Evolutionary and conservation biology of the Finnish house sparrow

Recently it has been recognized that evolutionary aspects play a major role in conservation issues of a species. In this thesis I have combined evolutionary research with conservation studies to provide new insight into these fields. The study object of this thesis is the house sparrow, a species that has features that makes it interesting for this type of study. The house sparrow has been ubiquitous almost all over the world. Even though being still abundant, several countries have reported major declines. These declines have taken place in a relatively short time covering both urban and rural habitats. In Finland this species has declined by more than two thirds in just over two decades. In addition, as the house sparrow lives only in human inhabited areas it can also raise public awareness to conservation issues. I used both an extensive museum collection of house sparrows collected in 1980s from all over Finland as well as samples collected in 2009 from 12 of the previously collected localities. I used molecular techniques to study neutral genetic variation within and genetic differentiation between the study populations. This knowledge I then combined with data gathered on morphometric measurements. In addition I analyzed eight heavy metals from the livers of house sparrows that lived in either rural or urban areas in the 1980s and evaluated the role of heavy metal pollution as a possible cause of the declines. Even though dispersal of house sparrows is limited I found that just as the declines started in 1980s the house sparrows formed a genetically panmictic population on the scale of the whole Finland. When compared to Norway, where neutral genetic divergence has been found even with small geographic distances, I concluded that this difference would be due to contrasting landscapes. In Finland the landscape is rather homogeneous facilitating the movements of these birds and maintaining gene flow even with the low dispersal. To see whether the declines have had an effect on the neutral genetic variation of the populations I did a comparison between the historical and contemporary genetic data. I showed that even though genetic diversity has not decreased due to the drastic declines the populations have indeed become more differentiated from each other. This shows that even in a still quite abundant species the declines can have an effect on the genetic variation. It is shown that genetic diversity and differentiation may approach their new equilibriums at different rates. This emphasizes the importance of studying both of them and if the latter has increased it should be taken as a warning sign of a possible loss of genetic diversity in the future. One of the factors suggested to be responsible for the house sparrow declines is heavy metal pollution. When studying the livers of house sparrows from 1980s I discovered higher levels of heavy metal concentrations in urban than rural habitats, but the levels of the metals were comparatively low and based on that heavy metal pollution does not seem to be a direct cause for the declines in Finland. However, heavy metals are known to decrease the amount of insects in urban areas and thus in the cities heavy metals may have an indirect effect on house sparrows. Although neutral genetic variation is an important tool for conservation genetics it does not tell the whole story. Since neutral genetic variation is not affected by selection, information can be one-sided. It is possible that even neutral genetic differentiation is low, there can be substantial variation in additive genetic traits indicating local adaptation. Therefore I performed a comparison between neutral genetic differentiation and phenotypic differentiation. I discovered that two traits out of seven are likely to be under directional selection, whereas the others could be affected by random genetic drift. Bergmann s rule may be behind the observed directional selection in wing length and body mass. These results highlight the importance of estimating both neutral and adaptive genetic variation.

SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Heated Nebulizer Microchips for Mass Spectrometry

