Molecular epidemiology of human rhinoviruses

The first part of this work investigates the molecular epidemiology of a human enterovirus (HEV), echovirus 30 (E-30). This project is part of a series of studies performed in our research team analyzing the molecular epidemiology of HEV-B viruses. A total of 129 virus strains had been isolated in different parts of Europe. The sequence analysis was performed in three different genomic regions: 420 nucleotides (nt) in the VP4/VP2 capsid protein coding region, the entire VP1 capsid protein coding gene of 876 nt, and 150 nt in the VP1/2A junction region. The analysis revealed a succession of dominant sublineages within a major genotype. The temporally earlier genotypes had been replaced by a genetically homogenous lineage that has been circulating in Europe since the late 1970s. The same genotype was found by other research groups in North America and Australia. Globally, other cocirculating genetic lineages also exist. The prevalence of a dominant genotype makes E-30 different from other previously studied HEVs, such as polioviruses and coxsackieviruses B4 and B5, for which several coexisting genetic lineages have been reported. The second part of this work deals with molecular epidemiology of human rhinoviruses (HRVs). A total of 61 field isolates were studied in the 420-nt stretch in the capsid coding region of VP4/VP2. The isolates were collected from children under two years of age in Tampere, Finland. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades. Seasonal clustering was found. Also, several distinct serotype-like clusters were found to co-circulate during the same epidemic season. Reappearance of a cluster after disappearing for a season was observed. The molecular epidemiology of the analyzed strains turned out to be complex, and we decided to continue our studies of HRV. Only five previously published complete genome sequences of HRV prototype strains were available for analysis. Therefore, all designated HRV prototype strains (n=102) were sequenced in the VP4/VP2 region, and the possibility of genetic typing of HRV was evaluated. Seventy-six of the 102 prototype strains clustered in HRV genetic group A (HRV-A) and 25 in group B (HRV-B). Serotype 87 clustered separately from other HRVs with HEV species D. The field strains of HRV represented as many as 19 different genotypes, as judged with an approximate demarcation of a 20% nt difference in the VP4/VP2 region. The interserotypic differences of HRV were generally similar to those reported between different HEV serotypes (i.e. about 20%), but smaller differences, less than 10%, were also observed. Because some HRV serotypes are genetically so closely related, we suggest that the genetic typing be performed using the criterion "the closest prototype strain". This study is the first systematic genetic characterization of all known HRV prototype strains, providing a further taxonomic proposal for classification of HRV. We proposed to divide the genus Human rhinoviruses into HRV-A and HRV-B. The final part of the work comprises a phylogenetic analysis of a subset (48) of HRV prototype strains and field isolates (12) in the nonstructural part of the genome coding for the RNA-dependent RNA polymerase (3D). The proposed division of the HRV strains in the species HRV-A and HRV-B was also supported by 3D region. HRV-B clustered closer to HEV species B, C, and also to polioviruses than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D coding region than in the VP4/VP2 coding region, in contrast to HEV. Moreover, the diversity of HRV in 3D exceeded that of HEV. One group of HRV-A, designated HRV-A', formed a separate cluster outside other HRV-A in the 3D region. It formed a cluster also in the capsid region, but located within HRV-A. This may reflect a different evolutionary history of distinct genomic regions among HRV-A. Furthermore, the tree topology within HRV-A in the 3D region differed from that in the VP4/VP2, suggesting possible recombination events in the evolution of the strains. No conflicting phylogenies were observed in any of the 12 field isolates. Possible recombination was further studied using the Similarity and Bootscanning analyses of the complete genome sequences of HRV available in public databases. Evidence for recombination among HRV-A was found, as HRV2 and HRV39 showed higher similarity in the nonstructural part of the genome. Whether HRV2 and HRV39 strains - and perhaps also some other HRV-A strains not yet completely sequenced - are recombinants remains to be determined.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

