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.

Transfers of Family Businesses to Non-Family Buyers - The Selling Business Family Perspective

This study focuses on business families and how they handle transitions such as business transfers. It also tries to shift the balance of research away from successions and towards business transfers as a key topic for family business researchers. In addition, it contributes to the family business research field by further highlighting the importance of the various different contributions in the family business from business family members other than the entrepreneurial founder. Based on interviews with both business family members and business brokers, it appears as important for business families who are selling their family business that it is managed in a similar way in the future regardless of the shift in ownership and management. It is also important that the employees can stay with the business. However, employees are seldom regarded as potential buyers of the family business; most preferably, from the point of view of business family members, this should be somebody who is similar to themselves. Business transfers can be lengthy processes, but once the family business is sold, previous owners most often want to leave the family business. This disengagement can be difficult for business family members if they have not managed to build up some other identity outside the family business environment. Money may compensate for the loss in the short run, but something else is needed in the long run, since the management of money is usually not perceived as that interesting. A family business transfer can have great influence on the members of the business family who is selling, and therefore it is suggested that personal due diligence could be of some help when planning the transfer. That tool can help business family members to analyse their own personal situation, but it may also make it easier to understand how the other business family members feel about the forthcoming change. Everyone is influenced in different ways during a family business transfer, and awareness of this fact may make it easier for the whole business family to adjust to their new environment.

Global, Local and Vector-Valued Tb Theorems on Non-Homogeneous Metric Spaces

Various Tb theorems play a key role in the modern harmonic analysis. They provide characterizations for the boundedness of Calderón-Zygmund type singular integral operators. The general philosophy is that to conclude the boundedness of an operator T on some function space, one needs only to test it on some suitable function b. The main object of this dissertation is to prove very general Tb theorems. The dissertation consists of four research articles and an introductory part. The framework is general with respect to the domain (a metric space), the measure (an upper doubling measure) and the range (a UMD Banach space). Moreover, the used testing conditions are weak. In the first article a (global) Tb theorem on non-homogeneous metric spaces is proved. One of the main technical components is the construction of a randomization procedure for the metric dyadic cubes. The difficulty lies in the fact that metric spaces do not, in general, have a translation group. Also, the measures considered are more general than in the existing literature. This generality is genuinely important for some applications, including the result of Volberg and Wick concerning the characterization of measures for which the analytic Besov-Sobolev space embeds continuously into the space of square integrable functions. In the second article a vector-valued extension of the main result of the first article is considered. This theorem is a new contribution to the vector-valued literature, since previously such general domains and measures were not allowed. The third article deals with local Tb theorems both in the homogeneous and non-homogeneous situations. A modified version of the general non-homogeneous proof technique of Nazarov, Treil and Volberg is extended to cover the case of upper doubling measures. This technique is also used in the homogeneous setting to prove local Tb theorems with weak testing conditions introduced by Auscher, Hofmann, Muscalu, Tao and Thiele. This gives a completely new and direct proof of such results utilizing the full force of non-homogeneous analysis. The final article has to do with sharp weighted theory for maximal truncations of Calderón-Zygmund operators. This includes a reduction to certain Sawyer-type testing conditions, which are in the spirit of Tb theorems and thus of the dissertation. The article extends the sharp bounds previously known only for untruncated operators, and also proves sharp weak type results, which are new even for untruncated operators. New techniques are introduced to overcome the difficulties introduced by the non-linearity of maximal truncations.

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.