Higher-order cognitive functions, such as executive function (EF) and intelligence, are crucial to the everyday functioning of human beings. Gaining knowledge about these functions is important for our general understanding of human nature as well as for our ability to help those who may not develop these processes optimally. The present thesis focused particularly on the EF component working memory (WM), described as the ability to maintain informa-tion in consciousness during short time periods with the purpose of using that information in complex cognition. The major aims of the thesis were to increase our understanding of higher-order cognition in children as well as of deficiencies in intelligence found in children with Attention Deficit Hyperactivity Disorder (ADHD). We approached these aims by studying the interrelations among EF-related components in terms of their independent contributions to intellectual functioning. We also studied whether the lower intelligence in children with ADHD was mediated by fundamental EF-related components or whether these deficiencies went beyond the weaknesses in these specific cognitive functions. Interpreting the present data, we suggest that intellectual functioning in children is best viewed as representing a system of primarily independent parts that may be accompanied by an overarching common mechanism. The multiple components involve, but are surely not limited to, WM functions, inhibitory functions, sustained attention, and processing speed. One of these functions, WM, was found to be further partitioned into domain-specific executive WM processes and domain-specific short-term storage processes, all of which constitute important aspects of higher-order cognitive functioning. We have further learned that deficits in fluid intelligence in children with ADHD may entail more than weaknesses in specific central cognitive functions. This additional deficit is cautiously interpreted as involving supe-rior executive attention functions setting the stage for the development and integration of the EF system as well as the “intelligence system”.

Calmodulin is an essential EF-hand protein with a helix-loop-helix calcium binding motif. Understanding Ca(II) dependent activation of calmodulin and other EF-hand proteins is limited by Ca(II)-induced conformational change, multiple and cooperative binding of Ca(II) ions, and interactions between the paired EF-hand motifs. The goal of this research project is to probe key determinants for calcium binding properties and pairing interactions at the site specific level using a grafting approach and high resolution NMR. An individual Ca(II) binding site of the EF-hand motifs of calmodulin was grafted into a non-calcium dependent protein, CD2, to bypass limitations associated with natural EF-hand proteins and peptide fragments. Using high resolution NMR, we have shown that the grafted EF-loop III of calmodulin in the host protein retains its native conformation with a strong loop and β-conformation preference. Grafted ligand residues in the engineered protein are directly involved in binding of Ca(II) and La(III). The NMR studies support our hypothesis that both ligand arrangement and dynamic properties play essential role in tuning Ca(II) binding affinities. Using pulse-field diffusion NMR and protein engineering, we further demonstrated that grafted EF- loop remains as a monomer. Although the EF-loop with flanking helices dimerizes in the presence of Ca(II). Additionally, removal of conserved hydrophobic residues at the flanking helices of the EF-hand motif leads to be monomer in the absence and presence of metal ions. Our results suggest that conserved hydrophobic residues are essential for the pair-paired interaction in the coupled EF-hand protein. We have shown that our developed grafting approach can be applied to probe intrinsic Ca(II) binding affinities of different Ca(II) binding sites.

Calpains are intracellular enzymes that merge cysteine protease and calcium sensing activities together in one molecule. They respond to Ca2+ signals and modify the activity of their targets by selective proteolysis. Calpains are involved in normal cellular process like cell migration and apoptosis. The over-activation of calpain due to disturbances in Ca2+ homeostasis or inactivation due to mutations, contribute to diseases like ischemic injury and muscular dystrophy. The classical calpains 1 and 2 are heterodimeric enzymes containing a large (80 kDa) subunit and a small subunit (28 kDa). Dimerization occurs through the 5th EF-hand of penta-EF-hand (PEF) domains present in both large and small subunits. In this study, I have used structural genomics approaches to explore the PEF and C2-like regulatory domains of some of the other 12 human calpain isoforms. I have shown that recombinant PEF domain of skeletal muscle-specific calpain 3 exists as a stable homodimer when produced alone. Modelling studies suggest that there would be no barriers for dimerization of the full-length enzyme through the PEF domains which would place the protease cores at opposite ends of the dimer. Co-expression studies using small subunit were performed with PEF domains of calpains 1, 3, 8, 9, 11, 12 and 13. A differential tagging system was devised to differentiate heterodimers from homodimers. The PEF domains of calpains 1, 3, 9 and 13 co-expressed with the small subunit, while the others failed to express. The PEF domains of calpains 1 and 9 formed heterodimers. Conversely, the PEF domain of calpain 3 formed a homodimer and that of calpain 13 predominantly formed a homodimer with a small amount of heterodimer. Homodimerization of calpains implies they are less-likely to be inhibited by the endogenous calpain inhibitor, calpastatin. C2-like regulatory domains of calpains 5-13 were also studied. The structure of the distal C2-like domain of calpain 7 was solved. It is markedly different from canonical C2 domains and may not bind Ca2+. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-02-11 12:30:29.18

