Deriving structural information from non-coding ribonucleic acids by mass spectrometry

The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.

Pathogenic Mechanisms of Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy (PLOSL)

PATHOGENIC MECHANISMS OF PLOSL Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease, is a recessively inherited disease of brain and bone. PLOSL manifests as early-onset progressive dementia and bone fractures. Mutations in the TYROBP (DAP12) and TREM2 genes have been identified as the primary cause of PLOSL. DAP12 and TREM2 encode important signalling molecules in cells of the innate immune system. The mechanism by which loss-of-function of the DAP12/TREM2 signalling complex leads to PLOSL is currently unknown. The aim of this thesis work was to gain insight into the pathogenic mechanisms behind PLOSL. To first identify the central nervous system (CNS) cell types that express both Dap12 and Trem2, the expression patterns of Dap12 and Trem2 in mouse CNS were analyzed. Dap12 and Trem2 expression was seen from embryonic stage to adulthood and microglial cells and oligodendrocytes were identified as the major Dap12/Trem2 producing cells of the CNS. To subsequently identify the pathways and biological processes associated with DAP12/TREM2 mediated signalling in human cells, genome wide transcript analysis of in vitro differentiated dendritic cells (DCs) of PLOSL patients representing functional knockouts of either DAP12 or TREM2 was performed. Both DAP12 and TREM2 deficient cells differentiated into DCs and responded to pathogenic stimuli. However, the DCs showed morphological differences compared to control cells due to defects in the actin filaments. Transcript profiles of the patient DCs showed differential expression of genes involved in immune response and for genes earlier associated with other disorders of the CNS as well as genes involved in the remodeling of bone, linking the findings with the tissue phenotype of PLOSL patients. To analyze the effect of Dap12 deficiency in the CNS, genome wide expression analysis of Dap12 deficient mouse brain and Dap12 deficient microglia as well as functional analysis of Dap12 deficient microglia was performed. Regulation of several pathways involved in synaptic function and transcripts coding for the myelin components was seen in Dap12 knockout mice. Decreased migration, morphological changes and shortened lifespan of the Dap12 knockout microglia was further observed. Taken together, this thesis work showed that both Dap12 and Trem2 are expressed by CNS microglia and that Dap12 deficiency results in functional defects of these cells. Lack of Dap12 in the CNS also leads to synaptic abnormalities even before pathological changes are seen in the tissue level.This work further showed that loss-of-function of DAP12 or TREM2 leads to changes in morphology and gene expression in human dendritic cells. These data underline the functional diversity of the molecules of the innate immune system and implies their significant contribution also in demyelinating CNS disorders, including those resulting in dementia.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

