Recovery of Yeast Saccharomyces cerevisiae after Thermal Insult

All organisms have evolved mechanisms to acquire thermotolerance. A moderately high temperature activates heat shock genes and triggers thermotolerance towards otherwise lethal high temperature. The focus of this work is the recovery mechanisms ensuring survival of Saccharomyces cerevisiae yeast cells after thermal insult. Yeast cells, first preconditioned at 37˚C, can survive a short thermal insult at 48-50˚C and are able to refold heat-denatured proteins when allowed to recover at physiological temperature 24˚C. The cytoplasmic chaperone Hsp104 is required for the acquisition of thermotolerance and dissolving protein aggregates in the cytosol with the assistance of disaccharide trehalose. In the present study, Hsp104 and trehalose were shown to be required for conformational repair of heat-denatured secretory proteins in the endoplasmic reticulum. A reporter protein was first accumulated in the lumen of endoplasmic reticulum and heat-denatured by thermal insult, and then failed to be repaired to enzymatically active and secretion-competent conformation in the absence of Hsp104 or trehalose. The efficient transport of a glycoprotein CPY, accumulated in the endoplasmic reticulum, to the vacuole after thermal insult also needed the presence of Hsp104 and trehalose. However, proteins synthesized after thermal insult at physiological temperature were secreted with similar kinetics both in the absence and in the presence of Hsp104 or trehalose, demonstrating that the secretion machinery itself was functional. As both Hsp104 and trehalose are cytosolic, a cross-talk between cytosolic and luminal chaperone machineries across the endoplasmic reticulum membrane appears to take place. Global expression profiles, obtained with the DNA microarray technique, revealed that the gene expression was shut down during thermal insult and the majority of transcripts were destroyed. However, the transcripts of small cytosolic chaperones Hsp12 and Hsp26 survived. The first genes induced during recovery were related to refolding of denatured proteins and resumption of de novo protein synthesis. Transcription factors Spt3p and Med3p appeared to be essential for acquisition of full thermotolerance. The transcription factor Hac1p was found to be subject to delayed up-regulation at mRNA level and this up-regulation was diminished or delayed in the absence of Spt3p or Med3p. Consequently, production of the chaperone BiP/Kar2p, a target gene of Hac1p, was diminished and delayed in Δspt3 and Δmed3 deletion strains. The refolding of heat-denatured secretory protein CPY to a transport-competent conformation was retarded, and a heat-denatured reporter enzyme failed to be effectively reactivated in the cytoplasm of the deletion strains.

Pathological changes at the target tissue level in Sjögren's syndrome and their effect on the exocrine function of the salivary glands

Sjögren s syndrome (SS) is a common autoimmune disease affecting the lacrimal and salivary glands. SS is characterized by a considerable female predominance and a late age of onset, commonly at the time of adreno- and menopause. The levels of the androgen prohormone dehydroepiandrosterone-sulphate (DHEA-S) in the serum are lower in patients with SS than in age- and sex-matched healthy control subjects. The eventual systemic effects of low androgen levels in SS are not currently well understood. Basement membranes (BM) are specialized layers of extracellular matrix and are composed of laminin (LM) and type IV collagen matrix networks. BMs deliver messages to epithelial cells via cellular LM-receptors including integrins (Int) and Lutheran blood group antigen (Lu). The composition of BMs and distribution of LM-receptors in labial salivary glands (LSGs) of normal healthy controls and patients with SS was assessed. LMs have complex and highly regulated distribution in LSGs. LMs seem to have specific tasks in the dynamic regulation of acinar cell function. LM-111 is important for the normal acinar cell differentiation and its expression is diminished in SS. Also LM-211 and -411 seem to have some acinar specific functional tasks in LSGs. LM-311, -332 and -511 seem to have more general structure maintaining and supporting roles in LSGs and are relatively intact also in SS. Ints α3β1, α6β1, α6β4 and Lu seem to supply structural basis for the firm attachment of epithelial cells to the BM in LSGs. The expression of Ints α1β1 and α2β1 differed clearly from other LM-receptors in that they were found almost exclusively around the acini and intercalated duct cells in salivons suggesting some type of acinar cell compartment-specific or dominant function. Expression of these integrins was lower in SS compared to healthy controls suggesting that the LM-111 and -211-to-Int α1β1 and α2β1 interactions are defective in SS and are crucial to the maintenance of the acini in LSGs. DHEA/DHEA-S concentration in serum and locally in saliva of patients with SS seems to have effects on the salivary glands. These effects were first detected using the androgen-dependent CRISP-3 protein, the production and secretion of which were clearly diminished in SS. This might be due to the impaired function of the intracrine DHEA prohormone metabolizing machinery, which fails to successfully convert DHEA into its active metabolites in LSGs. The progenitor epithelial cells from the intercalated ductal area of LSGs migrate to the acinar compartment and then undergo a phenotype change into secretory acinar cells. This migration and phenotype change seem to be regulated by the LM-111-to-Int α1β1/Int α2β1 interactions. Lack of these interactions could be one factor limiting the normal remodelling process. Androgens are effective stimulators of Int α1β1 and α2β1 expression in physiologic concentrations. Addition of DHEA to the culture medium had effective stimulating effect on the Int α1β1 and α2β1 expression and its effect may be deficient in the LSGs of patients with SS.