Ekokardiografi är en mycket användbar teknik som bland annat kan bedöma vänsterkammarensfunktion hos hjärtat med hjälp av ejektionsfraktion (EF) och global longitudinell strain (GLS). EF är idag den metoden som används mest vid bedömning av vänsterkammarfunktionen och beräknas enligt Simpsons biplanmetod. GLS är en ny metod som mäter myokardiets deformation och har i tidigare studier visat sig vara en känslig metod och kan upptäcka förändringar i myokardiet tidigare än EF. Vid behandling av bröstcancer kan hjärtat påverkas toxiskt och därmed genomgår denna patientgrupp regelbundna kontroller med ekokardiografi.   Syftet med denna studie är att jämföra två ekokardiografiska metoder hos 20 stycken kvinnliga bröstcancerpatienter. Jämförelser har gjorts mellan EF beräknat med Simpsons biplanmetod (EFbi) och GLS samt mellan EFbi och EF beräknat från GLS (EFGLS). Detta är en kvantitativ retrospektiv studie där data insamlats mellan oktober-december 2014 på avdelningen klinisk fysiologi vid Länssjukhuset Ryhov i Jönköping. Den analysmetod som använts är McNemar´s test samt kappavärde. Resultatet av studien visar att EFbi och GLS gav en dålig överenstämmelse mellan metoderna och en rimlig överenstämmelse sågs mellan EFbi och EFGLS. Slutsatsen har dragits utifrån kappavärdet. / Echocardiography is a useful technique which can value the function of the left chamber of the heart by using ejection fraction (EF) and global longitudinal strain (GLS). Today EF is the most common method in assessments of the left chamber function and is calculated by Simpson´s biplanemethod. GLS is a new method that measures the myocardial deformation. It has been proved to be a sensitive method and can detect changes in the myocardium earlier than EF.   The purpose of this study is to compare two echocardiographic methods among 20 female breast cancer patients. Comparisons have been made between EF calculated with Simpson´s biplanemethod (EFbi) and GLS, and between EFbi and EF calculated by GLS (EFGLS). This is a quantitative retrospective study. The data used in this study was collected between October – December 2014 at the Department of Clinical Physiology at the Hospital Ryhov in Jönköping. The method used to analyse the results was McNemar´s test and Kappa value. The results of the study showed that EFbi and GLS gave a modest correspondence between the methods. A reasonable correspondence were observed between EFbi and EFGLS. The conclusion has been drawn from the kappa value.

The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme. We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode. RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

Un modèle d’endommagement dédié aux composites bobinés est développé à partir des outils de lamécanique de l’endommagement continu, de la thermodynamique des processus irréversibles et dela théorie de représentation des fonctions tensorielles. La particularité de ce modèle est l’utilisationd’une approche à directions fixes de l’endommagement qui associe à chaque mode de dégradationdes variables internes scalaires et des tenseurs directionnels. La rupture des fibres (considéréecomme probabiliste), les fissurations tant matricielles, que hors – plan ou provoquées par lecisaillement sont ainsi prises en compte. Le modèle est capable de reproduire, dans un contextetridimensionnel imposé par les fortes épaisseurs de composite, la perte de rigidité, l’interaction entreanisotropies initiale et induite, la non linéarité du comportement, les déformations résiduelles et laviscosité en cisaillement. Afin de valider cette approche, le comportement thermomécaniqued’éprouvettes issues de structures bobinées a été caractérisé grâce à des essais de traction multi –instrumentés (vidéo – traction, émission acoustique,...). On montre que le modèle est capable nonseulement de simuler la réponse mécanique macroscopique de ces échantillons, mais également dereproduire l’émission acoustique enregistrée, de distinguer les différentes formesd’endommagement et de prédire précisément l’éclatement des réservoirs hyperbares. / A damage model dedicated to wound composite is developed by using the tools of continuousdamage mechanics, irreversible processes thermodynamics and the representation theory of tensorfunctions. The particularity of this model is the use of a fixed directions approach of damage whichassociates each degradation mode to internal scalar variables and directional tensors. Fiber breakage(considered probabilistic), matrix cracking (transverse, out-of-plane or caused by shear) are thustaken into account. The model is able to reproduce, in a three-dimensional context imposed bythick composite layers, loss of strength, interaction between initial and induced anisotropy, nonlinearitybehavior, residual strain and shear viscosity. To validate this approach, thermo mechanicalbehavior of specimens from the wound structures was characterized by tensile multi - instrumentedtests (video - traction, acoustic emission, ...). We show that the model is able to simulate not onlythe macroscopic mechanical response of these samples, but also to reproduce the recorded acousticemission to distinguish the various forms of damage and to accurately predict the burst ofhyperbaric tanks.