SPECT-aided diagnostics and genetic risk factors in Parkinson's disease

Hypokinesia, rigidity, tremor, and postural instability are the cardinal symptoms of Parkinson s disease (PD). Since these symptoms are not specific to PD the diagnosis may be uncertain in early PD. Etiology and pathogenesis of PD remain unclear. There is no neuroprotective therapy. Genetic findings are expected to reveal metabolic routes in PD pathogenesis and thereby eventually lead to therapeutic innovations. In this thesis, we first aimed to study the usefulness and accuracy of 123I-b-CIT SPECT in the diagnosis of PD in a consecutive clinic-based material including various movement disorders. We subsequently a genetic project to identify genetic risk factors for sporadic PD using a candidate gene approach in a case-control setting including 147 sporadic PD patients and 137 spouse controls. Dopamine transporter imaging by 123I-b-CIT SPECT could distinguish PD from essential tremor, drug-induced parkinsonism, dystonia and psychogenic parkinsonism. However, b-CIT uptake in Parkinson plus syndromes (PSP and multiple system atrophy) and dementia with Lewy bodies was not significantly different from PD. 123I-b-CIT SPECT could not reliably differentiate PD from vascular parkinsonism. 123I-b-CIT SPECT was 100% sensitive and specific in the diagnosis of PD in patients younger than 55 years but less specific in older patients, due to differential distribution of the above conditions in the younger and older age groups. 123I-b-CIT SPECT correlated with symptoms and detected bilateral nigrostriatal defect in patients whose PD was still in unilateral stage. Thus, in addition to as a differential diagnostic aid, 123I-b-CIT SPECT may be used to detect PD early, even pre-symptomatically in at-risk individuals. 123I-b-CIT SPECT was used to aid in the collection of patients to the genetic studies. In the genetic part of this thesis we found an association between PD and a polymorphic CAG-repeat in POLG1 gene encoding the catalytic subunit of mitochondrial polymerase gamma. The CAG-repeat encodes a polyglutamine tract (polyQ), the two most common lengths of which are 10Q (86-90%) and 11Q. In our Finnish material, the rarer non-10Q or non-11Q length variants (6Q-9Q, 12Q-14Q, 4R+9Q) were more frequent in patients than in spouse controls (10% vs. 3.5 %, p=0.003), or population controls (p=0.001). Therefore, we performed a replication study in 652 North American PD patients and 292 controls. Non-10/11Q alleles were more common in the US PD patients compared to the controls but the difference did not reach statistical significance (p=0.07). This larger data suggested our original definition of variant length allele might need reconsideration. Most previous studies on phenotypic effects of POLG1 polyQ have defined 10Q as the only normal allele. Non-10Q alleles were significantly more common in patients compared to the controls (17.3% vs. 12.3 %, p= 0.005). This association between non-10Q length variants and PD remained significant when compared to a larger set of 1541 literature controls (p=0.00005). In conclusion, POLG1 polyQ alleles other than 10Q may predispose to PD. We did not find association between PD and parkin or DJ-1, genes underlying autosomal recessive parkinsonism. The functional Val158Met polymorphism, which affects the catalytic effect of COMT enzyme, and another coding polymorphism in COMT were not associated with PD in our patient material. The APOE e2/3/4 polymorphism modifies risk for Alzheimer s disease and prognosis of for example brain trauma. APOE promoter and enhancer polymorphisms 219G/T and +113G/C, and APOE e3 haplotypes, have also been shown to modify the risk of Alzheimer s disease but not reported in PD. No association was found between PD and APOE e2/3/4 polymorphism, the promoter or enhancer polymorphisms, or the e3 haplotypes.

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.

Complex trait of acute pancreatitis : Studies of candidate genes and adipokines

Acute pancreatitis (AP) is a common disease. Mild disease resolves spontaneously in a few days. Severe forms of the disease can lead to local complications, necrosis, and abscesses in and around the pancreas. Systemic inflammation in severe AP is associated with distant organ failures. The aim of this study is to identify genetically determined prognostic factors involved in the clinical features of AP. The study employs a candidate-gene approach, and the genes are involved in trysinogen activation in the initiation phase of the disease, as well as in the systemic inflammation as the disease proceeds. The last study examines adipokines, fat-derived hormones characterized with the capacity to modify inflammation. SPINK 1 is a gene coding trypsin activation inhibitor. Mutations N34S and P55N were determined by minisequencing methods in 371 AP patients and in 459 controls. The mutation N34S was more common in AP patients (7.8%) than in controls (2.6%). This suggests that SPINK 1 gene mutation N34S is a risk factor for AP. In the fourth study, in 12 matched pairs of patients with severe and mild AP, levels of adipokines, adiponectin, and leptin were evaluated. Plasma adipokine levels did not differ between patients with mild and severe AP. The results suggest that in AP, adipokine plasma levels are not factors predisposing to organ failures. This study identified the SPINK 1 mutation N34S to be a risk factor for AP in the general population. As AP is a multifactorial disease, and extensive genetic heterogeneity is likely, further identification of genetic factors in the disease requires larger future studies with more advanced genetic study models. Further identification of the patient characteristics associated with organ failures offers another direction of the study to achieve more detailed understanding of the severe form of AP.

Patient exposure monitoring and radiation qualities in two-dimensional digital x-ray imaging

