Micropillar Array Based Microchips for Electrospray Ionization Mass Spectrometry, Microreactors, and Liquid Chromatographic Separation

This dissertation deals with the design, fabrication, and applications of microscale electrospray ionization chips for mass spectrometry. The microchip consists of microchannel, which leads to a sharp electrospray tip. Microchannel contain micropillars that facilitate a powerful capillary action in the channels. The capillary action delivers the liquid sample to the electrospray tip, which sprays the liquid sample to gas phase ions that can be analyzed with mass spectrometry. The microchip uses a high voltage, which can be utilized as a valve between the microchip and mass spectrometry. The microchips can be used in various applications, such as for analyses of drugs, proteins, peptides, or metabolites. The microchip works without pumps for liquid transfer, is usable for rapid analyses, and is sensitive. The characteristics of performance of the single microchips are studied and a rotating multitip version of the microchips are designed and fabricated. It is possible to use the microchip also as a microreactor and reaction products can be detected online with mass spectrometry. This property can be utilized for protein identification for example. Proteins can be digested enzymatically on-chip and reaction products, which are in this case peptides, can be detected with mass spectrometry. Because reactions occur faster in a microscale due to shorter diffusion lengths, the amount of protein can be very low, which is a benefit of the method. The microchip is well suited to surface activated reactions because of a high surface-to-volume ratio due to a dense micropillar array. For example, titanium dioxide nanolayer on the micropillar array combined with UV radiation produces photocatalytic reactions which can be used for mimicking drug metabolism biotransformation reactions. Rapid mimicking with the microchip eases the detection of possibly toxic compounds in preclinical research and therefore could speed up the research of new drugs. A micropillar array chip can also be utilized in the fabrication of liquid chromatographic columns. Precisely ordered micropillar arrays offer a very homogenous column, where separation of compounds has been demonstrated by using both laser induced fluorescence and mass spectrometry. Because of small dimensions on the microchip, the integrated microchip based liquid chromatography electrospray microchip is especially well suited to low sample concentrations. Overall, this work demonstrates that the designed and fabricated silicon/glass three dimensionally sharp electrospray tip is unique and facilitates stable ion spray for mass spectrometry.

A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Diastereomeric Drug Glucuronides: Enzymatic Glucuronidation and Analysis by Capillary Electrophoresis and Liquid Chromatography-Mass Spectrometry

Foreign compounds, such as drugs are metabolised in the body in numerous reactions. Metabolic reactions are divided into phase I (functionalisation) and phase II (conjugation) reactions. Uridine diphosphoglucuronosyltransferase enzymes (UGTs) are important catalysts of phase II metabolic system. They catalyse the transfer of glucuronic acid to small lipophilic molecules and convert them to hydrophilic and polar glucuronides that are readily excreted from the body. Liver is the main site of drug metabolism. Many drugs are racemic mixtures of two enantiomers. Glucuronidation of a racemic compound yields a pair of diastereomeric glucuronides. Stereoisomers are interesting substrates in glucuronidation studies since some UGTs display stereoselectivity. Diastereomeric glucuronides of O-desmethyltramadol (M1) and entacapone were selected as model compounds in this work. The investigations of the thesis deal with enzymatic glucuronidation and the development of analytical methods for drug metabolites, particularly diastereomeric glucuronides. The glucuronides were analysed from complex biological matrices, such as urine or from in vitro incubation matrices. Various pretreatment techniques were needed to purify, concentrate and isolate the analytes of interest. Analyses were carried out by liquid chromatography (LC) with ultraviolet (UV) or mass spectrometric (MS) detection or with capillary electromigration techniques. Commercial glucuronide standards were not available for the studies. Enzyme-assisted synthesis with rat liver microsomes was therefore used to produce M1 glucuronides as reference compounds. The glucuronides were isolated by LC/UV and ultra performance liquid chromatography (UPLC)/MS, while tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed in structural characterisation. The glucuronides were identified as phenolic O-glucuronides of M1. To identify the active UGT enzymes in (±)-M1 glucuronidation recombinant human UGTs and human tissue microsomes were incubated with (±)-M1. The study revealed that several UGTs can catalyse (±)-M1 glucuronidation. Glucuronidation in human liver microsomes like in rat liver microsomes is stereoselective. The results of the studies showed that UGT2B7, most probably, is the main UGT responsible for (±)-M1 glucuronidation in human liver. Large variation in stereoselectivity of UGTs toward (±)-M1 enantiomers was observed. Formation of M1 glucuronides was monitored with a fast and selective UPLC/MS method. Capillary electromigration techniques are known for their high resolution power. A method that relied on capillary electrophoresis (CE) with UV detection was developed for the separation of tramadol and its free and glucuronidated metabolites. The suitability of the method to identify tramadol metabolites in an authentic urine samples was tested. Unaltered tramadol and four of its main metabolites were detected in the electropherogram. A micellar electrokinetic chromatography (MEKC) /UV method was developed for the separation of the glucuronides of entacapone in human urine. The validated method was tested in the analysis of urine samples of patients. The glucuronides of entacapone could be quantified after oral entacapone dosing.

