RNA metabolism includes all the processes required for RNA synthesis, maturation, and degradation in living cells. Ribonucleases (RNases) are involved in RNA maturation and degradation, two essential processes in gene expression and regulation in both prokaryotes and eukaryotes. Oligoribonuclease (Orn) has an important role in eliminating small oligonucleotides (nano-RNA), the last step in mRNA degradation. In E. coli, Orn is the only essential exoribonuclease. The enzyme has been shown to form a stable dimer, both in solution and in the crystalline form. Analysis of the three-dimensional structure of Orn allowed us to hypothesize that dimerization is essential for enzyme catalysis. In order to test the hypothesis, I analyzed a number of deletion and point mutants of Orn and determined that tryptophan 143 is essential for dimerization. A W143A mutant is unable to dimerize and has very little activity, similar to that of an active site mutant (D162A). The atomic structure of the W143A mutant, solved at a resolution of 1.9 Å, showed that although the overall three-dimensional fold is similar to that of the wild-type protein, minor differences exist that could account for the monomeric behavior in solution. A flexible Arg174 is repositioned into the cavity created by the missing Trp143. In this new orientation Arg174 protrudes into a hydrophobic pocket in the dimerization interface and is proposed to produce sufficient unfavorable interactions to keep the monomers apart in solution. All these data suggest that dimerization of Orn is essential for its activity. The human homolog of Orn, also known as small fragment nuclease (Sfn), has been shown to degrade short single-stranded RNA, the last step in mRNA decay. In order to determine the mechanism of action of Sfn and its role in the cell, we solved the crystal structure of a truncated form of Sfn at a resolution of 2.6 Å. This mutant form of Sfn lacks the C-terminal 21 amino acids (Sfn-∆C21) yet is as efficient as full length Sfn on model substrates. Interestingly, Sfn is not as active as E. coli Orn in in vitro assays. Analysis of the atomic structure revealed that the active site cleft in Sfn is narrower than the corresponding active site in E. coli. We propose a model for how this narrower cleft may explain the lower in vitro activity. Bacillus subtilis does not have an Orn homolog and until recently, the enzyme responsible for nano-RNA degradation in this organism was unknown. YtqI (also termed nrnA or nanoRNase), a protein unrelated to E. coli Orn, was recently shown to be responsible for the digestion of oligonucleotides in B. subtilis. In order to better understand the mechanism of action of YtqI, I solved its crystal structure at a resolution of 2.0 Å. The nuclease has a RecJ-like fold with two globular domains connected via a flexible linker that forms a central groove. On one side of the groove, the larger N-terminal domain harbors the putative active site, while on the opposite side, the C-terminal domain includes a putative RNA binding domain. The structure of YtqI provides insights into how this enzyme binds and digests oligoribonucleotides. The studies described here provide a better understanding of the mechanism of action for several exoribonucleases that act on nano-RNA oligonucleotides - Oligoribonuclease from E. coli, its close homolog in humans (Small fragment nuclease), as well as a functional homolog in Bacillus (YtqI). This work is relevant to understanding RNA metabolism, which is an essential process for survival of both eukaryotic and prokaryotic organisms.

Thesis (Ph. D.)--University of Helsinki. / Includes bibliographical references (p. 86-93).

Thesis (M.A.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 113-120). Also available on the World Wide Web.

Two deeply buried, well-stratified, and well-dated components dating to the Early Holocene period were excavated at the Richard Beene site (41BX831) in Bexar County, Texas. This thesis utilizes both qualitative (interpretation of maps) and quantitative (unconstrained clustering) spatial analysis techniques to identify site structure and assess post-depositional disturbance by analyzing patterns among artifact categories, selected artifacts, and features from these components. Results of spatial analysis are compared to expectations of the archaeological record based on previous research. Each component revealed a distinct pattern. The Lower Medina component (ca. 6900 B.P.) is well preserved and spatial analysis showed clear distinctions between domestic and peripheral zones. The Upper Perez component (8800 B.P.) is a fluvial lag deposit of displaced artifacts and fire-cracked rock features. Results of spatial analysis confirmed that most, if not all, of this component is disturbed, revealing no site structure.

