<p>Research on biological motion, using point-light displays to present the motions, have been unravelling what information factors are still embedded in those impoverished stimuli and which of these factors are essential in visual processing of biological motion. Earlier studies suggest that orientation is a crucial factor in biological motion processing. The short-term priming experiment presented in this paper will further investigate the legitimacy of the primacy of orientation and suggest different solutions based on contradicting findings in previously published studies.</p><p>In a serial two-choice reaction-time task, participants were presented with a patch-light display of a human engaged in one of three possible actions: climbing up a rope, jumping jacks, and walking. Participants had to identify the in-plane orientation of the human figure emerging from the moving patch-lights. Reliable facilitation effect was established for transitions containing same-oriented upright trails and same-oriented inverted trials. Interestingly, transitions of same-oriented upright trials produced significantly greater facilitation effect than transitions of same-oriented inverted trials.</p>

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 189-202).

Betalaktamaser med utvidgat spektrum så kallade Extended Spectrum Beta-Lactamase (ESBL) är enzymer som produceras av bakterier och som har en avgörande roll ur kliniskt perspektiv då de blir resistenta mot de flesta antibiotika vilket leder till begränsade behandlingsalternativ. För att selektera fram dessa bakterier användes kromogena agarplattor vid odling, vilka selekterade bort jästsvampar och grampositiva bakterier och underlättar detektionen av ESBL-positiva stammar. Syftet med studien var att jämföra tre olika selektiva agarplattor CHROMagar ESBL, ChromID ESBL och CHROMagar C3GR genom att utvärdera deras specificitet och sensitivitet. Totalt användes 130 olika patientprover från feces, blod, sår och urin, vilka valdes ut slumpmässigt ur den rutinmässiga diagnostiken. Proverna odlades på de tre agarplattor. De bakteriestammar som växte fram artidentifierades med IVD Maldi Biotyper och resistensbestämdes med VITEK. Den totala sensitiviteten för ESBL, med ett 95 % konfidensintervall, efter 16 timmars aerob inkubering i 37° C var 96,7 % (KI 95% 81,0 – 99,9 %) för de tre agarplattorna. Specificiteten var 94,0 % (KI 95%  86,9 – 97,5%) för CHROMagar ESBL, 93,1 % (KI 95%  85,6– 96,9%) för ChromID ESBL och 73,0 % (KI 95% 63, 0- 81,2 %) för CHROMagar C3GR. De tre agarplattor uppvisade jämförbar sensitivitet men skillnaden i specificitet där ansåg CHROMagar ESBL och ChromID ESBL erhöll likvärdiga resultat. / Bacteria that produce enzymes with extended spectrum, Extended Spectrum Beta-Lactamase (ESBLs), have a major role in a clinical perspective since they became resistant to most antibiotics, leading to limited treatment options. In order to detect the bacteria, chromogenic agar plates were used for culture in order to inhibit growth of yeast and gram positive bacteria and enables the detection of ESBL-positive strains. The aim of this study was to compare three chromogenic agar plates CHROMagar ESBL, ChromID ESBL and CHROMagar C3GR regarding their specificity and sensitivity. A total of 130 different samples from faeces, blood, wounds and urine were randomly selected for the routine diagnosis. The samples were grown on the three chromogenic agar plates and were species identified by IVD Maldi biotypes and resistance determined with VITEK. The overall sensitivity for ESBLs with 95% confidence interval, after 16 hours of aerobic incubation at 37° C was 96.7 % (CI 81.0-99.9 %) for the three agar plates. The specificity showed 94.0 % (CI 86.9-97.5 %) for CHROMagar ESBL, 93.1 % (CI 85,6- 96.9 %) for ChromID ESBL and 73.0 % (CI 63, 0- 81 , 2 %) for CHROMagar C3GR. All three chromogenic agar plates were equally sensitive but the specificity differed. CHROMagar ESBL and ESBL ChromID were considered equivalent.

