Measurement of the ttbar production cross section with an in situ calibration of b-jet identification efficiency

A measurement of the top-quark pair-production cross section in ppbar collisions at sqrt{s}=1.96 TeV using data corresponding to an integrated luminosity of 1.12/fb collected with the Collider Detector at Fermilab is presented. Decays of top-quark pairs into the final states e nu + jets and mu nu + jets are selected, and the cross section and the b-jet identification efficiency are determined using a new measurement technique which requires that the measured cross sections with exactly one and multiple identified b-quarks from the top-quark decays agree. Assuming a top-quark mass of 175 GeV/c^2, a cross section of 8.5+/-0.6(stat.)+/-0.7(syst.) pb is measured.

Berry phenolics: isolation, analysis, identification, and antioxidant properties

The main objectives in this thesis were to isolate and identify the phenolic compounds in wild (Sorbus aucuparia) and cultivated rowanberries, European cranberries (Vaccinium microcarpon), lingonberries (Vaccinium vitis-idaea), and cloudberries (Rubus chamaemorus), as well as to investigate the antioxidant activity of phenolics occurring in berries in food oxidation models. In addition, the storage stability of cloudberry ellagitannin isolate was studied. In wild and cultivated rowanberries, the main phenolic compounds were chlorogenic acids and neochlorogenic acids with increasing anthocyanin content depending on the crossing partners. The proanthocyanidin contents of cranberries and lingonberries were investigated, revealing that the lingonberry contained more rare A-type dimers than the European cranberry. The liquid chromatography mass spectrometry (LC-MS) analysis of cloudberry ellagitannins showed that trimeric lambertianin C and sanguiin H-10 were the main ellagitannins. The berries, rich in different types of phenolic compounds including hydroxycinnamic acids, proanthocyanidins, and ellagitannins, showed antioxidant activity toward lipid oxidation in liposome and emulsion oxidation models. All the different rowanberry cultivars prevented lipid oxidation in the same way, in spite of the differences in their phenolic composition. In terms of liposomes, rowanberries were slightly more effective antioxidants than cranberry and lingonberry phenolics. Greater differences were found when comparing proanthocyanidin fractions. Proanthocyanidin dimers and trimers of both cranberries and lingonberries were most potent in inhibiting lipid oxidation. Antioxidant activities and antiradical capacities were also studied with hydroxycinnamic acid glycosides. The sinapic acid derivatives of the hydroxycinnamic acid glycosides were the most effective at preventing lipid oxidation in emulsions and liposomes and scavenging radicals in DPPH assay. In liposomes and emulsions, the formation of the secondary oxidation product, hexanal, was inhibited more than that of the primary oxidation product, conjugated diene hydroperoxides, by hydroxycinnamic acid derivatives. This indicates that they are principally chain-breaking antioxidants rather than metal chelators, although they possess chelating activity as well. The storage stability test of cloudberry ellagitannins was performed by storing ellagitannin isolate and ellagitannins encapsulated with maltodextrin at different relative vapor pressures. The storage stability was enhanced by the encapsulation when higher molecular weight maltodextrin was used. The best preservation was achieved when the capsules were stored at 0 or 33% relative vapor pressures. In addition, the antioxidant activities of encapsulated cloudberry extracts were followed during the storage period. Different storage conditions did not alter the antioxidant activity, even though changes in the ellagitannin contents were seen. The current results may be of use in improving the oxidative stability of food products by using berries as natural antioxidants.