Prognostic markers in head and neck carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Well-known risk factors include tobacco smoking and alcohol consumption. Overall survival has improved, but is still low especially in developing countries. One reason for this is the often advanced stage of the disease at the time of diagnosis, but also lack of reliable prognostic tools to enable individualized patient treatment to improve outcome. To date, the TNM classification still serves as the best disease evaluation criterion, although it does not take into account the molecular basis of the tumor. The need for surrogate molecular markers for more accurate disease prediction has increased research interests in this field. We investigated the prevalence, physical status, and viral load of human papillomavirus (HPV) in HNSCC to determine the impact of HPV on head and neck carcinogenesis. The prevalence and genotyping of HPV were assessed with an SPF10 PCR microtiter plate-based hybridization assay (DEIA), followed by a line probe-based genotyping assay. More than half of the patients had HPV DNA in their tumor specimens. Oncogenic HPV-16 was the most common type, and coinfections with other oncogenic and benign associated types also existed. HPV-16 viral load was unevenly distributed among different tumor sites; the tonsils harbored significantly greater amounts of virus than other sites. Episomal location of HPV-16 was associated with large tumors, and both integrated and mixed forms of viral DNA were detected. In this series, we could not show that the presence of HPV DNA correlated with survival. In addition, we investigated the prevalence and genotype of HPV in laryngeal carcinoma patients in a prospective Nordic multicenter study based on fresh-frozen laryngeal tumor samples to determine whether the tumors were HPV-associated. These patients were also examined and interviewed at diagnosis for known risk factors, such as tobacco smoking and alcohol consumption, and for several other habituations to elucidate their effects on patient survival. HPV analysis was performed with the same protocols as in the first study. Only 4% of the specimens harbored HPV DNA. Heavy drinking was associated with poor survival. Heavy drinking patients were also younger than nonheavy drinkers and had a more advanced stage of disease at diagnosis. Heavy drinkers had worse oral hygiene than nonheavy drinkers; however, poor oral hygiene did not have prognostic significance. History of chronic laryngitis, gastroesophageal reflux disease, and orogenital sex contacts were rare in this series. To clarify why vocal cord carcinomas seldom metastasize, we determined tumor lymph vessel (LVD) and blood vessel (BVD) densities in HNSCC patients. We used a novel lymphatic vessel endothelial marker (LYVE-1 antibody) to locate the lymphatic vessels in HNSCC samples and CD31 to detect the blood microvessels. We found carcinomas of the vocal cords to harbor less lymphatic and blood microvessels than carcinomas arising from sites other than vocal cords. The lymphatic and blood microvessel densities did not correlate with tumor size. High BVD was strongly correlated with high LVD. Neither BVD nor LVD showed any association with survival in our series. The immune system plays an important role in tumorigenesis, as neoplastic cells have to escape the cytotoxic lymphocytes in order to survive. Several candidate HLA class II alleles have been reported to be prognostic in cervical carcinomas, an epithelial malignancy resembling HNSCC. These alleles may have an impact on head and neck carcinomas as well. We determined HLA-DRB1* and -DQB1* alleles in HNSCC patients. Healthy organ donors served as controls. The Inno-LiPA reverse dot-blot kit was used to identify alleles in patient samples. No single haplotype was found to be predictive of either the risk for head and neck cancer, or the clinical course of the disease. However, alleles observed to be prognostic in cervical carcinomas showed a similar tendency in our series. DRB1*03 was associated with node-negative disease at diagnosis. DRB1*08 and DRB1*13 were associated with early-stage disease; DRB1*04 had a lower risk for tumor relapse; and DQB1*03 and DQB1*0502 were more frequent in controls than in patients. However, these associations reached only borderline significance in our HNSCC patients.

Molecular Genetics of Age-related Macular Degeneration

Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.

Observations on the first steps of atmospheric particle formation and growth

Atmospheric aerosol particles have a significant impact on air quality, human health and global climate. The climatic effects of secondary aerosol are currently among the largest uncertainties limiting the scientific understanding of future and past climate changes. To better estimate the climatic importance of secondary aerosol particles, detailed information on atmospheric particle formation mechanisms and the vapours forming the aerosol is required. In this thesis we studied these issues by applying novel instrumentation in a boreal forest to obtain direct information on the very first steps of atmospheric nucleation and particle growth. Additionally, we used detailed laboratory experiments and process modelling to determine condensational growth properties, such as saturation vapour pressures, of dicarboxylic acids, which are organic acids often found in atmospheric samples. Based on our studies, we came to four main conclusions: 1) In the boreal forest region, both sulphurous compounds and organics are needed for secondary particle formation, the previous contributing mainly to particle formation and latter to growth; 2) A persistent pool of molecular clusters, both neutral and charged, is present and participates in atmospheric nucleation processes in boreal forests; 3) Neutral particle formation seems to dominate over ion-mediated mechanisms, at least in the boreal forest boundary layer; 4) The subcooled liquid phase saturation vapour pressures of C3-C9 dicarboxylic acids are of the order of 1e-5 1e-3 Pa at atmospheric temperatures, indicating that a mixed pre-existing particulate phase is required for their condensation in atmospheric conditions. The work presented in this thesis gives tools to better quantify the aerosol source provided by secondary aerosol formation. The results are particularly useful when estimating, for instance, anthropogenic versus biogenic influences and the fractions of secondary aerosol formation explained by neutral or ion-mediated nucleation mechanisms, at least in environments where the average particle formation rates are of the order of some tens of particles per cubic centimeter or lower. However, as the factors driving secondary particle formation are likely to vary depending on the environment, measurements on atmospheric nucleation and particle growth are needed from around the world to be able to better describe the secondary particle formation, and assess its climatic effects on a global scale.

