Matrix metalloproteinases as biomarkers in premalignant and malignant tumors of the human skin

The incidence of non-melanoma skin cancer is increasing worldwide. Basal cell carcinoma followed by squamous cell carcinoma and malignant melanoma are the most frequent skin tumors. Immunosuppressed patients have an increased risk of neoplasia, of which non-melanoma skin cancer is the most common. Matrix metalloproteinases (MMPs) are proteolytic enzymes that collectively are capable of degrading virtually all components of the extracellular matrix. MMPs can also process substrates distinct from extracellular matrix proteins and influence cell proliferation, differentiation, angiogenesis, and apoptosis. MMP activity is regulated by their natural inhibitors, tissue inhibitors of metallopro-teinases (TIMPs). In this study, the expression patterns of MMPs, TIMPs, and certain cancer-related molecules were investigated in premalignant and malignant lesions of the human skin. As methods were used immunohistochemisty, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) from the cell cultures. Our aim was to evaluate the expression pattern of MMPs in extramammary Paget's disease in order to find markers for more advanced tumors, as well as to shed light on the origin of this rare neoplasm. Novel MMPs -21, -26, and -28 were studied in melanoma cell culture, in primary cutaneous melanomas, and their sentinel nodes. The MMP expression profile in keratoacanthomas and well-differentiated squamous cell carcinomas was analyzed to find markers to differentiate benign keratinocyte hyperproliferation from malignantly transformed cells. Squamous cell carcinomas of immunosuppressed organ transplant recipients were compared to squamous cell carcinomas of matched immunocompetent controls to investigate the factors explaining their more aggressive nature. We found that MMP-7 and -19 proteins are abundant in extramammary Paget's disease and that their presence may predict an underlying adenocarcinoma in these patients. In melanomas, MMP-21 was upregulated in early phases of melanoma progression, but disappeared from the more aggressive tumors with lymph node metastases. The presence of MMP-13 in primary melanomas and lymph node metastases may relate to more aggressive disease. In keratoacanthomas, the expression of MMP-7 and -9 is rare and therefore should raise a suspicion of well-differentiated squamous cell carcinomas. Furthermore, MMP-19 and p16 were observed in benign keratinocyte hyperproliferation of keratoacanthomas, whereas they were generally lost from malignant keratinocytes of SCCs. MMP-26 staining was significantly stronger in squamous cell carcinomas and Bowen s disease samples of organ transplant recipients and it may contribute to the more aggressive nature of squamous cell carcinomas in immunosuppressed patients. In addition, the staining for MMP-9 was significantly stronger in macrophages surrounding the tumors of the immunocompetent group and in neutrophils of those patients on cyclosporin medication. In conclusion, based on our studies, MMP-7 and -19 might serve as biomarkers for more aggressive extramammary Paget's disease and MMP-21 for malignant transformation of melanocytes. MMP -7, -9, and -26, however, could play an important role in the pathobiology of keratinocyte derived malignancies.

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.

Tactile processing in human somatosensory and auditory cortices

Tactile sensation plays an important role in everyday life. While the somatosensory system has been studied extensively, the majority of information has come from studies using animal models. Recent development of high-resolution anatomical and functional imaging techniques has enabled the non-invasive study of human somatosensory cortex and thalamus. This thesis provides new insights into the functional organization of the human brain areas involved in tactile processing using magnetoencephalography (MEG) and functional magnetic resonance imaging (fMRI). The thesis also demonstrates certain optimizations of MEG and fMRI methods. Tactile digit stimulation elicited stimulus-specific responses in a number of brain areas. Contralateral activation was observed in somatosensory thalamus (Study II), primary somatosensory cortex (SI; I, III, IV), and post-auditory belt area (III). Bilateral activation was observed in secondary somatosensory cortex (SII; II, III, IV). Ipsilateral activation was found in the post-central gyrus (area 2 of SI cortex; IV). In addition, phasic deactivation was observed within ipsilateral SI cortex and bilateral primary motor cortex (IV). Detailed investigation of the tactile responses demonstrated that the arrangement of distal-proximal finger representations in area 3b of SI in humans is similar to that found in monkeys (I). An optimized MEG approach was sufficient to resolve such fine detail in functional organization. The SII region appeared to contain double representations for fingers and toes (II). The detection of activations in the SII region and thalamus improved at the individual and group levels when cardiac-gated fMRI was used (II). Better detection of body part representations at the individual level is an important improvement, because identification of individual representations is crucial for studying brain plasticity in somatosensory areas. The posterior auditory belt area demonstrated responses to both auditory and tactile stimuli (III), implicating this area as a physiological substrate for the auditory-tactile interaction observed in earlier psychophysical studies. Comparison of different smoothing parameters (III) demonstrated that proper evaluation of co-activation should be based on individual subject analysis with minimal or no smoothing. Tactile input consistently influenced area 3b of the human ipsilateral SI cortex (IV). The observed phasic negative fMRI response is proposed to result from interhemispheric inhibition via trans-callosal connections. This thesis contributes to a growing body of human data suggesting that processing of tactile stimuli involves multiple brain areas, with different spatial patterns of cortical activation for different stimuli.