The methods for estimating patient exposure in x-ray imaging are based on the measurement of radiation incident on the patient. In digital imaging, the useful dose range of the detector is large and excessive doses may remain undetected. Therefore, real-time monitoring of radiation exposure is important. According to international recommendations, the measurement uncertainty should be lower than 7% (confidence level 95%). The kerma-area product (KAP) is a measurement quantity used for monitoring patient exposure to radiation. A field KAP meter is typically attached to an x-ray device, and it is important to recognize the effect of this measurement geometry on the response of the meter. In a tandem calibration method, introduced in this study, a field KAP meter is used in its clinical position and calibration is performed with a reference KAP meter. This method provides a practical way to calibrate field KAP meters. However, the reference KAP meters require comprehensive calibration. In the calibration laboratory it is recommended to use standard radiation qualities. These qualities do not entirely correspond to the large range of clinical radiation qualities. In this work, the energy dependence of the response of different KAP meter types was examined. According to our findings, the recommended accuracy in KAP measurements is difficult to achieve with conventional KAP meters because of their strong energy dependence. The energy dependence of the response of a novel large KAP meter was found out to be much lower than with a conventional KAP meter. The accuracy of the tandem method can be improved by using this meter type as a reference meter. A KAP meter cannot be used to determine the radiation exposure of patients in mammography, in which part of the radiation beam is always aimed directly at the detector without attenuation produced by the tissue. This work assessed whether pixel values from this detector area could be used to monitor the radiation beam incident on the patient. The results were congruent with the tube output calculation, which is the method generally used for this purpose. The recommended accuracy can be achieved with the studied method. New optimization of radiation qualities and dose level is needed when other detector types are introduced. In this work, the optimal selections were examined with one direct digital detector type. For this device, the use of radiation qualities with higher energies was recommended and appropriate image quality was achieved by increasing the low dose level of the system.

Patient doses in ct, dental cone beam ct and projection radiography in Finland, with emphasis on paediatric patients

Diagnostic radiology represents the largest man-made contribution to population radiation doses in Europe. To be able to keep the diagnostic benefit versus radiation risk ratio as high as possible, it is important to understand the quantitative relationship between the patient radiation dose and the various factors which affect the dose, such as the scan parameters, scan mode, and patient size. Paediatric patients have a higher probability for late radiation effects, since longer life expectancy is combined with the higher radiation sensitivity of the developing organs. The experience with particular paediatric examinations may be very limited and paediatric acquisition protocols may not be optimised. The purpose of this thesis was to enhance and compare different dosimetric protocols, to promote the establishment of the paediatric diagnostic reference levels (DRLs), and to provide new data on patient doses for optimisation purposes in computed tomography (with new applications for dental imaging) and in paediatric radiography. Large variations in radiation exposure in paediatric skull, sinus, chest, pelvic and abdominal radiography examinations were discovered in patient dose surveys. There were variations between different hospitals and examination rooms, between different sized patients, and between imaging techniques; emphasising the need for harmonisation of the examination protocols. For computed tomography, a correction coefficient, which takes individual patient size into account in patient dosimetry, was created. The presented patient size correction method can be used for both adult and paediatric purposes. Dental cone beam CT scanners provided adequate image quality for dentomaxillofacial examinations while delivering considerably smaller effective doses to patient compared to the multi slice CT. However, large dose differences between cone beam CT scanners were not explained by differences in image quality, which indicated the lack of optimisation. For paediatric radiography, a graphical method was created for setting the diagnostic reference levels in chest examinations, and the DRLs were given as a function of patient projection thickness. Paediatric DRLs were also given for sinus radiography. The detailed information about the patient data, exposure parameters and procedures provided tools for reducing the patient doses in paediatric radiography. The mean tissue doses presented for paediatric radiography enabled future risk assessments to be done. The calculated effective doses can be used for comparing different diagnostic procedures, as well as for comparing the use of similar technologies and procedures in different hospitals and countries.

Shifting the perspective after the patient's response to an interpretation

Psychoanalytic interpretation is normally understood as a sequence of two utterances: the analyst gives an interpretation and the patient responds to it. This paper suggests that, in the interpretative sequence, there is also a third utterance where psychoanalytic work takes place. This third interpretative turn involves the analyst’s action after the patient’s response to the interpretation. Using conversation analysis as method in the examination of audio-recorded psychoanalytic sessions, the paper will explicate the psychoanalytic work that gets done in third interpretative turns. Through it, the analyst takes a stance towards the patient’s understandings of the interpretation, which are shown in the patient’s response to the interpretation. The third interpretative turns on one hand ratify and accept the patient’s understandings, but, in addition to that, they also introduce a shift of perspective relative to them. In most cases, the shift of perspective is implicit but sometimes it is made explicit. The shifts of perspective bring to the foreground aspects or implications of the interpretation that were not incorporated in the patient’s response. They recast the description of the patient’s experience by showing new layers or more emotional intensity in it. The results are discussed in the light of Faimberg’s concept of listening to listening and Schlesinger’s concept of follow-up interpretation.