and military use. Nowadays, IMS has received increasing attention in environmental and biological analysis, and in food quality determination. This thesis consists of literature review of suitable sample preparation and introduction methods for liquid matrices applicable to IMS from its early development stages to date. Thermal desorption, solid phase microextraction (SPME) and membrane extraction were examined in experimental investigations of hazardous aquatic pollutants and potential pollutants. Also the effect of different natural waters on the extraction efficiency was studied, and the utilised IMS data processing methods are discussed. Parameters such as extraction and desorption temperatures, extraction time, SPME fibre depth, SPME fibre type and salt addition were examined for the studied sample preparation and introduction methods. The observed critical parameters were extracting material and temperature. The extraction methods showed time and cost effectiveness because sampling could be performed in single step procedures and from different natural water matrices within a few minutes. Based on these experimental and theoretical studies, the most suitable method to test in the automated monitoring system is membrane extraction. In future an IMS based early warning system for monitoring water pollutants could ensure the safe supply of drinking water. IMS can also be utilised for monitoring natural waters in cases of environmental leakage or chemical accidents. When combined with sophisticated sample introduction methods, IMS possesses the potential for both on-line and on-site identification of analytes in different water matrices.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.

Methods for construction and analysis of computational models in systems biology : applications to the modelling of the heat shock response and the self-assembly of intermediate filaments

Systems biology is a new, emerging and rapidly developing, multidisciplinary research field that aims to study biochemical and biological systems from a holistic perspective, with the goal of providing a comprehensive, system- level understanding of cellular behaviour. In this way, it addresses one of the greatest challenges faced by contemporary biology, which is to compre- hend the function of complex biological systems. Systems biology combines various methods that originate from scientific disciplines such as molecu- lar biology, chemistry, engineering sciences, mathematics, computer science and systems theory. Systems biology, unlike “traditional” biology, focuses on high-level concepts such as: network, component, robustness, efficiency, control, regulation, hierarchical design, synchronization, concurrency, and many others. The very terminology of systems biology is “foreign” to “tra- ditional” biology, marks its drastic shift in the research paradigm and it indicates close linkage of systems biology to computer science. One of the basic tools utilized in systems biology is the mathematical modelling of life processes tightly linked to experimental practice. The stud- ies contained in this thesis revolve around a number of challenges commonly encountered in the computational modelling in systems biology. The re- search comprises of the development and application of a broad range of methods originating in the fields of computer science and mathematics for construction and analysis of computational models in systems biology. In particular, the performed research is setup in the context of two biolog- ical phenomena chosen as modelling case studies: 1) the eukaryotic heat shock response and 2) the in vitro self-assembly of intermediate filaments, one of the main constituents of the cytoskeleton. The range of presented approaches spans from heuristic, through numerical and statistical to ana- lytical methods applied in the effort to formally describe and analyse the two biological processes. We notice however, that although applied to cer- tain case studies, the presented methods are not limited to them and can be utilized in the analysis of other biological mechanisms as well as com- plex systems in general. The full range of developed and applied modelling techniques as well as model analysis methodologies constitutes a rich mod- elling framework. Moreover, the presentation of the developed methods, their application to the two case studies and the discussions concerning their potentials and limitations point to the difficulties and challenges one encounters in computational modelling of biological systems. The problems of model identifiability, model comparison, model refinement, model inte- gration and extension, choice of the proper modelling framework and level of abstraction, or the choice of the proper scope of the model run through this thesis.

Application of morphometry, static DNA ploidy analysis, and steroid receptor expression in diagnosis and prognosis of Libyan breast cancer