Uterine cervix nitric oxide and human papillomavirus infection in women

Most women acquire genital high risk human papillomavirus (HPV) infection during their lifetime, but seldom the infection persists and leads to cervical cancer. However, currently it is not possible to identify the women who will develop HPV mediated cervical cancer and this often results to large scale follow-up and overtreatment of the likely spontaneously regressing infection. Thus, it is important to obtain more information on the course of HPV and find markers that could help to identify HPV infected women in risk for progression of cervical lesions and ultimately cancer. Nitric oxide is a free radical gas that takes part both in immune responses and carcinogenesis. Nitric oxide is produced also by cervical cells and therefore, it is possible that cervical nitric oxide could affect also HPV infection. In the present study, including 801 women from the University of Helsinki between years of 2006 and 2011, association between HPV and cervical nitric oxide was evaluated. The levels of nitric oxide were measured as its metabolites nitrate and nitirite (NOx) by spectrophotometry and the expression of nitric oxide producing enzymes endothelial and inducible synthases (eNOS, iNOS) by Western blotting. Women infected with HPV had two-times higher cervical fluid NOx levels compared with non-infected ones. The expression levels of both eNOS and iNOS were higher in HPV-infected women compared with non-infected. Another sexually transmitted disease Chlamydia trachomatis that is an independent risk factor for cervical cancer was also accompanied with elevated NOx levels, whereas vaginal infections, bacterial vaginosis and candida, did not have any effect on NOx levels. The meaning of the elevated HPV related cervical nitric oxide was evaluated in a 12 months follow-up study. It was revealed that high baseline cervical fluid NOx levels favored HPV persistence with OR 4.1. However, low sensitivity (33%) and high false negative rate (67%) restrict the clinical use of the current NOx test. This study indicated that nitric oxide favors HPV persistence and thus it seems to be one of the cofactor associated with a risk of carcinogenesis.

Studies of fungi associated with Tomicus piniperda L. in Finland

The Scots pine bark beetle, Tomicus piniperda is a secondary colonizer of pine and other conifers. It is a native species from Europe and Asia that was recently introduced in North America. Although it is necessary to understand this insect's interactions with other organisms, few studies have focussed on its fungal associates. This study focused on the effect of latitude in the occurrence of fungi associated with T. piniperda. T. piniperda were collected from Pinus sylvestris in Northern (Rovaniemi) and Southern (Hyytiala) Finland. Both endo- and epi- mycota were isolated. The fungi were identified using a combination of morphological features and molecular data. The results revealed a great diversity of fungi species associated with T. piniperda, with a total of 3073 isolates representing 23 species. The most frequently isolated fungi in the bark beetles from Northern Finland were Beauvaria bassiana, Kuraishia sp. and Penicillium sp. whereas P. brevicompactum and Mortierella sp. were mostly observed in the South. Ophiostoma canum and O. minus were also observed. The number of isolates per insect in the north was 2.83 epi- and 2.38 for endo-mycota fungus. In the south, the number of isolates per insect was 4.1 for epi- and 3.5 for endo-mycota. Statistical analysis indicated that there was significant differences in fungal populations associated with the beetles in Southern and Northern Finland. There was however no significant difference between the epi- and endo-mycota fungal populations. The highest richness and diversity of the fungal species was observed in the South. However, the overall fungal diversity index analysis revealed that the mycobiota was undersampled.

