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The giant protein titin is the third myofilament in the cardiac sarcomere. It is responsible for generating passive forces in stretched myocardium and maintaining sarcomere structure. The force generation properties of titin are determined by titin's elastic springlike elements, and this dissertation focuses on the determination of the physical properties of these springlike elements using atomic force microscopy. The primary project of this dissertation investigates the link between a single point mutation in one of titin's subdomains and arrhythmogenic cardiomyopathy.

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 78-81).

Melanoblasts are the embryonic precursors of melanocytes, the pigment producing cells of the skin and hair. Melanoblasts are of key interest to developmental biologists for numerous reasons, including their ability to migrate throughout the body from a single origin in the neural crest (NC). Current methods for the study of the melanocyte lineage are limited by the heavy reliance on animal models. To challenge this, a platform of in vitro tools were designed to replace and complement current studies. A major obstacle is the transition from 2D cultures, which provide only limited behavioural information, to 3D models which are able to recapitulate the environmental conditions. 3D cultures are regularly created using tissue samples and synthetic matrices for attachment, but building a model from cell lines only has not been achieved. A co-culture model using immortalised keratinocyte (COCA) and melanoblast cell lines proved unsuitable for observing developmental processes, due to lack of movement at high cell densities, but may be practical in pigmentation research. Other methods were explored to examine melanoblast behaviour, including the use of cell derived matrices (CDMs) integrated with melanoblast cell lines, and aggregates formed by hanging drop (HD) culture. CDMs were successfully generated from the COCA line, as well as NIH3T3 fibroblasts which has been shown previously. These structures are denuded of cells to leave the deposited extracellular matrix (ECM) components intact, representative of the dermal (fibroblast) and epidermal (keratinocyte) layers of the skin. HDs were prepared from cultured melanoblast cell lines, and form tight aggregates which disseminate when plated, in a manner similar to the dissemination of cells from the NC in explant cultures. The receptor tyrosine kinase KIT and its ligand (KITL), are vital for melanoblast development. Previous study of this signalling complex has often focussed on the haematopoietic lineage and spermatogenesis, where they perform essential roles. KITL is expressed in a membrane localised form found on the surface of keratinocytes thought to promote melanoblast/melanocyte survival, and a soluble isoform found sequestered in the ECM which promotes cell migration. Cell lines expressing fluorescently tagged KIT and KITL were created to visualise their interactions using live-cell confocal imaging. Firstly, cell lines were generated to perform co-culture experiments with KIT and KITL, and we showed that these constructs are able to interact by uptake of KITL into KIT cells. Secondly, tandem fluorescent protein timers of KIT and KITL were generated which were used to observe protein kinetics. We showed that these protein timers can be manipulated using cycloheximide to block protein production, or by increasing ligand availability. These protein timers reveal that soluble KITL (sKITL) has a faster turnover than membrane bound KITL (mKITL), and that in all three proteins, there is distinct change in spatial localisation as the proteins age. Using a novel melanoblast reporter mouse, Pmel-CMN, primary mouse melanoblasts between E12.5 and E14.5 were isolated for RNA sequencing. This time period is the earliest reported for melanoblast isolation for use in gene expression analysis. We show that within this time course, there are significant changes in the RNA expression profiles, including decreasing expression of other NC cell markers, and huge increasing expression of pigmentation genes. To assess the biological relevance of using in vitro assays, cells of the immortalised melanoblast cell line, melb-a, were cultured under different conditions and examined via RNA sequencing. Results reveal differences in several areas between primary cells and those in culture, including loss of melanocyte specificity. The different tools described in this thesis provide a platform on which to study various aspects of cell behaviour, including migration, morphology and cell adhesion at both the individual cell and population levels.

Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

<p>En studie i hur känsligheten avtar från ytan hos biosensorer med höga frekvenser presenteras. Med ny teknologi som avancerade elektroakustiska tunnfilms komponenter, så kallade FBARs, blir tidigare outforskade områden som decay längden möjliga att studera.</p><p>För att undersöka hur frekvenssvaret och känsligheten påverkas av interaktioner långt ut från en sensoryta används proteinkemi. Ett protokoll har optimerats innehållande aktivering med EDC/NHS och fibrinogen för att säkerställa en jämn tjocklek och fördelning av ett adsorberat proteinlager över en yta.</p><p>Dessa ytor kontrollerades först med hjälp av ellipsometri och sedan i ett QCM instrument. Alla experiment med de högfrekventa FBAR sensorerna utfördes vid Ångströmslaboratoriet i Uppsala där pågående forskning inom området finns.</p><p>Resultaten bekräftar teorin om en avtagande känslighet i och med ett ökat avstånd från ytan. En experimentell genomförd och beräknad tjocklek för decay längden uppskattades som inte helt stämde överens med den teoretiskt beräknade.</p><p>En ny term föreslås då frekvenssvaret hos en biosensor planar ut. Detta är en effekt som sker vid dubbla tjockleken av den teoretisk beräknade tjockleken av decay längden och har fått namnet; detection length. Efter denna längd eller gräns observeras en inverterad signal som det än så länge inte finns någon förklaring till.</p> / <p>A study of the sensitivity decrease of biosensors working at high frequencies is presented. With new technology such as film bulk acoustic resonators (FBAR), issues like the decay length is no longer irrelevant theory but may cause limitation in the system as well as it offers new detection possibilities.</p><p>To investigate the frequency response and sensitivity, layer-on-layer construction chemistry was used. A protocol involving activation with EDC/NHS and coupling chemistry with fibrinogen was optimized to ensure accurate thickness and uniformly distribution of each layer over the surface.</p><p>Surfaces were characterized using null ellipsometry and the protocol was tested in a traditional quartz crystal microbalance (QCM). Experiments with the FBAR were preformed at the Ångström laboratory in Uppsala were there is ongoing research and development in FBAR technology.</p><p>The results confirmed the theory of decreasing frequency and sensitivity further out from the surface. An experimental and estimated thickness was calculated which to some extent correlates to the theoretically calculated decay length.</p><p>A new terminology is suggested when the frequency levels off. It occurs approximately at twice the distance and thickness of the theoretically calculated decay length and is given the name; detection length. Beyond the detection length an inverted signal is observed which cannot yet be explained for.</p>

En studie i hur känsligheten avtar från ytan hos biosensorer med höga frekvenser presenteras. Med ny teknologi som avancerade elektroakustiska tunnfilms komponenter, så kallade FBARs, blir tidigare outforskade områden som decay längden möjliga att studera. För att undersöka hur frekvenssvaret och känsligheten påverkas av interaktioner långt ut från en sensoryta används proteinkemi. Ett protokoll har optimerats innehållande aktivering med EDC/NHS och fibrinogen för att säkerställa en jämn tjocklek och fördelning av ett adsorberat proteinlager över en yta. Dessa ytor kontrollerades först med hjälp av ellipsometri och sedan i ett QCM instrument. Alla experiment med de högfrekventa FBAR sensorerna utfördes vid Ångströmslaboratoriet i Uppsala där pågående forskning inom området finns. Resultaten bekräftar teorin om en avtagande känslighet i och med ett ökat avstånd från ytan. En experimentell genomförd och beräknad tjocklek för decay längden uppskattades som inte helt stämde överens med den teoretiskt beräknade. En ny term föreslås då frekvenssvaret hos en biosensor planar ut. Detta är en effekt som sker vid dubbla tjockleken av den teoretisk beräknade tjockleken av decay längden och har fått namnet; detection length. Efter denna längd eller gräns observeras en inverterad signal som det än så länge inte finns någon förklaring till. / A study of the sensitivity decrease of biosensors working at high frequencies is presented. With new technology such as film bulk acoustic resonators (FBAR), issues like the decay length is no longer irrelevant theory but may cause limitation in the system as well as it offers new detection possibilities. To investigate the frequency response and sensitivity, layer-on-layer construction chemistry was used. A protocol involving activation with EDC/NHS and coupling chemistry with fibrinogen was optimized to ensure accurate thickness and uniformly distribution of each layer over the surface. Surfaces were characterized using null ellipsometry and the protocol was tested in a traditional quartz crystal microbalance (QCM). Experiments with the FBAR were preformed at the Ångström laboratory in Uppsala were there is ongoing research and development in FBAR technology. The results confirmed the theory of decreasing frequency and sensitivity further out from the surface. An experimental and estimated thickness was calculated which to some extent correlates to the theoretically calculated decay length. A new terminology is suggested when the frequency levels off. It occurs approximately at twice the distance and thickness of the theoretically calculated decay length and is given the name; detection length. Beyond the detection length an inverted signal is observed which cannot yet be explained for.