The aim of this study was to describe the demographic, clinicopathological, biological and morphometric features of Libyan breast cancer patients. The supporting value of nuclear morphometry and static image cytometry in the sensitivity for detecting breast cancer in conventional fine-needle aspiration biopsies were estimated. The findings were compared with findings in breast cancer in Finland and Nigeria. In addation, the value of ER and PR were evaluated. There were 131 histological samples, 41 cytological samples, and demographic and clinicopathological data from 234 Libyan patients. The Libyan breast cancer is dominantly premenopausal and in this feature it is similar to breast cancer in sub-Saharan Africans, but clearly different from breast cancer in Europeans, whose cancers are dominantly postmenopausal in character. At presention most Libyan patients have locally advanced disease, which is associated with poor survival rates. Nuclear morphometry and image DNA cytometry agree with earlier published data in the Finnish population and indicate that nuclear size and DNA analysis of nuclear content can be used to increase the cytological sensitivity and specificity in doubtful breast lesions, particularly when free cell sampling method is used. Combination of the morphometric data with earlier free cell data gave the following diagnostic guidelines: Range of overlap in free cell samples: 55 μm2 -71 μm2. Cut-off values for diagnostic purposes: Mean nuclear area (MNA) >54 μm2 for 100% detection of malignant cases (specificity 84 %), MNA < 72 μm2 for 100% detection of benign cases (sensitivity 91%). Histomorphometry showed a significant correlation between the MNA and most clinicopathological features, with the strongest association observed for histological grade (p <0.0001). MNA seems to be a prognosticator in Libyan breast cancer (Pearson’s test r = - 0.29, p = 0.019), but at lower level of significance than in the European material. A corresponding relationship was not found in shape-related morphometric features. ER and PR staining scores were in correlation with the clinical stage (p= 0.017, and 0.015, respectively), and also associated with lymph node negative patients (p=0.03, p=0.05, respectively). Receptor-positive (HR+) patients had a better survival. The fraction of HR+ cases among Libyan breast cancers is about the same as the fraction of positive cases in European breast cancer. The study suggests that also weak staining (corresponding to as few as 1% positive cells) has prognostic value. The prognostic significance may be associated with the practice to use antihormonal therapy in HR+ cases. The low survival and advanced presentation is associated with active cell proliferation, atypical nuclear morphology and aneuploid nuclear DNA content in Libyan breast cancer patients. The findings support the idea that breast cancer is not one type of disease, but should probably be classified into premenopausal and post menopausal types.

Disturbance and ecosystem functioning : the role of changes in benthic biological traits

Rapid changes in biodiversity are occurring globally, as a consequence of anthropogenic disturbance. This has raised concerns, since biodiversity is known to significantly contribute to ecosystem functions and services. Marine benthic communities participate in numerous functions provided by soft-sedimentary ecosystems. Eutrophication-induced oxygen deficiency is a growing threat against infaunal communities, both in open sea areas and in coastal zones. There is thus a need to understand how such disturbance affects benthic communities, and what is lost in terms of ecosystem functioning if benthic communities are harmed. In this thesis, the status of benthic biodiversity was assessed for the open Baltic Sea, a system severely affected by broad-scale hypoxia. Long-term monitoring data made it possible to establish quantitative biodiversity baselines against which change could be compared. The findings show that benthic biodiversity is currently severely impaired in large areas of the open Baltic Sea, from the Bornholm Basin to the Gulf of Finland. The observed reduction in biodiversity indicates that benthic communities are structurally and functionally impoverished in several of the sub-basins due to the hypoxic stress. A more detailed examination of disturbance impacts (through field studies and -experiments) on benthic communities in coastal areas showed that changes in benthic community structure and function took place well before species were lost from the system. The degradation of benthic community structure and function was directed by the type of disturbance, and its specific temporal and spatial characteristics. The observed shifts in benthic trait composition were primarily the result of reductions in species’ abundances, or of changes in demographic characteristics, such as the loss of large, adult bivalves. Reduction in community functions was expressed as declines in the benthic bioturbation potential and in secondary biomass production. The benthic communities and their degradation accounted for a substantial proportion of the changes observed in ecosystem multifunctionality. Individual ecosystem functions (i.e. measures of sediment ecosystem metabolism, elemental cycling, biomass production, organic matter transformation and physical structuring) were observed to differ in their response to increasing hypoxic disturbance. Interestingly, the results suggested that an impairment of ecosystem functioning could be detected at an earlier stage if multiple functions were considered. Importantly, the findings indicate that even small-scale hypoxic disturbance can reduce the buffering capacity of sedimentary ecosystem, and increase the susceptibility of the system towards further stress. Although the results of the individual papers are context-dependent, their combined outcome implies that healthy benthic communities are important for sustaining overall ecosystem functioning as well as ecosystem resilience in the Baltic Sea.

