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Methanopyrrolidine-5-carboxylic acids (MetPyr-5-acids), or 5-syn-carboxy-2- azabicyclo[2.1.1]hexanes are building blocks for β-peptides that cannot form backbone hydrogen bonds. To introduce functionality to this ring system, 6-syn-benzyloxymethyl and 6-syn-phenyl substituted derivatives have been prepared by an efficient synthetic procedure. Addition of appropriately substituted allyl amines to 3-butynone, amide protection, and irradiation afford mainly 5-acetyl-2-azabicyclo[2.1.1]hexanes. Haloform oxidation leads to the desired 6-substituted MetPyr-5-acids. A 1-ethoxycarbonyl-MetPyr-5-acid also was prepared in high yield. Condensation of ally amine with ethyl 2,4-dioxopentanoate afforded ethyl 2-(allylamino)-4-oxopent-2- enoate, and this was protected to give ethyl 2-[allyl(tert-butoxycarbonyl)amino]-4- oxopent-2-enoate. Irradiation afforded 5-syn-acetyl-1-ethoxycarbonyl-2- azabicyclo[2.1.1]hexane with high stereoselectivity and oxidation of the acetyl group afforded the desired 1-ethoxycarbonyl-MetPyr-5-acid. Resolutions of (±)-6-syn-benzyloxymethyl-MetPyr-5-acid and (±)-1- ethoxycarbonyl-MetPyr-5-acid were carried out (> 98% ee) by a classical resolution method using (S)-(-)-α-methylbenzylamine. The absolute configurations of (1S,4R,5R,6S)-(-)-6-benzyloxymethyl-MetPyr-5-acid and (1R,4S,5S)-(+)- 1- ethoxycarbonyl-MetPyr-5-acid were determined by X-ray analysis of their 5-(S)-(-)-α- methylbenzylamide. A prior X-ray analysis of N-Boc-(MetPyr)4-CO2Me indicated all amides to be trans oriented with all 5-syn-carbonyl groups directed toward Carbon-4 of the methanopyrrolidine. These structures were assigned as T4T4T4T4 or [T4]n (n = 4). The solution structure was not determined. Homooligomers of (1S,4R,5R)-5-syn-carboxy-2- azabicyclo[2.1.1]hexane (MPCA) terminally protected as N-Boc methylesters were constructed by EDC/HOBt coupling of terminal ester N-deprotected free amine units and N-Boc free acid units. To facilitate NMR analysis of the secondary structures of homooligomers, N-Boc was replaced by N-isobutyryl. NMR experiments indicated that N-isobutyryl-(MetPyr)n-CO2Me, (n = 2, 3, 4) have major favored [T4]n-1T where the orientation of the terminal ester carbonyl could not be determined. / Chemistry

We have explored correlations between the measured efficiency of the RNAi process and several computed signatures that characterize equilibrium secondary structure of the participating mRNA, siRNA, and their complexes. A previously published data set of 609 experimental points was used for the analysis. While virtually no correlation with the computed structural signatures are observed for individual data points, several clear trends emerge when the data is averaged over 10 bins of N ~ 60 data points per bin. The strongest trend is a positive linear (r² = 0.87) correlation between ln(remaining mRNA) and ΔG_ms, the combined free energy cost of unraveling the siRNA and creating the break in the mRNA secondary structure at the complementary target strand region. At the same time, the free energy change ΔG_total of the entire process mRNA + siRNA → (mRNA – siRNA)_complex is not correlated with RNAi efficiency, even after averaging. These general findings appear to be robust to details of the computational protocols. The correlation between computed ΔG_ms and experimentally observed RNAi efficiency can be used to enhance the ability of a machine learning algorithm based on a support vector machine (SVM) to predict effective siRNA sequences for a given target mRNA. Specifically, we observe modest, 3 to 7%, but consistent improvement in the positive predictive value (PPV) when the SVM training set is pre- or post-filtered according to a ΔG_ms threshold. / Master of Science

