Introduction of high-content screening method for studying drug-induced mitochondrial dysfunction in hepatocytes

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.

Terveyteen liittyvä elämänlaatu laitoskuntoutuksen vaikuttavuuden arvioinnissa : RAND-36-mittarin soveltuvuus työikäisten laitoskuntoutuksen ongelmaprofiilin määrittämiseen ja kuntoutuksen vaikutusten arvioimiseen

The aim of the present research was to examine the validity of the RAND-36 measure of health-related quality of life among the working age rehabilitation clients. The research included two cross-sectional studies and one follow-up study. The subjects of the first study (n = 794) participated in the first period of the five following types of rehabilitation: occupationally oriented medical rehabilitation, musculoskeletal rehabilitation, medical rehabilitation for job burnout, rehabilitation for supporting the work ability and capacity of disabled subjects (vocational rehabilitation) and individualized rehabilitation between October 2000 and October 2001. The subjects of the second study (n = 990) participated in the same rehabilitation during their first rehabilitation period between May 2007 and May 2008. The first subjects participated in a follow-up period no later than May 2003 with the exception of the individual rehabilitation clients (n = 588). Based on the ICF classification, the RAND-36 provides a diverse measure of the health-related quality of life and of the capacity for subjective, perceived physical and psycho-social functioning. The construct properties of the RAND-36 measure proved to be very consistent on the basis of both the cluster and confirmatory factor analyses. At the group level, the RAND-36 measure was shown to be illustrative and sensitive in differentiating the clients’ rehabilitation needs. The results of cluster analyses with the two cross-sectional data indicated a consistent five-cluster solution of rehabilitation groups on the basis of the eight subscales of health-related quality of life. Each of these clusters represented a clear difference in their need for rehabilitation. The RAND-36 measure proved to be sensitive to change. The changes observed in the pre- and post-conditions in relation to all the subscales of quality of life were statistically significant. Depending on the rehabilitation type, different changes in the subscales of the measure were observed, and these changes corresponded to the different emphasis and goals of the specific type of rehabilitation intervention. Similarly, changes in the subscales of the measure were observed in relation to the RAND groups formed by cluster analysis, which were logical and corresponded to the problem profiles of these groups. The confirmatory factor analysis indicated a two-factor solution: an index of the capacity for physical functioning (self-rated general health, bodily pain, physical functioning, physical role functioning) and an index of the capacity for psycho-social functioning (psychological well-being, social functioning, psychological role functioning and energy). These two indices describing functional capacity proved also to be sensitive to change. This two-factor solution seems to be usable for group level analyses when assessing the effects of rehabilitation. The moderately strong correlation between the RAND-36 and work ability index suggests that they partly measure the same phenomenon: perceived health-related quality of life, subjective capacity for activity and perceived work ability have strong links. As expected, the capacity for physical functioning had a stronger correlation with work ability index than with the capacity for psycho-social functioning. According to the present research, the RAND-36 measure can be considered as a screening method for rehabilitation orientation in relation to rehabilitation needs and as a follow-up measure for the health-related quality of life among the working age clients. The RAND-36 measure is also shown to be a useful instrument in estimating the benefits of rehabilitation as well as in effectiveness research.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

Early life determinants of atopy : A 20-year prospective follow-up study on unselected, healthy newborns