First Measurement of the Ratio of Branching Fractions B(Lambda_b to Lambda_c mu nu)/B(Lambda_b to Lambda_c pi)

The analysis uses data from an integrated luminosity of approximately 172 pb-1 of ppbar collisions at sqrt(s)=1.96 TeV, collected with the CDF II detector at the Fermilab Tevatron. The Lambda_b and B0 relative branching fractions are measured to be: B(Lambda_b to Lambda_c+ mu nu)/B(Lambda_b to Lambda_c+ pi) = 16.6 +- 3.0 (stat) +- 1.0 (syst) +2.6 -3.4 (PDG) +- 0.3 (EBR), B(B0 to D+ mu nu)/B(B0 to D+ pi) = 9.9 +- 1.0 (stat) +- 0.6 (syst) +- 0.4 (PDG) +- 0.5 (EBR), B(B0 to D*+ mu nu)/B(B0 to D*+ pi) = 16.5 +- 2.3 (stat) +- 0.6 (syst) +- 0.5 (PDG) +- 0.8 (EBR) This article also presents measurements of the branching fractions of four new Lambda_b semileptonic decays: Lambda_b to Lambda_c(2595)+ mu nu, Lambda_b to Lambda_c(2625)+ mu nu, Lambda_b to Sigma_c(2455)0 pi mu nu, Lambda_b to Sigma_c(2455)++ pi mu nu, relative to the branching fraction of the Lambda_b to Lambda_c mu nu decay. Finally, the transverse-momentum distribution of Lambda_b baryons produced in p-pbar collisions is measured and found to be significantly different from that of B0 mesons.

First observation of the decay Bs --> Ds K and measurement of the ratio of branching fractions Br(Bs --> DsK)/Br(Bs --> Ds pi)

A combined mass and particle identification fit is used to make the first observation of the decay Bs --> Ds K and measure the branching fraction of Bs --> Ds K relative to Bs --> Ds pi. This analysis uses 1.2 fb^-1 integrated luminosity of pbar-p collisions at sqrt(s) = 1.96 TeV collected with the CDF II detector at the Fermilab Tevatron collider. We observe a Bs --> Ds K signal with a statistical significance of 8.1 sigma and measure Br(Bs --> Ds K)/Br(Bs --> Ds pi) = 0.097 +- 0.018(stat) +- 0.009(sys).

First Measurement of the b-Jet Cross Section in Events with a W Boson in pp̅ Collisions at √s=1.96 TeV

The cross section for jets from b quarks produced with a W boson has been measured in ppbar collision data from 1.9/fb of integrated luminosity recorded by the CDF II detector at the Tevatron. The W+b-jets process poses a significant background in measurements of top quark production and prominent searches for the Higgs boson. We measure a b-jet cross section of 2.74 +- 0.27(stat.) +- 0.42(syst.) pb in association with a single flavor of leptonic W boson decay over a limited kinematic phase space. This measured result cannot be accommodated in several available theoretical predictions.

First measurement of the ratio of branching fractions B(Λb0→Λc+μ-ν̅ μ)/B(Λb0→Λc+π-)

This article presents the first measurement of the ratio of branching fractions B(Λb0→Λc+μ-ν̅ μ)/B(Λb0→Λc+π-). Measurements in two control samples using the same technique B(B̅ 0→D+μ-ν̅ μ)/B(B̅ 0→D+π-) and B(B̅ 0→D*(2010)+μ-ν̅ μ)/B(B̅ 0→D*(2010)+π-) are also reported. The analysis uses data from an integrated luminosity of approximately 172  pb-1 of pp̅ collisions at √s=1.96  TeV, collected with the CDF II detector at the Fermilab Tevatron. The relative branching fractions are measured to be B(Λb0→Λc+μ-ν̅ μ)/B(Λb0→Λc+π-)=16.6±3.0(stat)±1.0(syst)+2.6/-3.4(PDG)±0.3(EBR), B(B̅ 0→D+μ-ν̅ μ)/B(B̅ 0→D+π-)= 9.9±1.0(stat)±0.6(syst)±0.4(PDG)±0.5(EBR), and B(B̅ 0→D*(2010)+μ-ν̅ μ)/B(B̅ 0→D*(2010)+π-)=16.5±2.3(stat)± 0.6(syst)±0.5(PDG)±0.8(EBR). The uncertainties are from statistics (stat), internal systematics (syst), world averages of measurements published by the Particle Data Group or subsidiary measurements in this analysis (PDG), and unmeasured branching fractions estimated from theory (EBR), respectively. This article also presents measurements of the branching fractions of four new Λb0 semileptonic decays: Λb0→Λc(2595)+μ-ν̅ μ, Λb0→Λc(2625)+μ-ν̅ μ, Λb0→Σc(2455)0π+μ-ν̅ μ, and Λb0→Σc(2455)++π-μ-ν̅ μ, relative to the branching fraction of the Λb0→Λc+μ-ν̅ μ decay. Finally, the transverse-momentum distribution of Λb0 baryons produced in pp̅ collisions is measured and found to be significantly different from that of B̅ 0 mesons, which results in a modification in the production cross-section ratio σΛb0/σB̅ 0 with respect to the CDF I measurement.