Dental identification and aspects of medico-legal investigations of the Finnish victims of the Sumatra-Andaman earthquake on 26 December 2004

Tsunami waves of the Sumatra-Andaman earthquake on 26 December 2004 claimed approximately 230 000 lives and started the biggest identification operation in Interpol's history. The aim of this study was to resolve methods of the identification and results received. The viewpoint is mainly that of forensic odontology, but also includes other means of identification and results of the medico-legal examination performed in Finland. Of the 5395 victims in Thailand, approximately 2 400 were foreigners from 36 nations including 177 Finnish nationals. Additionally, a Finnish woman perished in Sri Lanka and a severely injured man after the evacuation in a hospital. The final numbers of missing persons and dead bodies registered in the Information Management Centre in Phuket,Thailand, were 3 574 ante-mortem (AM) and 3 681 post-mortem (PM) files. The number of identifications by December 2006 was 3 271 or 89% of the victims registered. Of Finnish victims, 172 have been identified in Thailand and 163 repatriated to Finland. One adult and four children are still missing. For AM data, a list of Finnish missing persons including 178 names was published on 30 December 2004. By February 2005 all useful dental AM data were available. Five persons on the list living in Finland lacked records. Based on the AM database, for the children under age 18 years (n=60) dental identification could be established for 12 (20%). The estimated number for adults (n=112) was 96 (86%). The final identification rate, based on PM examinations in Finland, was 14 (25%) for children (n= 56) and 98 (90%) for adults (n= 109). The number of Finnish victims identified by dental methods, 112 (68%), was high compared to all examined in Thailand (43%). DNA was applied for 26 Finnish children and for 6 adults, fingerprints for 24 and 7, respectively. In 12 cases two methods were applied. Every victim (n=165) underwent in Finland a medico-legal investigation including an autopsy with sampling specimens for DNA, the toxicological and histological investigation. Digital radiographs and computed tomography were taken of the whole body to verify autopsy findings and bring out changes caused by trauma, autolysis, and sampling for DNA in Thailand. Data for identification purposes were also noted. Submersion was the cause of death for 101 of 109 adults (92.7%), and trauma for 8 (7.3%). Injuries were 33 times contributing factors for submersion and 3 times for trauma-based death. Submersion was the cause of death for 51 (92.7%) children and trauma for 4 (7.3%). Injuries were in 3 cases contributing factors in submersion and once in trauma-based death. The success of the dental identification of Finnish victims is mainly based on careful registration of dental records, and on an education program from 1999 in forensic odontology.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

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.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.

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.

Biomarkers in acute respiratory failure

Acute respiratory failure (ARF) is the most common type of organ failure leading to the need for intensive care. It is often secondary to acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). ARF, and especially ALI and ARDS, cause increased morbidity, and mortality rates remain high (up to 40%). These disorders are characterised by inflammatory reaction and tissue damage. In some cases, inflammation continues and leads to an overwhelming repair process with ongoing fibrosis, accompanied by organ dysfunction and eventually a loss of function. Measuring the magnitude of the inflammation, and the repair process, would theoretically offer information concerning outcome. Early identification of patients whose disease process is likely to proceed unfavourably, would help clinicians to optimise their treatment. The aim of this study was to evaluate the epidemiology of ARF, its treatment, and outcome in Finland, with special interest in biomarkers, and their value in the prediction of mortality. Altogether, 958 adult patients treated with ventilatory support were prospectively included in this study during an eight week period in 2007 in 25 intensive care units. Plasma aminoterminal pro-brain natriuretic peptide (NT-pro-BNP) was assessed in 602 patients, and plasma cell-free DNA in 580 patients, to evaluate their prognostic value in ARF. Markers of collagen metabolism were studied in longitudinal serum samples in 68 patients in order to evaluate their evolution in ARF and the association to multiple organ dysfunction (MOD). Ventilatory support was used in 39% of all ICU patients. The estimated incidence of ARF was 149.5/100 000 per year. Median tidal volumes used were higher than recommended. Overall mortality at 90 days was 31%. Plasma NT-pro-BNP and cell-free DNA were highly increased in the majority of patients. Both markers were independent predictors of 90-day mortality, but their discriminative power was at most moderate when used separately. The mortality was highest in those patients, in whom both biomarkers were over their separate cut-off values. Thus, combined use of these biomarkers may increase their clinical value in the mortality prediction. The markers of collagen metabolism changed significantly over time in surviving patients. None of these markers did associate with MOD in these patients.