RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold. To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity. The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the iv collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues. In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n. Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.

A trend in parallel computer architecture is that systems with a large shared memory are becoming more and more popular. A shared memory system can be either a uniform memory architecture (UMA) or a cache coherent non-uniform memory architecture (cc-NUMA). In the present thesis, the performance of parallel PDE solvers on cc-NUMA computers is studied. In particular, we consider the shared namespace programming model, represented by OpenMP. Since the main memory is physically, or geographically distributed over several multi-processor nodes, the latency for local memory accesses is smaller than for remote accesses. Therefore, the geographical locality of the data becomes important. The focus of the present thesis is to study multithreaded PDE solvers on cc-NUMA systems, in particular their memory access pattern with respect to geographical locality. The questions posed are: (1) How large is the influence on performance of the non-uniformity of the memory system? (2) How should a program be written in order to reduce this influence? (3) Is it possible to introduce optimizations in the computer system for this purpose? The main conclusion is that geographical locality is important for performance on cc-NUMA systems. This is shown experimentally for a broad range of PDE solvers as well as theoretically using a model involving characteristics of computer systems and applications. Geographical locality can be achieved through migration directives that are inserted by the programmer or — possibly in the future — automatically by the compiler. On some systems, it can also be accomplished by means of transparent, hardware initiated migration and replication. However, a necessary condition that must be fulfilled if migration is to be effective is that the memory access pattern must not be "speckled", i.e. as few threads as possible shall make accesses to each memory page. We also conclude that OpenMP is competitive with MPI on cc-NUMA systems if care is taken to get a favourable data distribution.

The recent development of methods for constructing andhandling large collections (libraries) of proteins, from whichvariants with desired traits can be isolated, hasrevolutionized the field of protein engineering. Key elementsof such methods are the various ways in which the genotypes(the genes) and the phenotypes (the encoded proteins) arephysically linked during the process. In one section of thework underlying this thesis, one such technique (phagedisplay), was used to isolateand identify protein librarymembers based on their catalytic or target molecule-bindingproperties. In a first study, phage display libraries of the lipolyticenzyme Lipolase from Thermomyces lanuginosa were constructed,the objective being to identify variants with improvedcatalytic efficiency in the presence of detergents. Toconstruct the libraries, nine positions were targeted for codonrandomization, all of which are thought to be involved in theconformational change-dependent enzyme activation that occursat water-lipid interfaces. The aim was to introduce two tothree amino acid mutations at these positions per lipase gene.After confirming that the wt enzyme could be functionallydisplayed on phage, selections with the library were performedutilizing a mechanism-based biotinylated inhibitor in thepresence of a detergent formulation. According to rhodamineB-based activity assays, the fraction of active clonesincreased from 0.2 to 90 % over three rounds of selection.Although none of the variants selected using this approachshowed increased activity, in either the presence or absence ofdetergent compared to the wild type enzyme, the resultsdemonstrated the possibility of selecting variants of theenzyme based on catalytic activity. In the following work, phage libraries of the StaphylococcalProtein A (SPA)-derived Z-domain, constructed by randomizationof 13 surface-located positions, were used to isolate Z domainvariants (affibodies) with novel binding specificities. Astargets for selections, the parental SPA domains as well as twopreviously selected affibodies directed against two unrelatedtarget proteins were used. Binders of all three targets wereisolated with affinities (KD) in the range of 2-0.5 µM.One SPA binding affibody (ZSPA-1) was shown to bind to each of the fivehomologous native IgG-binding domains of SPA, as well as theZdomain used as the scaffold for library constructions.Furthermore, the ZSPA-1affibody was shown to compete with one of thenative domains of SPA for binding to the Fc part of humanantibodies, suggesting that the ZSPA-1affibody bound to the Fc-binding surface ofthe Z domain. The majority of the affibodies isolated in theother two selections using two different affibodies as targets,showed very little or no binding to unrelated affibodies,indicating that the binding was directed to the randomizedsurface of their respective targets, analogously toanti-idiotypic antibodies. The structure of the wild type Z domain/ZSPA-1affibody co-complex was determined by x-raycrystallography, which confirmed the earlier findings in thatthe affibody ZSPA-1affibody was shown to bind to the Fc bindingsurface of the Z domain. Further, both the Z domain and the ZSPA-1affibody had very similar three helix-bundletopologies, and the interaction surface involved ten out of thethirteen randomized residues, with a central hydrophobic patchsurrounded by polar residues. In addition, the interactionsurface showed a surprisingly high shape complementarity, giventhe limited size of the library used for selections. The ZSPA-1affibody was further investigated for use invarious biotechnological applications. In one study, the ZSPA-1affibody was successfully recruited as a novelaffinity gene fusion partner for production, purification anddetection of cDNA-encoded recombinant proteins using anSPA-based medium for affinity chromatography. Further, the SPAbinding capability of the ZSPA-1affibody was employed for site-specific andreversible docking of ZSPA-1affibody-tagged reporter proteins onto an SPAfusion protein anchored to a cellulose surface via acellulose-binding moiety. These generated protein complexesresembles the architecture of so-called cellulosomes observedin cellulolytic bacteria. The results suggest it may bepossible to use anti-idiotypic affibody-binding protein pairsas modules to build other self-assembling types of proteinnetworks. Keywords:phage display, selection, mechanism-basedinhibitor, affinity domains, crystal structure, Staphylococcusaureus protein A, affinity chromatography, anti-idiotypicbinding pairs, affibody, combinatorial, protein engineering,lipase, cellulosome, assembly.