The objective of this longitudinal study was to study the relationship between changes in prevalence of resistant enteric bacteria associated with mean monthly doses (MMD) of various antibiotics used in each of two host species. From January 2004 – January 2007, monthly composite swine fecal samples and human wastewater samples representing various production and occupational cohorts, respectively, were collected from 19 geographically unique locations in east- and south-central Texas. Bacterial isolates cultivated on CHROMagar-E.coliTM and DifcoTM mEnterococcus (ME) were tested for susceptibility to multiple antibiotics by microbroth dilution using the SensititreTM system. The relationship between the prevalence of resistant bacteria, sampling period, and antibiotic use within each host species was assessed in a generalized linear model adjusted for the dependence of responses within location using a binomial distribution and logit link function in STATA® ver. 9.2. For the swine E. coli isolates, the relationship between tetracycline resistance and level of chlortetracycline (CTC) use in swine illustrated a dose-response relationship, with odds ratios (OR) of 1.20 and 1.81 (P < 0.05) for second- and third-level categories of MMD relative to baseline (zero-use) respectively. When considered by swine production groups, intake boar isolates had an elevated relative odds of resistance to tetracycline (OR = 1.51, P < 0.05), and the nursery units had an elevated odds (OR = 2.61, P < 0.05) of exhibiting resistance to ceftiofur, relative to pigs housed in the farrowing barns. Regarding swine Enterococci isolates, those swine from locations that utilized tylosin had an elevated OR of 3.54 (P < 0.05) of exhibiting resistance to tylosin, relative to those locations that used no tylosin. At this juncture, an apparent occupational risk of harboring tetracycline-resistant E. coli, and the apparent sparing effect (Enterococcus spp.) associated with exposure to swine production, remain unexplained. This study demonstrated that the prevalences of tetracycline- and tylosin-resistant enteric bacteria swine were dependent on CTC and tylosin use in feed, respectively. Swine production group-effects on the prevalence of tetracycline, ceftiofur, and erythromycin resistance were also important. This study provides a better understanding of the relationships between antibiotic prescribing practices at the ecologic level and the relative odds of carriage of resistant bacteria within two host species in a vertically integrated agri-food system.

Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c. The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes. In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed. The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.

