Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.

Optimal auctions for multi-unit procurement with volume discount bids

Our attention, is focused on designing an optimal procurement mechanism which a buyer can use for procuring multiple units of a homogeneous item based on bids submitted by autonomous, rational, and intelligent suppliers. We design elegant optimal procurement mechanisms for two different situations. In the first situation, each supplier specifies the maximum quantity that can be supplied together with a per unit price. For this situation, we design an optimal mechanism S-OPT (Optimal with Simple bids). In the more generalized case, each supplier specifies discounts based on the volume of supply. In this case, we design an optimal mechanism VD-OPT (Optimal with Volume Discount, bids). The VD-OPT mechanism uses the S-OPT mechanism as a building block. The proposed mechanisms minimize the cost to the buyer, satisfying at the same time, (a) Bayesian, incentive compatibility and (b) interim individual rationality.

Regulation of Reproduction in the Primitively Eusocial Wasp Ropalidia marginata: on the Trail of the Queen Pheromone

Queens and workers are not morphologically differentiated in the primitively eusocial wasp, Ropalidia marginata. Upon removal of the queen, one of the workers becomes extremely aggressive, but immediately drops her aggression if the queen is returned. If the queen is not returned, this hyper-aggressive individual, the potential queen (PQ), will develop her ovaries, lose her hyper-aggression, and become the next colony queen. Because of the non-aggressive nature of the queen, and because the PQ loses her aggression by the time she starts laying eggs, we hypothesized that regulation of worker reproduction in R marginata is mediated by pheromones rather than by physical aggression. Based on the immediate loss of aggression by the PQ upon return of the queen, we developed a bioassay to test whether the queen's Dufour's gland is, at least, one of the sources of the queen pheromone. Macerates of the queen's Dufour's gland, but not that of the worker's Dufour's gland, mimic the queen in making the PQ decrease her aggression. We also correctly distinguished queens and workers of R. marginata nests by a discriminant function analysis based on the chemical composition of their respective Dufour's glands.

Action regulation across the adult lifespan (ARAL): A metatheory of work and aging

The goals of this article are to integrate action regulation theory (ART) with the lifespan developmental perspective and to outline tenets of a new metatheory of work and aging. The action regulation across the adult lifespan (ARAL) theory explains how workers influence, and are influenced by, their environment across different time spans. First, the basic concepts of ART are described, including the sequential and hierarchical structure of actions, complete tasks and actions, foci of action regulation, and the action-regulating mental model. Second, principles of the lifespan developmental perspective are delineated, including development as a lifelong and multidirectional process, the joint occurrence of gains and losses, intraindividual plasticity, historical embeddedness, and contextualism. Third, propositions of ARAL theory are derived by analyzing workers’ action regulation from a lifespan developmental perspective (i.e., effects of aging on action regulation), and by analyzing aging and development in the work context from an ART perspective (i.e., effects of action regulation on age-related changes in cognition and personality). Fourth, we develop further propositions to integrate ART with lifespan theories of motivation and socioemotional experience. Finally, we discuss implications for future research and practice based on ARAL theory.

Volume change behaviour of fine grained solis

Soils in and and semi-arid zones undergoes volume changes due to wetting. Depending upon the type of clay minerals present, degree of saturation, externally applied load and bonding, the fine grained soils either swells or compresses. One of the parameter that affects the volume change behaviour is the primary clay mineral present in their clay size fraction. A simple method of identifying the same has been presented. It has been brought out that in an expansive unsaturated undisturbed soil, the diffuse double layer repulsion, the stress state and the bonding play significant role in their volume change behaviour. In non-expansive fine grained unsaturated undisturbed soils, the shearing resistance at particle level (including the matrix suction and bonding) and fabric play a significant role in influencing the volume change behaviour. While both the mechanism co-exist, one of them play a dominant role depending upon the primary clay mineral is swelling or non swelling.

Handling sectional views in volume-based approach to automatically construct 3D solid from 2D views

This paper presents an algorithm for solid model reconstruction from 2D sectional views based on volume-based approach. None of the existing work in automatic reconstruction from 2D orthographic views have addressed sectional views in detail. It is believed that the volume-based approach is better suited to handle different types of sectional views. The volume-based approach constructs the 3D solid by a boolean combination of elementary solids. The elementary solids are formed by sweep operation on loops identified in the input views. The only adjustment to be made for the presence of sectional views is in the identification of loops that would form the elemental solids. In the algorithm, the conventions of engineering drawing for sectional views, are used to identify the loops correctly. The algorithm is simple and intuitive in nature. Results have been obtained for full sections, offset sections and half sections. Future work will address other types of sectional views such as removed and revolved sections and broken-out sections. (C) 2004 Elsevier Ltd. All rights reserved.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

The regulation of nitrate reductas enad catalase by amino acids in Neurospora crassa

The induction of nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) by nitrate in Neurospora crassa and its control by amino acids have been studied. The growth-inhibitory amino acids, isoleucine and cysteine as well as the growth-promotory ones, glutamine, asparagine, arginine, histidine and NH4+, repress nitrate reductase effectively. Methionine, tryptophan, proline, aspartic acid and glutamic acid exert little control on nitrate reductase. The repression of nitrate reductase by cysteine, isoleucine, glutamine and asparagine is accompanied by inactivation of the enzyme present initially. The nitrate-induced NADPH-cytochrome c reductase (NADPH:cytochrome c oxidoreductase, EC 1.6.2.3) is also repressed by amino acids which control nitrate reductase, providing further evidence to show that these two enzyme activities may reside in the same protein. Catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) has been found to be induced subsequent to the induction of nitrate reductase by nitrate in N. crassa. The induction of catalase is probably by its substrate H2O2 which would be formed by the interaction of the flavine component of nitrate reductase with oxygen. The amino acids which control nitrate reductase, repress catalase also. The catalase level appears to be determined by the nitrate reductase activity of the mycelia.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.