Human health risk assessment of heavy metals in urban stormwater

Toxic chemical pollutants such as heavy metals (HMs) are commonly present in urban stormwater. These pollutants can pose a significant risk to human health and hence a significant barrier for urban stormwater reuse. The primary aim of this study was to develop an approach for quantitatively assessing the risk to human health due to the presence of HMs in stormwater. This approach will lead to informed decision making in relation to risk management of urban stormwater reuse, enabling efficient implementation of appropriate treatment strategies. In this study, risks to human health from heavy metals were assessed as hazard index (HI) and quantified as a function of traffic and land use related parameters. Traffic and land use are the primary factors influencing heavy metal loads in the urban environment. The risks posed by heavy metals associated with total solids and fine solids (<150µm) were considered to represent the maximum and minimum risk levels, respectively. The study outcomes confirmed that Cr, Mn and Pb pose the highest risks, although these elements are generally present in low concentrations. The study also found that even though the presence of a single heavy metal does not pose a significant risk, the presence of multiple heavy metals could be detrimental to human health. These findings suggest that stormwater guidelines should consider the combined risk from multiple heavy metals rather than the threshold concentration of an individual species. Furthermore, it was found that risk to human health from heavy metals in stormwater is significantly influenced by traffic volume and the risk associated with stormwater from industrial areas is generally higher than that from commercial and residential areas.

Psychoacoustic sampling as a reliable, non-invasive method to monitor orthopteran species diversity in tropical forests

We evaluated trained listener-based acoustic sampling as a reliable and non-invasive method for rapid assessment of ensiferan species diversity in tropical evergreen forests. This was done by evaluating the reliability of identification of species and numbers of calling individuals using psychoacoustic experiments in the laboratory and by comparing psychoacoustic sampling in the field with ambient noise recordings made at the same time. The reliability of correct species identification by the trained listener was 100% for 16 out of 20 species tested in the laboratory. The reliability of identifying the numbers of individuals correctly was 100% for 13 out of 20 species. The human listener performed slightly better than the instrument in detecting low frequency and broadband calls in the field, whereas the recorder detected high frequency calls with greater probability. To address the problem of pseudoreplication during spot sampling in the field, we monitored the movement of calling individuals using focal animal sampling. The average distance moved by calling individuals for 17 out of 20 species was less than 1.5 m in half an hour. We suggest that trained listener-based sampling is preferable for crickets and low frequency katydids, whereas broadband recorders are preferable for katydid species with high frequency calls for accurate estimation of ensiferan species richness and relative abundance in an area.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

The Evolving Roles of the Human Resource Function: Understanding Role Changes in the Context of Large-Scale Mergers

The human resource (HR) function is under pressure both to change roles and to play a large variety of roles. Questions of change and development in the HR function become particularly interesting in the context of mergers and acquisitions when two corporations are integrated. The purpose of the thesis is to examine the roles played by the HR function in the context of large-scale mergers and thus to understand what happens to the HR function in such change environments, and to shed light on the underlying factors that influence changes in the HR function. To achieve this goal, the study seeks first to identify the roles played by the HR function before and after the merger, and second, to identify the factors that affect the roles played by the HR function. It adopts a qualitative case study approach including ten focal case organisations (mergers) and four matching cases (non-mergers). The sample consists of large corporations originating from either Finland or Sweden. HR directors and members of the top management teams within the case organisations were interviewed. The study suggests that changes occur within the HR function, and that the trend is for the HR function to become increasingly strategic. However, the HR function was found to play strategic roles only when the HR administration ran smoothly. The study also suggests that the HR function has become more versatile. An HR function that was perceived to be mainly administrative before the merger is likely after the merger to perform some strategically important activities in addition to the administrative ones. Significant changes in the roles played by the HR function were observed in some of the case corporations. This finding suggests that the merger integration process is a window of opportunity for the HR function. HR functions that take a proactive and leading role during the integration process might expand the number of roles played and move from being an administrator before the merger to also being a business partner after integration. The majority of the HR functions studied remained mainly reactive during the organisational change process and although the evidence showed that they moved towards strategic tasks, the intra-functional changes remained comparatively small in these organisations. The study presents a new model that illustrates the impact of the relationship between the top management team and the HR function on the role of the HR function. The expectations held by the top management team for the HR function and the performance of the HR function were found to interact. On a dimension reaching from tactical to strategic, HR performance is likely to correspond to the expectations held by top management.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