Interpreting marine benthic ecosystem functioning in coastal waters: validating the biological trait concept

Coastal areas harbour high biodiversity, but are simultaneously affected by rapid degradations of species and habitats due to human interactions. Such alterations also affect the functioning of the ecosystem, which is primarily governed by the characteristics or traits expressed by the organisms present. Marine benthic fauna is nvolved in numerous functions such as organic matter transformation and transport, secondary production, oxygen transport as well as nutrient cycling. Approaches utilising the variety of faunal traits to assess benthic community functioning have rapidly increased and shown the need for further development of the concept. In this thesis, I applied biological trait analysis that allows for assessments of a multitude of categorical traits and thus evaluation of multiple functional aspects simultaneously. I determined the functional trait structure, diversity and variability of coastal zoobenthic communities in the Baltic Sea. The measures were related to recruitment processes, habitat heterogeneity, large-scale environmental and taxonomic gradients as well as anthropogenic impacts. The studies comprised spatial scales from metres to thousands of kilometres, and temporal scales spanning one season as well as a decade. The benthic functional structure was found to vary within and between seagrass landscape microhabitats and four different habitats within a coastal bay, in papers I and II respectively. Expressions of trait categories varied within habitats, while the density of individuals was found to drive the functional differences between habitats. The findings in paper III unveiled high trait richness of Finnish coastal benthos (25 traits and 102 cateogries) although this differed between areas high and low in salinity and human pressure. In paper IV, the natural reduction in taxonomic richness across the Baltic Sea led to an overall reduction in function. However, functional richness in terms of number of trait categories remained comparatively high at low taxon richness. Changes in number of taxa within trait categories were also subtle and some individual categories were maintained or even increased. The temporal analysis in papers I and III highlighted generalities in trait expressions and dominant trait categories in a seagrass landscape as well as a “type organism” for the northern Baltic Sea. Some initial findings were made in all four papers on the role of common and rare species and traits for benthic community functioning. The findings show that common and rare species may not always express the same trait categories in relation to each other. Rare species in general did not express unique functional properties. In order to advance the understanding of the approach, I also assessed some issues concerning the limitations of the concept. This was conducted by evaluating the link between trait category and taxonomic richness using especially univariate measures. My results also show the need to collaborate nationally and internationally on safeguarding the utility of taxonomic and trait data. The findings also highlight the importance of including functional trait information into current efforts in marine spatial planning and biomonitoring.

Data analysis tools and methods for DNA microarray and high-throughput sequencing data

The recent rapid development of biotechnological approaches has enabled the production of large whole genome level biological data sets. In order to handle thesedata sets, reliable and efficient automated tools and methods for data processingand result interpretation are required. Bioinformatics, as the field of studying andprocessing biological data, tries to answer this need by combining methods and approaches across computer science, statistics, mathematics and engineering to studyand process biological data. The need is also increasing for tools that can be used by the biological researchers themselves who may not have a strong statistical or computational background, which requires creating tools and pipelines with intuitive user interfaces, robust analysis workflows and strong emphasis on result reportingand visualization. Within this thesis, several data analysis tools and methods have been developed for analyzing high-throughput biological data sets. These approaches, coveringseveral aspects of high-throughput data analysis, are specifically aimed for gene expression and genotyping data although in principle they are suitable for analyzing other data types as well. Coherent handling of the data across the various data analysis steps is highly important in order to ensure robust and reliable results. Thus,robust data analysis workflows are also described, putting the developed tools andmethods into a wider context. The choice of the correct analysis method may also depend on the properties of the specific data setandthereforeguidelinesforchoosing an optimal method are given. The data analysis tools, methods and workflows developed within this thesis have been applied to several research studies, of which two representative examplesare included in the thesis. The first study focuses on spermatogenesis in murinetestis and the second one examines cell lineage specification in mouse embryonicstem cells.