Functionalized locked nucleic acid for therapeutic and diagnostic purposes /by Michael E. {Ostrok}stergaard.

Conventional drugs target proteins while oligonucleotide (ON) drugs can target mRNA, which is present in a cell in a lower copy number than proteins (i.e. less drug load is necessary) and ON drug design is simple due to specific Watson-Crick base pairing (i.e. rapid drug development). ON drugs are, however, currently rarely used by patients due to limited target affinity, specificity and sub-optimal pharmacokinetics. Accordingly, chemically modified nucleotides are widely used to improve ON drug efficacy. Locked Nucleic Acid (LNA) exhibits very favorable hybridization properties, enhanced enzymatic stability and is commercial available. Hence LNA is widely used by multiple research groups and several ON drugs employing LNA modifications are currently being evaluated in clinical trials.;Herein, C5-functionalized LNA uridines, a new class of LNA analogs, were characterized. C5-functionalized LNA uridines were synthesized using a straightforward synthetic strategy employing only 2-3 additional steps compared to the highly optimized industry process for synthesizing LNA. When incorporated into ONs C5-functionalized LNA uridines exhibit favorable hybridization properties; small C5-substituents result in highly thermally stabilized duplexes which even surpass conventional LNA while exhibiting excellent target specificity. Large, hydrophobic, non-fluorescent C5-substituents lead to small changes in thermostability relative to unmodified DNA while preserving pronounced target specificity. Furthermore, the larger the C5-substituent the better the enzymatic stability, i.e. the C5-substituent is acting as a steric shield with the largest C5-functionalities (stearic acid and cholesterol) bestowing nuclease immunity to the ONs. CS-functionalized LNA uridines are thus promising building blocks for use in antisense ONs (i.e. targeting of mRNA) to optimize RNA affinity, target specificity, enzymatic stability and pharmacokinetics of the drug.;880-01Initial studies of 2'- N -(pyren-1-yl)carbonyl-2'-amino-LNA also called Glowing LNA has previously shown promise as a fluorescent probe signaling duplex formation. Preliminary data showed that Glowing LNA probes exhibit favorable target affinity, specificity and brightly fluorescent duplexes. Herein, Glowing LNA probes were further investigated by: (1) probe design optimization, (2) incorporation into a quencher-free molecular bacon, and (3) detection of RNA in a living cell. Overall, Glowing LNA probes exhibit low sequence dependency and high biostability rendering them promising probes for diagnostic purposes as well as a research tool to study RNA trafficking in cells.;This report highlights the advantages of combining short rigid linkers with conformationally restricted nucleotides to position functionalities precisely in a duplex. Precise spatial control of functionalities in a duplex will lead to improved diagnostic probes. Precise positional control, however, can also lead to improved ON drugs as rigid positioning of functionalities can have a significant influence upon interactions with proteins in vivo, which will result in altered pharmacokinetics.Thesis (Ph. D., Chemistry)--University of Idaho, July 2010

Influence of the blood bacterial load on the meningeal inflammatory response in <it>Streptococcus pneumoniae </it>meningitis

<p>Abstract</p> <p>Background</p> <p>Despite bacteraemia is present in the majority of patients with pneumococcal, little is known about the influence of the systemic infection on the meningeal inflammatory response.</p> <p>Methods</p> <p>To explore the role of systemic infection on the meningeal inflammation, experimental meningitis was induced by intracisternal injection of ~1 × 10⁶CFU <it>Streptococcus pneumoniae</it>, type 3, and the 26 rabbits were either provided with ~1 × 10⁶CFU <it>S. pneumoniae </it>intravenously at 0 hour ("bacteraemic" rabbits, n = 9), immunized with paraformaldehyde-killed <it>S. pneumoniae </it>for 5 weeks prior to the experiment ("immunized" rabbits", n = 8), or not treated further ("control" rabbits, n = 9). WBC and bacterial concentrations were determined in CSF and blood every second hour during a 16 hours study period together with CSF IL-8 and protein levels. We also studied CSF and blood WBC levels in 153 pneumococcal meningitis patients with and without presence of bacteraemia.</p> <p>Results</p> <p>As designed, blood bacterial concentrations were significantly different among three experimental groups during the 16 hours study period (Kruskal Wallis test, <it>P </it>< 0.05), whereas no differences in CSF bacterial levels were observed (<it>P </it>> 0.05). Blood WBC decreased in bacteraemic rabbits between ~10–16 hours after the bacterial inoculation in contrast to an increase for both the immunized rabbits and controls (<it>P </it>< 0.05). The CSF pleocytosis was attenuated in bacteraemic rabbits as compared to the two other groups between 12–16 hours from time of infection (<it>P </it>< 0.017), despite accelerated CSF IL-8 levels in bacteraemic rabbits.</p> <p>In patients with pneumococcal meningitis, no significant difference in CSF WBC was observed between patients with or without bacteraemia at admission (n = 103, 1740 cells/μL (123–4032) vs. n = 50, 1961 cells/μL (673–5182), respectively, <it>P </it>= 0.18), but there was a significant correlation between CSF and blood WBC (n = 127, Spearman rho = 0.234, <it>P </it>= 0.008).</p> <p>Conclusion</p> <p>Our results suggest that a decrease in peripheral WBC induced by enhanced bacteraemia in pneumococcal meningitis results in an attenuated CSF pleocytosis.</p

Additional file 1: of Effectiveness and implementation of interventions to increase commuter cycling to school: a quasi-experimental study

Table A. Unadjusted changes in leisure time physical activity and cycling behaviour. Table B. Detailed information on the school based multifaceted interventions. Table C. Detailed information on the distribution of available information (i.e. the degree of missing) at baseline questionnaire assessed variables as well as variables assessed through physical testing by the three different regions. (DOCX 22 kb