As many genome sequencing efforts today are completed, we are now provided with the genetic maps for several organisms, including man. With these maps at hand, the scientific focus is now shifting towards investigations of the functionality of proteins. This task is even more challenging than the genomic field since proteins, in contrast to DNA, do not allow themselves to be specifically probed or amplified by easy and generic methods. However, to achieve knowledge regarding protein function, useful information includes where, when and how much certain proteins are expressed in an organism. Such information can be obtained if protein-specific binding molecules are available as tools. One such class of target specific binders are the antibody molecules, traditionally employed in a broad variety of biotechnical applications, including protein localization studies on both cellular and sub cellular levels. In a first serie of studies, new methodology for recombinant production and purification of antigens for generation of antibodies via immunization routes were investigated. Parallel affinity gene fusion-based expression systems were used for evaluation of different concepts for production of antigen and post-immunization antibody purification. Carefully designed protein antigens from different organisms were produced and used to raise antisera which were affinity purified on their respective antigens to obtain highly specific polyclonal antibodies (monospecific antibodies). One of the constructed expression systems includes an affinity handle, ZSPA-1, previously selected from a combinatorial protein library for its capacity to selectively bind protein A. This allows for convenient, non IgG-dependent, affinity purification of proteins on conventional protein A resins. A strategy where highly target specific antibody preparations could be affinity purified in a more streamlined setup is also presented. By this strategy it was possible to fractionate antibodies showing reactivity to different parts of the antigen into separate fractions. This resulted in affinity purified antibodies showing monospecific but still multi-epitope reactivity. Purified monospecific antibodies were used in different studies including Western blot immunofluorescence and recovery applications. For affinity purification of endogenous target from its native surrounding a selective elution strategy where the recombinant antigen was used to competitively elute the captured target was developed. / QC 20100824

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

BspA was first identified on the basis of being the major constituent of 5 M LiCl washes of whole Lactobacillus fermentum BR11 cells. The bspA gene is encoded within a putative ATP-binding cassette (ABC) transport operon, and sequence analysis revealed that it is a member of the family III solute binding proteins. Unlike the majority of solute binding proteins from Gram-positive bacteria, BspA is not tethered to a lipid anchor in the cell membrane, and hence is not a lipoprotein. Extraction of BspA with concentrated salt solutions such as 5 M LiCl is consistent with the notion that electrostatic interactions are responsible for securing it to the L. fermentum BR11 cell. L. fermentum PNG201 is a BspA negative mutant strain created by disrupting bspA. This strain was shown to be incapable of cystine uptake. Thus, the genetic and biochemical evidence strongly suggests BspA is a cystine binding protein of an ABC transporter. Measurement of the binding affinity between BspA and L-cystine has confirmed high affinity binding (dissociation constant is 0.2 µM), and high specificity (over 100-fold excess of non-target amino acids did not disrupt BspA / L-cystine binding). In addition, collagen did not appear to affect BspA/cystine binding, indicating extracellular matrix (ECM) binding capacity noted by other researchers may be unrelated to amino acid binding. An interesting phenotypic characteristic of L. fermentum PNG201 is its apparent increased sensitivity to oxygen and the superoxide-generating chemical - paraquat compared to the parent L. fermentum BR11 strain. Catalase supplemented aerobic cultures of L. fermentum BR11, and L. fermentum PNG201 were protected from oxidative stress, suggesting hydrogen peroxide is responsible for the observed oxidative stress. It was found that addition of cystine to aerobic cultures of L. fermentum BR11 or L. fermentum PNG201 protected both strains from oxidative stress, with L. fermentum BR11 able to utilize smaller concentrations of cystine compared to L. fermentum PNG201. Detection of hydrogen peroxide in aerobic cultures of L. fermentum BR11 and L. fermentum PNG201 confirmed the production of hydrogen peroxide is responsible for causing oxidative stress. The BspA mutant strain L. fermentum PNG201 consistently produced more hydrogen peroxide per optical density compared with the wild type, indicating it overproduced hydrogen peroxide. When 0.4 mM hydrogen peroxide has been accumulated by growing cell cultures, both L. fermentum BR11 and L. fermentum PNG201 enters stationary phase, suggesting both strains have a similar sensitivity to hydrogen peroxide. Small epitopes from the HIV gp41 protein and the Chlamydia psittaci major outer membrane protein have been successfully displayed on the cell surface of L. fermentum BR11 as fusion proteins to the BspA molecule. However, the capability of BspA in exporting larger polypeptides has not been tested. In this study, the large extracellular enzyme - glucosyltransferase (GtfJ) from Streptococcus salivarius ATCC 25975 was fused to BspA to demonstrate that this expression system is capable of exporting large functional enzymes to the cell surface of L. fermentum BR11. The native GtfJ is 160kDa in size and also contained an export signal, which was deleted in the cloning process and replaced with BspA, resulting in a fusion protein of 175kDa. Export of the BspA/GtfJ fusion protein is dependant entirely on BspA's export signal. Recombinant enzyme expression and glucosyltransferase activity were detected by measuring the glucan formed by sonicated cell extracts in acrylamide gels. Enzyme activity measurements on whole cells has revealed the recombinant Lactobacillus was incorporating 20-40 nmol of sucrose-derived-glucose into glucan per ml of cell culture per OD unit, which is comparable to activity levels exhibited by the native bacteria that expressed this enzyme. Comparison of GtfJ enzyme activity between whole cells and sonicated cell extracts of recombinant L. fermentum confirmed the extracellular location of BspA/GtfJ as enzyme activity was essentially identical.

Univ., Diss.--Trier, 2007.

Thesis (J.C.L.)--Catholic University of America, 2007. / Includes bibliographical references (leaves 60-64).

Thesis (M.S.)--University of Michigan, 1989.

Thesis (J.C.L.)--Catholic University of America, 2007. / Includes bibliographical references (leaves 60-64).