Atopy-related allergic diseases, i.e. allergic rhinoconjunctivitis, atopic dermatitis and asthma, have increased in frequency in the industrialized countries. In order to reverse this trend, effective preventive strategies need to be developed. This requires a better understanding of the early-life events leading to the expression of the atopic phenotype. The present study has aimed at defining early-life factors and markers associated with the subsequent development of allergic diseases in a cohort of 200 healthy, unselected Finnish newborns prospectively followed up from birth to age 20 years. Their mothers were encouraged to start and maintain exclusive breastfeeding as long as it was nutritionally sufficient for the infant. Consequently, all the infants received some duration of exclusive breastfeeding, 58% of the infants were on exclusive breastfeeding for the first 6 months of life, and 18% received this feeding at least for the first 9 months. Of the infants, 42% had a family history of allergy. After the first year of follow-up, the children were re-assessed at ages 5, 11 and 20 years with clinical examination, skin prick testing, and parental and personal interviews. Exclusive breastfeeding for over 9 months was associated with atopic dermatitis and symptoms of food hypersensitivity at age 5 years, and with symptoms of food hypersensitivity at age 11 years in the children with a familial allergy. Subjects with allergic symptoms or a positive skin prick test in childhood or adolescence had lower retinol concentrations during their infancy and childhood than others. An elevated cord serum immunoglobulin E concentration predicted subsequent atopic manifestations though with modest sensitivity. Children and adolescents with allergic symptoms, skin prick test positivity and an elevated IgE had lower total cholesterol levels in infancy and childhood than the nonatopic subjects. In conclusion, prolonging strictly exclusive breastfeeding for over 9 months of age was not of help in prevention of allergic symptoms; instead, it was associated with increased atopic dermatitis and food hypersensitivity symptoms in childhood. Due to the modest sensitivity, cord serum IgE is not an effective screening method for atopic predisposition in the general population. Retinol and cholesterol concentrations in infancy were inversely associated with the subsequent development of allergic symptoms. Based on these findings, it is proposed that there may be differences in the inborn regulation of retinol and cholesterol levels in children with and without a genetic susceptibility to atopy, and these may play a role in the development of atopic sensitization and allergic diseases.

Bentsodiatsepiinien ja amfetamiinijohdannaisten seulonta virtsasta mikropilarisähkösumutusmassaspektrometrian avulla

Väärinkäytettyjen aineiden seulontaan käytetyn menetelmän tulee olla herkkä, selektiivinen, yksinkertainen, nopea ja toistettava. Työn tavoitteena oli kehittää yksinkertainen, mutta herkkä, esikäsittelymenetelmä bentsodiatsepiinien ja amfetamiinijohdannaisten kvalitatiiviseen seulomiseen virtsasta mikropilarisähkösumutussirun (μPESI) avulla, mikä tarjoaisi vaihtoehdon seulonnassa käytetyille immunologisille menetelmille, joiden herkkyys ja selektiivisyys ovat puutteellisia. Tavoitteena oli samalla tarkastella mikropilarisähkösumutussirun toimivuutta biologisten näytteiden analyysissa. Esikäsittely optimoitiin erikseen bentsodiatsepiineille ja amfetamiinijohdannaisille. Käytettyjä esikäsittelymenetelmiä olivat neste-nesteuutto, kiinteäfaasiuutto Oasis HLB-patruunalla ja ZipTip®-pipetinkärjellä sekä laimennus ja suodatus ilman uuttoa. Mittausten perusteella keskityttiin optimoimaan ZipTip®-uuttoa. Optimoinnissa tutkittavia yhdisteitä spiikattiin 0-virtsaan niiden ennaltamääritetyn raja-arvon verran, bentsodiatsepiineja 200 ng/ml ja amfetamiinijohdannaisia 300 ng/ml. Bentsodiatsepiinien kohdalla optimoitiin kutakin uuton vaihetta ja optimoinnin tuloksena näytteen pH säädettiin arvoon 5, faasi kunnostettiin asetonitriililla, tasapainotettiin ja pestiin veden (pH 5) ja asetonitriilin (10 % v/v) seoksella ja eluoitiin asetonitriilin, muurahaishapon ja veden (95:1:4 v/v/v) seoksella. Amfetamiinijohdannaisten uutossa optimoitiin näytteen ja liuottimien pH-arvoja ja tuloksena näytteen pH säädettiin arvoon 10, faasi kunnostettiin veden ja ammoniumvetykarbonaatin(pH 10, 1:1 v/v) seoksella, tasapainotettiin ja pestiin asetonitriilin ja veden (1:5 v/v) seoksella ja eluoitiin metanolilla. Optimoituja uuttoja testattiin Yhtyneet Medix Laboratorioista toimitetuilla autenttisilla virtsanäytteillä ja saatuja tuloksia verrattiin kvantitatiivisen GC/MS-analyysin tuloksiin. Bentsodiatsepiininäytteet hydrolysoitiin ennen uuttoa herkkyyden parantamiseksi. Autenttiset näytteet analysoitiin Q-TOF-laitteella Viikissä. Lisäksi hydrolysoidut bentsodiatsepiininäytteet mitattiin Yhtyneet Medix Laboratorioiden TOF-laitteella. Kehitetty menetelmä vaatii tulosten perusteella lisää optimointia toimiakseen. Ongelmana oli etenkin toistoissa ilmennyt tulosten hajonta. Manuaalista näytteensyöttöä tulisi kehittää toistettavammaksi. Autenttisten bentsodiatsepiininäytteiden analyysissa ongelmana olivat virheelliset negatiiviset tulokset ja amfetamiinijohdannaisten analyysissa virheelliset positiiviset tulokset. Virheellisiä negatiivisia tuloksia selittää menetelmän herkkyyden puute ja virheellisiä positiivisia tuloksia mittalaitteen, sirujen tai liuottimien likaantuminen.