Classification of Protein Kinases on the Basis of Both Kinase and Non-Kinase Regions

Background: Protein phosphorylation is a generic way to regulate signal transduction pathways in all kingdoms of life. In many organisms, it is achieved by the large family of Ser/Thr/Tyr protein kinases which are traditionally classified into groups and subfamilies on the basis of the amino acid sequence of their catalytic domains. Many protein kinases are multidomain in nature but the diversity of the accessory domains and their organization are usually not taken into account while classifying kinases into groups or subfamilies. Methodology: Here, we present an approach which considers amino acid sequences of complete gene products, in order to suggest refinements in sets of pre-classified sequences. The strategy is based on alignment-free similarity scores and iterative Area Under the Curve (AUC) computation. Similarity scores are computed by detecting common patterns between two sequences and scoring them using a substitution matrix, with a consistent normalization scheme. This allows us to handle full-length sequences, and implicitly takes into account domain diversity and domain shuffling. We quantitatively validate our approach on a subset of 212 human protein kinases. We then employ it on the complete repertoire of human protein kinases and suggest few qualitative refinements in the subfamily assignment stored in the KinG database, which is based on catalytic domains only. Based on our new measure, we delineate 37 cases of potential hybrid kinases: sequences for which classical classification based entirely on catalytic domains is inconsistent with the full-length similarity scores computed here, which implicitly consider multi-domain nature and regions outside the catalytic kinase domain. We also provide some examples of hybrid kinases of the protozoan parasite Entamoeba histolytica. Conclusions: The implicit consideration of multi-domain architectures is a valuable inclusion to complement other classification schemes. The proposed algorithm may also be employed to classify other families of enzymes with multidomain architecture.

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.

Human Ecology in India Some Historical Perspectives

Before the spread of extensive settled cultivation, the Indian subcontinent would have been inhabited by territorial hunter–gatherers and shifting cultivators with cultural traditions of prudent resource use. The disruption of closed material cycles by export of agricultural produce to centres of non-agricultural population would have weakened these traditions. Indeed, the fire-based sacrificial ritual and extensive agricultural settlements might have catalysed the destruction of forests and wildlife and the suppression of tribal peoples during the agricultural colonization of the Gangetic plains. Buddhism, Jainism and later the Hindu sects may have been responses to the need for a reassertion of ecological prudence once the more fertile lands were brought under cultivation. British rule radically changed the focus of the country's resource use pattern from production of a variety of biological resources for local consumption to the production of a few commodities largely for export. The resulting ecological squeeze was accompanied by disastrous famines and epidemics between the 1860s and the 1920s. The counterflows to tracts of intensive agriculture have reduced such disasters since independence. However, these are quite inadequate to balance the state-subsidized outflows of resources from rural hinterlands. These imbalances have triggered serious environmental degradation and tremendous overcrowding of the niche of agricultural labour and marginal cultivator all over the country.

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.

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer gamma P-32]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of P-32]-inorganic phosphate ((32)Pi). Inclusion of UDP in the incubation medium resulted in conversion of gamma P-32]ATP to P-32]UTP, while inclusion of AMP resulted in conversion of gamma P-32]ATP to P-32]ADP. Ebselen markedly reduced P-32]UTP formation but displayed negligible effect on (32)Pi or P-32]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC50=6.9 +/- 2 mu M). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V-max of the reaction (K-i=7.6 +/- 3 mu M), having negligible effect on KM values. Our study demonstrates that ebselen is a potent noncompetitive inhibitor of extracellular NDPK.

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: Results of a pilot study

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar, The volunteers responded only to the first two immunizations, The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively, During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable, A 50 mu l aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of I-125-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively, The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded, Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging, Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles, There was no production of antibodies capable of interacting with non-specific tissues, It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen, This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.