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Life is anti-entropic and highly organized phenomenon with two characteristics reinforcing each other: homochirality and the stereospecific catalysis of chemical reactions. The exclusive presence of L-amino acids and R-sugars in living world well depict this. Hypothetically, the amino acids and sugars of reverse chirality could form a parallel kingdom which is highly orthogonal to the present world. The components from this mirror kingdom, such as protein or nucleic acid, will be much more resistant to the defensive mechanism of present living system, which could be of great value. Therefore, by gradually rewiring the present bio-machineries, we look to build a bridge leading us to the space of mirror-imaged biomolecules. We begin by investigating protein synthesis with mirror amino acid since most amino acids contain one chiral center to be inversed comparing to sugars. In this work, we analyzed three stages critical for the incorporation of D-amino acid into ribosomal protein synthesis: amino acylation, EF-Tu binding of amino acyl-tRNA and delivery bias, and ribosome catalyzed peptidyl transfer. We have demonstrated that the affinity between EF-Tu and amino acyl-tRNA plays critical role on D-amino acid incorporation, and built a platform aimed to select for ribosome tolerating D-amino acid better. / Chemistry and Chemical Biology

The main topic of this master thesis is creating, testing and implementation of algorithms for autofocus with Canon camera lens using CAN-EF interface, while one of the assignments is possibility to continuous focus to the vehicle in traffic monitoring. There are tested a number of criteria for the assessment if sharpness in the image and is designed automatic control system of the lens and camera.

Ribosomes translate the genetic information contained in mRNAs into protein by linking together amino acids with the help of aminoacyl-tRNAs. In bacteria, protein synthesis stalls when the ribosome reaches the 3'-end of truncated mRNA transcripts lacking a stop codon. Trans-translation is a conserved bacterial quality control process that rescues stalled ribosomes. Transfer-messenger RNA (tmRNA) and its protein partner SmpB mimic a tRNA by entering the A site of the ribosome and accepting the growing peptide chain. The ribosome releases the truncated mRNA and resumes translation on the tmRNA template. The open reading frame found on tmRNA encodes a peptide tag that marks the defective nascent peptide for proteolysis. A stop codon at the end of the open reading frame allows the ribosome to be recycled and engage in future rounds of translation.The entry of tmRNA into stalled ribosomes presents a challenge to our understanding of ribosome function because during the canonical decoding process, the ribosome specifically recognizes the codon-anticodon duplex formed between tRNA and mRNA in the A site. Recognition of proper base-pairing leads to conformational changes that accelerate GTP hydrolysis by EF-Tu and rapid accommodation of the tRNA into the ribosome for peptidyl transfer. The puzzle is that tmRNA enters stalled ribosomes and reacts with the nascent peptide in the absence of a codon-anticodon interaction. Instead, SmpB binding in the decoding center begins the rescue process, but it has been unclear how SmpB licenses tmRNA entry into stalled ribosomes. We analyzed a series of SmpB and ribosomal RNA mutants using pre-steady-state kinetic assays for EF-Tu activation and peptidyl transfer. Although the conserved 16S nucleotides A1492 and A1493 play an essential role in canonical decoding, they play little or no role in EF-Tu activation or peptidyl transfer to tmRNA. In contrast, a third nucleotide, G530, stacks with the side chain of SmpB residue His136, inducing conformational changes that lead to GTP hydrolysis by EF-Tu. A portion of the C-terminal tail forms a helix within the mRNA channel, monitoring the length of mRNA bound in the ribosome to avoid aborting productive protein synthesis. Helix formation in the mRNA channel is essential for accommodation and peptidyl transfer, but not for GTP hydrolysis. We show that conserved residues in the tail are essential for EF-Tu activation, accommodation, or translocation to the P site. Our findings lead to a clearer model of how the tmRNA-SmpB complex enters stalled ribosomes.