Influenza B virus causes a respiratory disease in people with a compromised immune system. The NS1B protein of influenza B virus is essential for virus growth and plays a crucial role in inhibiting the anti-viral responses mounted by the infected host cell. The N terminal 104 amino acids of NS1B bind a cellular protein called ISG15. ISG15 is an interferon induced 'ubiquitin-like' protein, and upon interferon induction, is conjugated to hundreds of targets. It has been found that both ISG15 and its conjugation inhibit many viruses. The focus of the current study was to characterize the interaction between NS1B and ISG15. Study of a recombinant influenza B virus which encoded a mutant NS1B protein that is unable to bind ISG15 revealed that ISG15 is mis-localized in cells infected with wild type but not the mutant influenza B virus. Further, such a mutant virus is attenuated in growth as compared to wild type virus in human cell lines but is not attenuated in canine cell lines. This result led to the discovery of the species specific nature of the interaction between NS1B and ISG15. Specifically, NS1B was found to bind ISG15 homologs from human and old world monkeys like Rhesus macaques and African green monkeys but not those from mouse or canines. These findings were extended by identifying the hinge between the N and C terminal domains of ISG15 as one of the major determinants of species specificity. These results highlight the importance of using human or primate cell culture models to study the effect of ISG15 on influenza B virus, and raises new possibilities on differences in the function of the ISG15 system in different species. / text

Human immunodeficiency virus reverse transcriptase (HIV RT) is a virally encoded polymerase responsible for replicating the HIV genome. Most HIV treatments include nucleotide RT inhibitors (NRTIs) which inhibit HIV RT replication by serving as a substrate for the polymerase reaction but then blocks subsequent polymerization after incorporation. However, resistance to these NRTIs may occur through specific mutations in HIV RT that increase the discrimination of HIV RT for natural nucleotides over NRTIs. The role of enzyme conformational dynamics in specificity and substrate selection was studied using transient kinetic methods on HIV RT enzymes that have been site-specifically labeled with a conformationally sensitive fluorophore, to measure the rates of binding and catalysis. First, HIV RT with the mutation of lysine to arginine at the residue position 65 (K65R) was examined for its resistance against the NRTI tenofovir diphosphate (TFV), an acyclic deoxyadenosine triphosphate (dATP) analog. It was found that HIV RT K65R resistance to TFV was achieved through decreased rates of catalysis and increased rates of dissociation for TFV over dATP when compared with the kinetics of wild-type HIV RT. Moreover, global fitting analysis confirmed a mechanism where a large conformational change, after initial ground state binding of the substrate, contributed significantly to enzyme specificity. This led to our investigation of the molecular basis for enzyme specificity using HIV RT as a model system. Again, transient kinetic methods were applied with the addition of molecular dynamics simulations. The simulated results were substantiated by the corroborating experimental results. It was found that a substrate-induced conformational change in the transition of HIV RT from an open nucleotide-bound state to a closed nucleotide-bound state was the major determinant in enzyme specificity. The molecular basis for substrate selection resulted from the molecular alignments of the substrate in the active-site, which induced the conformational change. When the correct nucleotide was bound, optimal molecular interactions in the active-site yielded a stably closed complex, which promoted nucleotide incorporation. In contrast, when an incorrect nucleotide was bound, the molecular interactions at the active-site were not ideal, which yielded an unstable closed complex, which promoted substrate dissociation rather than incorporation. / text