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.

Biocompatibility and biofunctionalization of mesoporous silicon particles

Several of the newly developed drug molecules experience poor biopharmaceutical behavior, which hinders their effective delivery at the proper site of action. Among the several strategies employed in order to overcome this obstacle, mesoporous silicon-based materials have emerged as promising drug carriers due to their ability to improve the dissolution behavior of several poorly water-soluble drugs compounds confined within their pores. In addition to improve the dissolution behavior of the drugs, we report that porous silicon (PSi) nanoparticles have a higher degree of biocompatibility than PSi microparticles in several cell lines studied. In addition, the degradation of the nanoparticles showed its potential to fast clearance in the body. After oral delivery, the PSi particles were also found to transit the intestines without being absorbed. These results constituted the first quantitative analysis of the behavior of orally administered PSi nanoparticles compared with other delivery routes in rats. The self-assemble of a hydrophobin class II (HFBII) protein at the surface of hydrophobic PSi particles endowed the particles with greater biocompatibility in different cell lines, was found to reverse their hydrophobicity and also protected a drug loaded within its pores against premature release at low pH while enabling subsequent drug release as the pH increased. These results highlight the potential of HFBII-coating for PSi-based drug carriers in improving their hydrophilicity, biocompatibility and pH responsiveness in drug delivery applications. In conclusion, mesoporous silicon particles have been shown to be a versatile platform for improving the dissolution behavior of poorly water-soluble drugs with high biocompatibility and easy surface modification. The results of this study also provide information regarding the biofunctionalization of the THCPSi particles with a fungal protein, leading to an improvement in their biocompatibility and endowing them with pH responsive and mucoadhesive properties.

Resistance of Norway spruce (Picea abies) to root and butt rot (Heterobasidion parviporum) in peatland and mineral soil

Root and butt rot is the most harmful fungal disease affecting Norway spruce in southern Finland. In approximately 90 % of cases the causal agent is Heterobasidion parviporum. Root and butt rot infections have not been reported in Finnish peatlands. However, the increase in logging operations in peatlands means there is a risk that the fungus will eventually spread to these areas. The aim of this study was to find out the impact of growing site on the resistance of Norway spruce to Heterobasidion parviporum infections. This was investigated by artificially inoculating H. parviporum to spruce trees in pristine mire, drained peatland and mineral soil and comparing the defence reactions. Additionally, the effect of genotype on resistance was studied by comparing the responses of spruce clones representing different geographic origins. The roots and stems of the trees to be sampled were wounded and inoculated with wood dowels pre-colonised by H. parviporum hyphae. The resulting necrosis around the point of inoculation was observed. It was presumed that increased length of necrosis indicates high susceptibility of the tree to the disease. The relationship between growth rate and host resistance was also studied. The results indicated that growing site does not have a statistically significant effect on host resistance. The average length of necrosis around the point of inoculation was 35 mm in pristine mire, 37 mm in drained peatland and 40 mm in mineral soil. It was observed that growth rate does not affect resistance, but that the genotype of the tree does have an effect. The most resistant spruce clone was the one with Russian origin. The results suggest that the spruce stands in peatlands are not more resistant to root and butt rot infections than those in mineral soil. These findings should be taken into consideration when logging peatland forests.