This thesis investigates the diversity of two silica-scaled protist groups, Paraphysomonadida and Thaumatomonadida by light and electron microscopical observations and sequencing (rDNA) on novel clonal cultures. Despite these groups of protist dominating pelagic, littoral as well as inland freshwater and soil habitats, they are taxonomically poorly understood to the extent that any progress in ecological theory is hampered. Now that environmental DNA sequencing is being carried out faster than we can characterise protists from culture it is important that we understand how molecular and physical diversity match up, especially because so many protists are morphospecies. Nearly one hundred isolates were cultured on which both morphological and molecular data was carried out in parallel to reveal around 50 new species of protist from eight different genera: two heterokont genera, Paraphysomonas and Incisomonas n. gen., and six cercozoan genera, Thaumatomonas, Allas, Reckertia, Thaumatospina n. gen., Cowlomonas n. gen., and Scutellomonas n. gen. These data make major contributions to taxonomy and understanding aspects of protist diversity where previously morphological diversity was heavily biased towards over- generalized morphotypes. This thesis quickly showed that gross lumping of morphospecies was true of Paraphysomonas, for which many of the isolates cultured herein might have been regarded as one species (not more than 20). The many cultured isolates exhibited varied cell and scale morphology, and by sequencing (rDNA), it was possible to see the evolution of scale morphology map on to trees. This marriage of molecular and morphological data made it possible to view distinct groups of species that shared scale detail that might have otherwise been overlooked had either method been used alone. This research has shed significant light on how scale morphology can be used as reliable taxonomic marker for protists, the insights of which can be applied to make taxonomic improvements to other silica-scaled protist groups.

Background: RNA secondary structure prediction by energy minimization is the central computational tool for the analysis of structural non-coding RNAs and their interactions. Sparsification has been successfully applied to improve the time efficiency of various structure prediction algorithms while guaranteeing the same result; however, for many such folding problems, space efficiency is of even greater concern, particularly for long RNA sequences. So far, spaceefficient sparsified RNA folding with fold reconstruction was solved only for simple base-pair-based pseudo-energy models. Results: Here, we revisit the problem of space-efficient free energy minimization. Whereas the space-efficient minimization of the free energy has been sketched before, the reconstruction of the optimum structure has not even been discussed. We show that this reconstruction is not possible in trivial extension of the method for simple energy models. Then, we present the time- and space-efficient sparsified free energy minimization algorithm SparseMFEFold that guarantees MFE structure prediction. In particular, this novel algorithm provides efficient fold reconstruction based on dynamically garbage-collected trace arrows. The complexity of our algorithm depends on two parameters, the number of candidates Z and the number of trace arrows T; both are bounded by n2, but are typically much smaller. The time complexity of RNA folding is reduced from O(n3) to O(n2 + nZ); the space complexity, from O(n2) to O(n + T + Z). Our empirical results show more than 80 % space savings over RNAfold [Vienna RNA package] on the long RNAs from the RNA STRAND database (≥2500 bases). Conclusions: The presented technique is intentionally generalizable to complex prediction algorithms; due to their high space demands, algorithms like pseudoknot prediction and RNA–RNA-interaction prediction are expected to profit even stronger than \"standard\" MFE folding. SparseMFEFold is free software, available at http://www.bioinf.unileipzig. de/~will/Software/SparseMFEFold.

Biomaterials requirements nowadays are becoming more and more specialized to meet increasingly demanding needs for biomedical applications such as matrices for tissue scaffolds. Among various useful classes of biomaterials, protein-based materials have been extensively pursued as they can offer a wide range of material properties to accommodate a broader spectrum of functional and performance requirements. The advent of genetic engineering and recombinant DNA technology has enabled the production of new protein-based biopolymers with precisely controlled amino acid sequence. As an example, silk-elastinlike protein (SELP) polymers consisting of polypeptide sequences from native silk of remarkable mechanical strength and polypeptide sequences from native elastin that is extremely durable and resilient have been produced. In this dissertation, a particular silk-elastinlike protein copolymer, SELP-47K, was cast into film form, and fully characterized for its material properties, including the mechanical property, secondary structure transition, optical transparency, surface, and other physical, chemical properties. The relationship between mechanical property and protein secondary structure was investigated as well. In addition, the material property tunability which can be induced by physical, mechanical, and chemical treatments has been explored. It is worth noting that the physically crosslinked SELP-47K films displayed mechanical properties comparable to those of native elastin obtained from bovine ligament. Secondary structure study through Raman and FTIR spectra showed that methanol treatment is capable of inducing theβ-sheet crystallization of silklike blocks, which act as physical crosslinks in the protein polymer chain network, thus stabilizing the protein structure and conferring the improved material integrity. The SELP-47K protein polymer thin films displayed excellent optical transparency. In particular, its excellent optical transmittance (over 90%) in visible light range may indicate SELPs can be a family of promising biomaterial candidate for ocular applications. Besides material property characterization, SELP-47K protein polymer has been fabricated into a variety of drug delivery devices to sustainably release a common ocular antibiotic, ciprofloxacin over a period of up to 220 h, with near-first order kinetics.