Miniaturized analytical devices, such as heated nebulizer (HN) microchips studied in this work, are of increasing interest owing to benefits like faster operation, better performance, and lower cost relative to conventional systems. HN microchips are microfabricated devices that vaporize liquid and mix it with gas. They are used with low liquid flow rates, typically a few µL/min, and have previously been utilized as ion sources for mass spectrometry (MS). Conventional ion sources are seldom feasible at such low flow rates. In this work HN chips were developed further and new applications were introduced. First, a new method for thermal and fluidic characterization of the HN microchips was developed and used to study the chips. Thermal behavior of the chips was also studied by temperature measurements and infrared imaging. An HN chip was applied to the analysis of crude oil – an extremely complex sample – by microchip atmospheric pressure photoionization (APPI) high resolution mass spectrometry. With the chip, the sample flow rate could be reduced significantly without loss of performance and with greatly reduced contamination of the MS instrument. Thanks to its suitability to high temperature, microchip APPI provided efficient vaporization of nonvolatile compounds in crude oil. The first microchip version of sonic spray ionization (SSI) was presented. Ionization was achieved by applying only high (sonic) speed nebulizer gas to an HN microchip. SSI significantly broadens the range of analytes ionizable with the HN chips, from small stable molecules to labile biomolecules. The analytical performance of the microchip SSI source was confirmed to be acceptable. The HN microchips were also used to connect gas chromatography (GC) and capillary liquid chromatography (LC) to MS, using APPI for ionization. Microchip APPI allows efficient ionization of both polar and nonpolar compounds whereas with the most popular electrospray ionization (ESI) only polar and ionic molecules are ionized efficiently. The combination of GC with MS showed that, with HN microchips, GCs can easily be used with MS instruments designed for LC-MS. The presented analytical methods showed good performance. The first integrated LC–HN microchip was developed and presented. In a single microdevice, there were structures for a packed LC column and a heated nebulizer. Nonpolar and polar analytes were efficiently ionized by APPI. Ionization of nonpolar and polar analytes is not possible with previously presented chips for LC–MS since they rely on ESI. Preliminary quantitative performance of the new chip was evaluated and the chip was also demonstrated with optical detection. A new ambient ionization technique for mass spectrometry, desorption atmospheric pressure photoionization (DAPPI), was presented. The DAPPI technique is based on an HN microchip providing desorption of analytes from a surface. Photons from a photoionization lamp ionize the analytes via gas-phase chemical reactions, and the ions are directed into an MS. Rapid analysis of pharmaceuticals from tablets was successfully demonstrated as an application of DAPPI.

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Mammographic mass-screening and future breast cancer burden in Finland

A population-based early detection program for breast cancer has been in progress in Finland since 1987. According to regulations during the study period 1987-2001, free of charge mammography screening was offered every second year to women aged 50-59 years. Recently, the screening service was decided to be extended to age group 50-69. However, the scope of the program is still frequently discussed in public and information about potential impacts of mass-screening practice changes on future breast cancer burden is required. The aim of this doctoral thesis is to present methodologies for taking into account the mass-screening invitation information in breast cancer burden predictions, and to present alternative breast cancer incidence and mortality predictions up to 2012 based on scenarios of the future screening policy. The focus of this work is not on assessing the absolute efficacy but the effectiveness of mass-screening, and, by utilizing the data on invitations, on showing the estimated impacts of changes in an existing screening program on the short-term predictions. The breast cancer mortality predictions are calculated using a model that combines incidence, cause-specific and other cause survival on individual level. The screening invitation data are incorporated into modeling of breast cancer incidence and survival by dividing the program into separate components (first and subsequent rounds and years within them, breaks, and post screening period) and defining a variable that gives the component of the screening program. The incidence is modeled using a Poisson regression approach and the breast cancer survival by applying a parametric mixture cure model, where the patient population is allowed to be a combination of cured and uncured patients. The patients risk to die from other causes than breast cancer is allowed to differ from that of a corresponding general population group and to depend on age and follow-up time. As a result, the effects of separate components of the screening program on incidence, proportion of cured and the survival of the uncured are quantified. According to the predictions, the impacts of policy changes, like extending the program from age group 50-59 to 50-69, are clearly visible on incidence while the effects on mortality in age group 40-74 are minor. Extending the screening service would increase the incidence of localized breast cancers but decrease the rates of non-localized breast cancer. There were no major differences between mortality predictions yielded by alternative future scenarios of the screening policy: Any policy change would have at the most a 3.0% reduction on overall breast cancer mortality compared to continuing the current practice in the near future.