Consequences of Supersymmetry in the Early Universe

Currently, we live in an era characterized by the completion and first runs of the LHC accelerator at CERN, which is hoped to provide the first experimental hints of what lies beyond the Standard Model of particle physics. In addition, the last decade has witnessed a new dawn of cosmology, where it has truly emerged as a precision science. Largely due to the WMAP measurements of the cosmic microwave background, we now believe to have quantitative control of much of the history of our universe. These two experimental windows offer us not only an unprecedented view of the smallest and largest structures of the universe, but also a glimpse at the very first moments in its history. At the same time, they require the theorists to focus on the fundamental challenges awaiting at the boundary of high energy particle physics and cosmology. What were the contents and properties of matter in the early universe? How is one to describe its interactions? What kind of implications do the various models of physics beyond the Standard Model have on the subsequent evolution of the universe? In this thesis, we explore the connection between in particular supersymmetric theories and the evolution of the early universe. First, we provide the reader with a general introduction to modern day particle cosmology from two angles: on one hand by reviewing our current knowledge of the history of the early universe, and on the other hand by introducing the basics of supersymmetry and its derivatives. Subsequently, with the help of the developed tools, we direct the attention to the specific questions addressed in the three original articles that form the main scientific contents of the thesis. Each of these papers concerns a distinct cosmological problem, ranging from the generation of the matter-antimatter asymmetry to inflation, and finally to the origin or very early stage of the universe. They nevertheless share a common factor in their use of the machinery of supersymmetric theories to address open questions in the corresponding cosmological models.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Sex steroids in Sjögren's syndrome

Sjögren s syndrome (SS) is a strongly female dominant autoimmune disease. SS targets mainly salivary and lacrimal glands and leads to loss of the secreting acinar cells of these glands. Accordingly, secretion of the affected glands is diminished and the main symptoms of SS, dryness of mouth and eyes, follow. In addition to these sicca symptoms, SS patients suffer from severe fatigue and can have various extraglandular symptoms. To date, the etiology of SS still remains unknown. Female dominance and the late onset of the disease simultaneously with remarkable hormonal changes in the body (menopause, adrenopause) encouraged us to hypothesize that sex steroids, especially androgens, are involved in the onset and progression of SS. We confirmed our hypothesis and showed that patients with SS suffer from androgen depletion both systemically and locally in the target tissue of SS, salivary glands. We especially focused on the local androgen environment in salivary glands and demonstrated that healthy salivary glands contain a complete enzymatic machinery for local synthesis of androgens and estrogens from pro-hormone dehydroepiandrosterone (DHEA). However, in SS salivary glands the enzymes catalyzing the local androgen synthesis are defective and, in a subgroup of patients, practically non-functional. Probably due to this local defect in DHEA processing, therapy with DHEA was found unbeneficial for SS patients in the treatment of fatigue. We also studied the effect of the local androgen depletion on salivary glands. We found that in salivary gland cells and healthy labial salivary glands androgens upregulate integrin subunits α1 and α2, which are important for the communication, differentiation and function of the acinar cells. On the contrary, in SS salivary glands DHEA failed to upregulate these signaling molecules, again probably due to defective processing of DHEA into active androgens. Our finding highlights the importance of the local androgen environment and local DHEA processing for the function and welfare of salivary glands. In conclusion, this study showed that patients with SS are androgen depleted both systemically and locally in salivary glands. SS patients also have a defective local sex steroid synthesizing enzymatic machinery further impairing the local androgen depletion. We also showed that the local androgen defect leads to decreased expression of acinar cell specific integrin molecules, which impairs the signaling between the acinar cells and basement membrane and might thus explain the acinar cell loss seen in SS salivary glands. By showing the importance of the local sex steroid imbalance in SS we have clarified some etiopathogenetic mechanisms of SS, which have thus far remained unknown.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