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.

Desorptio/fotoionisaatio ilmanpaineessa takavarikoitujen huumausaineiden, steroidien ja räjähdysaineiden tutkimuksessa

Tutkimuksen tarkoituksena oli selvittää desorptio/fotoionisaatio ilmanpaineessa tekniikan (engl. desorption atmospheric pressure photoionization, DAPPI) soveltuvuutta rikosteknisen laboratorion näytteiden analysointiin. DAPPI on nopea massaspektrometrinen ionisaatiotekniikka, jolla voidaan tutkia yhdisteitä suoraan erilaisilta pinnoilta. DAPPI:ssa käytetään lämmitettyä mikrosirua, joka suihkuttaa höyrystynyttä liuotin- ja kaasuvirtausta kohti näytettä. Näytteen pinnan komponentit desorboituvat lämmön vaikutuksesta, jonka jälkeen ionisoituminen tapahtuu VUV-lampun emittoimien fotonien avulla.DAPPI:lla tutkittiin takavarikoituja huumausaineita, anabolisia steroideja ja räjähdysaineita sekä niiden jäämiä erilaisilta pinnoilta. Lisäksi kartoitettiin DAPPI:n mahdollisuuksia ja rajoituksia erilaisille näytematriiseille ilman näytteiden esikäsittelyä. Takavarikoitujen huumausaineiden tutkimuksessa analysoitiin erilaisia tabletteja, jauheita, kasvirouheita, huumekasveja (khat, oopium, kannabis) ja sieniä. Anabolisia steroideja tunnistettiin tableteista sekä ampulleista, jotka sisälsivät öljymäistä nestettä. Jauheet ripoteltiin kaksipuoliselle teipille ja analysoitiin siltä. Muut näytteet analysoitiin sellaisenaan ilman minkäänlaista esikäsittelyä, paitsi nestemäisten näytteiden kohdalla näyte pipetoitiin talouspaperille, joka analysoitiin DAPPI:lla. DAPPI osoittautui nopeaksi ja yksinkertaiseksi menetelmäksi takavarikoitujen huumausaineiden ja steroidien analysoimisessa. Se soveltui hyvin rikoslaboratorion erityyppisten näytteiden rutiiniseulontaan ja helpotti erityisesti huumekasvien ja öljymäisten steroidiliuosten tutkimusta. Massaspektrometrin likaantuminen pystyttiin ehkäisemään säätämällä näytteen etäisyyttä sen suuaukosta. Likaantumista ei havaittu huolimatta näytteiden korkeista konsentraatioista ja useita kuukausia jatkuneista mittauksista. Räjähdysaineiden tutkimuksessa keskityttiin seitsemän eri räjähdysaineen DAPPI-MS-menetelmän kehitykseen; trinitrotolueeni (TNT), nitroglykoli (NK), nitroglyseriini (NG), pentriitti (PETN), heksogeeni (RDX), oktogeeni (HMX) ja pikriinihappoä Nämä orgaaniset räjähteet ovat nitraattiyhdisteitä, jotka voidaan jakaa rakenteen puolesta nitroamiineihin (RDX ja HMX), nitroaromaatteihin (TNT ja pikriinihappo) sekä nitraattiestereihin (PETN, NG ja NK). Menetelmäkehityksessä räjähdysainelaimennokset pipetoitiin polymetyylimetakrylaatin (PMMA) päälle ja analysoitiin siitä. DAPPI:lla tutkittiin myäs autenttisia räjähdysainejäämiä erilaisista matriiseista. DAPPI:lla optimoitiin jokaiselle räjähdysaineelle sopiva menetelmä ja yhdisteet saatiin näkymään puhdasaineina. Räjähdysainejäämien analysoiminen erilaisista rikospaikkamateriaaleista osoittautui haastavammaksi tehtäväksi, koska matriisit aiheuttivat itsessään korkean taustan spektriin, josta räjähdysaineiden piikit eivät useimmiten erottuneet tarpeeksi. Muut desorptioionisaatiotekniikat saattavat soveltua paremmin haastavien räjähdysainejäämien havaitsemiseksi.