P2-like bacteriophages integrate their genome into the E. coli host cell by a site-specific recombination event upon lysogenization. The integrative recombination occurs between a specific sequence in the phage genome, attP, and a specific sequence in the host genome, attB, generating the host-phage junctions attL and attR. The integration is mediated by the phage enzyme integrase (Int) and the host factor IHF. The excisive recombination takes place between attL and attR, and is mediated by Int, IHF and phage encoded protein Cox. For safe integration of foreign genes into eukaryotic chromosome a recombinases is necessary which can perform the integration site-specifically. P2-like phage integrases have the potential to become tools for safe gene therapy. Their target is simple but specific, and once integration has occurred it is very stable in the absence of the Cox protein. The site-specific recombination mechanism has to be understood at the molecular level. Therefore, I have initiated the characterization of the site-specific recombination system of the P2-like phage ΦD145. In this work, Int and IHF are shown to bind to the different attachment sites cooperatively. One of two possible inverted repeats in attP is shown to be the Int core recognition site. The attP core of this phage has high identity with a site on human chromosome, denoted as ΨattB. In this study we have shown that in in vivo recombination ΦD145 Int can accept ΨattB in both bacteria and in eukaryotic cells. Also shown that Int consists of an intrinsic nuclear localization signal. A study also reveled that ΦD145 Int activity was affected by the Tyr-phosphorylation. Attempts have been made to change the specificity of the other P2-like phage P2 and WΦ integrases and also structural and functional analysis was done. A study on comparative analysis of Cox proteins and Cox binding sites gave us the basic information about the recombination mechanism. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Until recently, multiple SPME fibres could not be automatically evaluated in a single sequence without manual intervention. This drawback had been a critical issue until recently, particularly during the analysis of numerous on-site samples. Recently, GERSTEL® has developed and commercialized a Multi-Fibre Exchanger (MFX) system designed to overcome this drawback. In this research, a critical evaluation of the MFX performance in terms of storage stability and long term operation is presented. It was established in the course of our research that the MFX can operate continuously and precisely for over 200 extraction/injection cycles. However, when the effect of residence time of commercial fibres on the MFX tray was evaluated, the results have shown that amongst the evaluated fibre coatings, carboxen/polydimethylsiloxane (CAR/PDMS) was the only coating capable of efficient storage on the MFX tray for up to 24 hours after field sampling without suffering significant loss of analytes. Additionally, the MFX system capability for high-throughput analysis was demonstrated by the unattended desorption of multiple fibres after on-site sampling of two different systems, indoor air and biogenic emissions. Subsequently, a protocol based on a new, fast, reproducible, reusable and completely automated method that enables quick assessment of SPME coatings was developed. The protocol consists of an innovative in-vial standard generator containing vacuum pump oil doped with McReynolds probes and subsequently mixed with a polystyrene-divinylbenzene resin. According to our results, the protocol has proven to be a useful tool for the quick assessment of inter-fibre reproducibility prior to their application in on-site analysis. The implications of such protocols include, but are not limited to: time-saving, assurance of reliable and reproducible data, and a dependable guide for novice users of the technique. Finally, an innovative, reusable and readily deployable pen-like diffusive sampler for needle traps (PDS-NT) is proposed. Results have shown that the new PDS-NT is effective for air analysis of benzene, toluene, and o-xylene (BTX). In addition, no statistically significant effects of pen geometry on the uptake of analytes were found.

Uppsatsen syftar till att ta reda på vad ungdomar uppmärksammar som offentlig konst och vad är det som gör att vissa konstverk får mer uppmärksamhet. Jag har genomfört intervjuer med personer som befinner sig på campus i Växjö eller runt Växjösjön som sedan analyseras. Den här uppsatsen har främst fokuserat på ungdomar. För att ungdomar i min studie ska uppfatta något som offentlig så måste den stå utomhus. Den ska finnas i en miljö där man lätt kan se den, även på långt håll ska man kunna urskilja att där står ett konstverk. Verket måste vara stort. Färgen på verket bör bryta mot miljön för att den ska vara mer utstickande. Om ett konstverk har en funktion blir detta ungdomars referens till föremålet, det vill säga att det inte ses som ett konstverk. Om meningen som konstnären har kring verket visas tydligt så kommer intresset att öka

Understanding the transport and retention of radionuclides in the environment is important for protecting freshwater supplies and minimizing impact to biologic systems. Technetium-99 (Tc⁹⁹) is a radionuclide of interest due to its long half-life (2.13 x 10⁵ years) and toxicity. In the form of pertechnetate (TcO₄⁻), Tc is expected to move nearly unretarded in the subsurface. Under reducing conditions Tc can precipitate in low solubility Tc oxide (TcO₂·nH₂O) and/or Tc sulfide (Tc₂S[subscript x]) phases. The studies presented in this dissertation investigate the physiochemical mechanisms for the transport and retention of Tc. Transport studies determined that TcO₄⁻ would move at pore water velocity in unsaturated sediments. Geochemical studies of contaminated sediments determined that nearly ~ 25 % of the total Tc was retained in phases associated with iron oxide and aluminosilicate minerals, thus reducing the mobility of Tc. Studies of Tc₂S[subscript x] mineral phases, generated using nano Zero Valent Iron (nZVI) and sulfide (HS-) in sediments, determined that Tc could be stabilized in mineral phases as Tc₂S[subscript x] that were slower to reoxidize than TcO₂·nH₂O phases. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Feb. 14, 2013 - Feb. 14, 2014