First Observation of B̅ s0→Ds±K∓ and Measurement of the Ratio of Branching Fractions B(B̅ s0→Ds±K∓)/B(B̅ s0→Ds+π-)

A combined mass and particle identification fit is used to make the first observation of the decay B̅ s0→Ds±K∓ and measure the branching fraction of B̅ s0→Ds±K∓ relative to B̅ s0→Ds+π-. This analysis uses 1.2  fb-1 integrated luminosity of pp̅ collisions at √s=1.96  TeV collected with the CDF II detector at the Fermilab Tevatron collider. We observe a B̅ s0→Ds±K∓ signal with a statistical significance of 8.1σ and measure B(B̅ s0→Ds±K∓)/B(B̅ s0→Ds+π-)=0.097±0.018(stat)±0.009(syst).

Significant effects of Pgi genotype and body reserves on lifespan in the Glanville fritillary butterfly

Individuals with a particular variant of the gene phosphoglucose isomerase (Pgi ) have been shown to have superior dispersal capacity and fecundity in the Glanville fritillary butterfly (Melitaea cinxia), raising questions about the mechanisms that maintain polymorphism in this gene in the field. Here, we investigate how variation in the Pgi genotype affects female and male life history under controlled conditions. The most striking effect is the longer lifespan of genotypes with high dispersal capacity, especially in nonreproducing females. Butterflies use body reserves for somatic maintenance and reproduction, but
different resources (in thorax versus abdomen) are used under dissimilar conditions, with some interactions with the Pgi genotype. These results indicate life-history trade-offs that involve resource allocation and genotype!environment interactions, and these trade-offs are likely to contribute to the maintenance of Pgi polymorphism in the natural populations.

First Measurement of the tt̅ Differential Cross Section dσ/dMtt̅ in pp̅ Collisions at √s=1.96 TeV

We present a measurement of the $\ttbar$ differential cross section with respect to the $\ttbar$ invariant mass, dSigma/dMttbar, in $\ppbar$ collisions at $\sqrt{s}=1.96$ TeV using an integrated luminosity of $2.7\invfb$ collected by the CDF II experiment. The $\ttbar$ invariant mass spectrum is sensitive to a variety of exotic particles decaying into $\ttbar$ pairs. The result is consistent with the standard model expectation, as modeled by \texttt{PYTHIA} with \texttt{CTEQ5L} parton distribution functions.

First simultaneous measurement of the top quark mass in the lepton+jets and dilepton channels at CDF

We present a measurement of the mass of the top quark using data corresponding to an integrated luminosity of 1.9fb^-1 of ppbar collisions collected at sqrt{s}=1.96 TeV with the CDF II detector at Fermilab's Tevatron. This is the first measurement of the top quark mass using top-antitop pair candidate events in the lepton + jets and dilepton decay channels simultaneously. We reconstruct two observables in each channel and use a non-parametric kernel density estimation technique to derive two-dimensional probability density functions from simulated signal and background samples. The observables are the top quark mass and the invariant mass of two jets from the W decay in the lepton + jets channel, and the top quark mass and the scalar sum of transverse energy of the event in the dilepton channel. We perform a simultaneous fit for the top quark mass and the jet energy scale, which is constrained in situ by the hadronic W boson mass. Using 332 lepton + jets candidate events and 144 dilepton candidate events, we measure the top quark mass to be mtop=171.9 +/- 1.7 (stat. + JES) +/- 1.1 (syst.) GeV/c^2 = 171.9 +/- 2.0 GeV/c^2.

First Observation of B̅ s0→Ds±K∓ and Measurement of the Ratio of Branching Fractions B(B̅ s0→Ds±K∓)/B(B̅ s0→Ds+π-)

A combined mass and particle identification fit is used to make the first observation of the decay Bs --> Ds K and measure the branching fraction of Bs --> Ds K relative to Bs --> Ds pi. This analysis uses 1.2 fb^-1 integrated luminosity of pbar-p collisions at sqrt(s) = 1.96 TeV collected with the CDF II detector at the Fermilab Tevatron collider. We observe a Bs --> Ds K signal with a statistical significance of 8.1 sigma and measure Br(Bs --> Ds K)/Br(Bs --> Ds pi) = 0.097 +- 0.018(stat) +- 0.009(sys).