Emerging bioenergy business at Finnish independent industrial sawmills

Finnish forest industry is in the middle of a radical change. Deepening recession and the falling demand of woodworking industry´s traditional products have forced also sawmilling industry to find new and more fertile solutions to improve their operational preconditions. In recent years, the role of bioenergy production has often been highlighted as a part of sawmills´ business repertoire. Sawmilling produces naturally a lot of by-products (e.g. bark, sawdust, chips) which could be exploited more effectively in energy production, and this would bring more incomes or maybe even create new business opportunities for sawmills. Production of bioenergy is also supported by government´s climate and energy policies favouring renewable energy sources, public financial subsidies, and soaring prices of fossil fuels. Also the decreasing production of domestic pulp and paper industry releases a fair amount of sawmills´ by-products for other uses. However, bioenergy production as a part of sawmills´ by-product utilization has been so far researched very little from a managerial point of view. The purpose of this study was to explore the relative significance of the main bioenergy-related processes, resources and factors at Finnish independent industrial sawmills including partnerships, cooperation, customers relationships and investments, and also the future perspectives of bioenergy business at these sawmills with the help of two resource-based approaches (resource-based view, natural-resource-based view). Data of the study comprised of secondary data (e.g. literature), and primary data which was attracted from interviews directed to sawmill managers (or equivalent persons in charge of decisions regarding bioenergy production at sawmill). While a literature review and the Delphi method with two questionnaires were utilized as the methods of the study. According to the results of the study, the most significant processes related to the value chain of bioenergy business are connected to raw material availability and procurement, and customer relationships management. In addition to raw material and services, the most significant resources included factory and machinery, personnel, collaboration, and geographic location. Long-term cooperation deals were clearly valued as the most significant form of collaboration, and especially in processes connected to raw material procurement. Study results also revealed that factors related to demand, subsidies and prices had highest importance in connection with sawmills´ future bioenergy business. However, majority of the respondents required that certain preconditions connected to the above-mentioned factors should be fulfilled before they will continue their bioenergy-related investments. Generally, the answers showed a wide divergence of opinions among the respondents which may refer to sawmills´ different emphases and expectations concerning bioenergy. In other words, bioenergy is still perceived as a quite novel and risky area of business at Finnish independent industrial sawmills. These results indicate that the massive expansion of bioenergy business at private sawmills in Finland is not a self-evident truth. The blocking barriers seem to be connected mainly to demand of bioenergy and money. Respondents´ answers disseminated a growing dissatisfaction towards the policies of authorities, which don´t treat equally sawmill-based bioenergy compared to other forms of bioenergy. This proposition was boiled down in a sawmill manager´s comment: “There is a lot of bioenergy available, if they just want to make use of it.” It seems that the positive effects of government´s policies favouring the renewables are not taking effect at private sawmills. However, as there anyway seems to be a lot of potential connected to emerging bioenergy business at Finnish independent industrial sawmills, there is also a clear need for more profound future studies over this topic.

Trade Blocs and the Cold War : The CMEA and the EC challenge, 1969-1976

The coherence of the Soviet bloc was seriously tested at the turn of the 1970s, as the Soviet Union and its allies engaged in intensive negotiations over their relations with the European Communities (EC). In an effort to secure their own national economic interests many East European countries began independent manoeuvres against the wishes of their bloc leader. However, much of the intra-bloc controversy was kept out of the public eye, as the battle largely took place behind the scenes, within the organisation for economic cooperation, the Council for Mutual Economic Assistance (CMEA). The CMEA policy-making process vis-à-vis the EC is described in this study with reference to primary archival materials. This study investigates the negotiating positions and powers of the CMEA member states in their efforts to deal with the economic challenge created by the progress of the EC, as it advanced towards the customs union. This entails an analysis of the functioning principles and performance of the CMEA machinery. The study traces the CMEA negotiations that began in 1970 over its policy toward the EC. The policy was finally adopted in 1974, and was followed by the first official meeting between the two organisations in early 1975. The story ends in 1976, when the CMEA s efforts to enter into working relations with the EC were seemingly frustrated by the latter. The first major finding of the study is that, contrary to much of the prior research, the Soviet Union was not in a hegemonic position vis-à-vis its allies. It had to use a lot of its resources to tame the independent manoeuvring of its smaller allies. Thus, the USSR was not the kind of bloc leader that the totalitarian literature has described. Because the Soviet Union had to spend so much attention on its own bloc-politics, it was not able to concentrate on formulating a policy vis-à-vis the EC. Thus, the Soviet leadership was dependent on its allies in those instances when the socialist countries needed to act as a bloc. This consequently opened up the possibility for the USSR s allies to manoeuvre. This study also argues that when the CMEA did manage to find a united position, it was a force that the EC had to reckon with in its policy-making. This was particularly the case in the implementation of the EC Common Commercial Policy. The other main finding of the study is that, although it has been largely neglected in the previous literature on the history of West European integration, the CMEA did in fact have an effect on EC decision-making. This study shows how for political and ideological reasons the CMEA members did not acknowledge the EC s supranational authority. Therefore the EC had no choice but to refrain from implementing its Common Commercial Policy in full.