Community forestry and environmental literacy in northern Thailand : Towards collaborative natural resource management and conservation

Conservation and sustainable management of tropical forests needs a holistic approach: in addition to ecological concerns, socio-economic issues including cultural aspects must be taken into consideration. An ability to adapt practices is a key to successful collaborative natural resource management. Achieving this requires local participation and understanding of local conceptions of the environment. This study examined these issues in the context of northern Thailand. Northern uplands are the home of much of the remaining natural forest in Thailand and several ethnic minority groups commonly referred to as hill tribes. The overall purpose of this study was to grasp a regional view of an ethnically diverse forested area and to elicit prospects to develop community forestry for conservation purposes and for securing people s livelihood. Conservation was a central goal of management as the forests in the area were largely designated as protected. The aim was to study local perceptions, objectives, values and practices of forest management, under the umbrella of the concept environmental literacy, as well as the effects of forest policy on community management goals and activities. Environmental literacy refers to holistic understanding of the environment. It was used as a tool to examine people s views, interests, knowledge and motivation associated to forests. The material for this study was gathered in six villages in Chiang Mai Province. Three minority groups were included in the study, the Karen, Hmong and Lawa, and also the Thai. Household and focus group interviews were conducted in the villages. In addition, officials at district, regional and national levels, workers of non-governmental organisations, and academics were interviewed, and some data were gathered from the students of a local school. The results showed that motivation for protecting the forests existed among each ethnic group studied. This was a result of culture and traditions evolved in the forest environment but also of a need to adapt to a changed situation and environment and to outside pressures. The consequences of deforestation were widely agreed on in the villages, and the impact of socio-economic changes on the forests and livelihood was also recognised. The forest was regarded as a source of livelihood providing land, products and services essential to the people inhabiting rural uplands. Traditions, fire control, cooperation, reforestation, separation of protected and utilisable areas, and rules were viewed as central for conservation. For the villagers, however, conservation meant sustainable use, whereas the government has tended to prefer strict restrictions on forest resource use. Thus, conflicts had arisen. Between communities, cooperation was more dominant than conflict. The results indicated that the heterogeneity of forest dwellers, although it has to be recognised, should not be overemphasised: ethnic diversity can be considered as no major obstacle for successful community forestry. Collaborative management is particularly important in protected areas in order to meet the conservation goals while providing opportunities for livelihood. Forest management needs more positive incentives and increased dialogue.

Integrating biodiversity conservation into forestry : an empirical analysis of institutional adaptation

Integrating biodiversity conservation into forest management in non-industrial private forests requires changes in the practices of those public and private actors that have implementing responsibilities and whose strategic and operational opportunities are at stake. Understanding this kind of context-dependent institutional adaptation requires bridging between two analytical approaches: policy implementation and organizational adaptation, backed up with empirical analysis. The empirical analyses recapitulated in this thesis summary address organizational competences, specialization, professional judgment, and organizational networks. The analyses utilize qualitative and quantitative data from public and private sector organizations as well as associations. The empirical analyses produced stronger signals of policy implementation than of organizational adaptation. The organizations recognized the policy and social demand for integrating biodiversity conservation into forest management and their professionals were in favor of conserving biodiversity. However, conservation was integrated to forest management so tightly that it could be said to be subsumed by mainstream forestry. The organizations had developed some competences for conservation but the competences did not differentiate among the organizations other than illustrating the functional differences between industry, administration and associations. The networks that organizations depended on consisted of traditional forestry actors and peers both in planning policy and at the operational level. The results show that he demand for biodiversity conservation has triggered incremental changes in organizations. They can be considered inert regarding this challenge. Isomorphism is advanced by hierarchical guidance and standardization, and by professional norms. Analytically, this thesis contributes to the understanding of organizational behavior across the public and private sector boundaries. The combination of a policy implementation approach inherent in analysis of public policies in hierarchical administration settings, and organizational adaptation typically applied to private sector organizations, highlights the importance of institutional interpretation. Institutional interpretation serves the understanding of the empirically identified diversions from the basic tenets of the two approaches. Attention to institutions allows identification of the overlap of the traditionally segregated approaches.