Mass spectrometry and n-in-one analytics in early drug discovery: Combinatorial chemistry libraries, lipophilicity and absorption screening

This thesis describes current and past n-in-one methods and presents three early experimental studies using mass spectrometry and the triple quadrupole instrument on the application of n-in-one in drug discovery. N-in-one strategy pools and mix samples in drug discovery prior to measurement or analysis. This allows the most promising compounds to be rapidly identified and then analysed. Nowadays properties of drugs are characterised earlier and in parallel with pharmacological efficacy. Studies presented here use in vitro methods as caco-2 cells and immobilized artificial membrane chromatography for drug absorption and lipophilicity measurements. The high sensitivity and selectivity of liquid chromatography mass spectrometry are especially important for new analytical methods using n-in-one. In the first study, the fragmentation patterns of ten nitrophenoxy benzoate compounds, serial homology, were characterised and the presence of the compounds was determined in a combinatorial library. The influence of one or two nitro substituents and the alkyl chain length of methyl to pentyl on collision-induced fragmentation was studied, and interesting structurefragmentation relationships were detected. Two nitro group compounds increased fragmentation compared to one nitro group, whereas less fragmentation was noted in molecules with a longer alkyl chain. The most abundant product ions were nitrophenoxy ions, which were also tested in the precursor ion screening of the combinatorial library. In the second study, the immobilized artificial membrane chromatographic method was transferred from ultraviolet detection to mass spectrometric analysis and a new method was developed. Mass spectra were scanned and the chromatographic retention of compounds was analysed using extract ion chromatograms. When changing detectors and buffers and including n-in-one in the method, the results showed good correlation. Finally, the results demonstrated that mass spectrometric detection with gradient elution can provide a rapid and convenient n-in-one method for ranking the lipophilic properties of several structurally diverse compounds simultaneously. In the final study, a new method was developed for caco-2 samples. Compounds were separated by liquid chromatography and quantified by selected reaction monitoring using mass spectrometry. This method was used for caco-2 samples, where absorption of ten chemically and physiologically different compounds was screened using both single and nin- one approaches. These three studies used mass spectrometry for compound identification, method transfer and quantitation in the area of mixture analysis. Different mass spectrometric scanning modes for the triple quadrupole instrument were used in each method. Early drug discovery with n-in-one is area where mass spectrometric analysis, its possibilities and proper use, is especially important.