Of the 430+ extant species of Gyrodactylus, ectoparasitic monogenetic flukes of aquatic vertebrates, Gyrodactylus salaris Malmberg, 1957 is arguably the most well-known. Following the introduction of this species into Norway in the 1970s with consignments of infected Atlantic salmon smolts, Salmo salar L., this species has had a devastating impact on the Norwegian Atlantic salmon population, decimating wild stocks in over 40 rivers. Gyrodactylus salaris is the only OIE (Office International des Epizooties) listed parasitic pathogen of fish and has been reported from 19 countries across Europe, though many of these records require confirmation. The UK, Ireland and some selected watersheds in Finland are currently recognised as G. salaris-free states; however, the threat that this notifiable parasite poses to the salmon industry in the UK and Ireland is of national concern. Current British contingency plans are based on the assumption that if G. salaris were to be introduced, the parasite would follow similar dynamics to those on salmonid stocks from across Scandinavia, i.e. that Atlantic strains of Atlantic salmon would be highly susceptible to infection, with mortalities resulting; that brown trout, Salmo trutta fario L., would be resistant and would lose their infection in a relatively short period of time; and that grayling, Thymallus thymallus (L.), would also be resistant to infection, but would carry parasites, at a low level, for up to 143 days. Two of the objectives of this study were to confirm the current distribution of G. salaris across Europe, and then, to investigate the relative susceptibility of British salmonids to G. salaris, to determine whether they would follow a similar pattern of infection to their Scandinavian counterparts or whether, given their isolation since the last glaciation and potential genetic differences, they would exhibit different responses. It has been almost six years since the distribution of G. salaris across Europe was last evaluated. Some of the European states identified as being G. salaris-positive, however, are ascribed this status based on misidentifications, on partial data resulting from either morphological or molecular tests, or according to records that have not been revisited. Additional Gyrodactylus material from selected salmonids was obtained from several countries to contribute to current understanding regarding the distribution of G. salaris across Europe. From the work conducted in the study, G. salaris is reported from Italy for the first time, alongside three other species, and appears to occur extensively throughout the central region without causing significant mortalities to their rainbow trout, Oncorhynchus mykiss (Walbaum), hosts. The analysis of archive material from G. salaris-positive farms would suggest that G. salaris has been in the country for at least 12 years. Material obtained from rainbow trout from Finland and Germany was confirmed as G. salaris supporting existing data for these countries. No specimens of G. salaris, however, were found in the additional Gyrodactylus material obtained from Portuguese and Spanish rainbow trout, only Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999, a morphologically similar species was found. Gyrodactylus salaris is now reported from 23 out of ~50 recognised states throughout Europe, only 17 of these however, have been confirmed by either morphology or by an appropriate molecular test, and only ten of these records have been confirmed by a combination of both methods. To assess the susceptibility of English and Welsh salmonids to G. salaris, a number of salmonid stocks of wild origin, were flown to the Norwegian Veterinary Institute (NVI) in Oslo, where they were experimentally challenged with G. salaris. Atlantic salmon from the Welsh River Dee, S. trutta fario from the English River Tyne and T. thymallus from the English River Nidd, raised from wild stock in government hatcheries, were flown out and subsequently challenged with G. salaris haplotype A. After acclimation, each fish was infected with ~50–70 G. salaris and marked, so that parasite numbers on individual fish could be followed. The dynamics on individual fish were followed against a control (Lierelva Atlantic salmon). The experiment found that the number of G. salaris on S. salar from the River Dee continued to rise exponentially to a mean intensity (m.i.) of ~3851 G. salaris fish-1 (day 40 post-infection). These salmon were highly susceptible, more so than the Norwegian salmon control (m.i. ~1989 G. salaris fish-1 d40 post-infection) and were unable to regulate parasite numbers. The S. trutta fario and T. thymallus populations, although initially susceptible, were able to control and reduce parasite burdens after 12 (m.i. ~146 G. salaris fish-1) and 19 (m.i. ~253 G. salaris fish-1) days, respectively when peak infections were seen. Although the latter two hosts were able to limit their G. salaris numbers, both hosts carried infections for up to 110 days (i.e. when the experiment was terminated). The ability of S. trutta fario and T. thymallus to carry an infection for long periods increases the window of exposure and the potential transfer of G. salaris to other susceptible hosts. The potential role that brown trout may play in the transmission and spread of G. salaris in the event of an outbreak, needs to be considered carefully, as well as the interpretation of the term “resistant” which is commonly used when referring to brown trout’s susceptibility to G. salaris. The current British surveillance programmes for G. salaris are focused on the screening of Atlantic salmon and on the monitoring of the rainbow trout movements. The findings from this study demonstrate that G. salaris can persist on brown trout for long periods, and suggest that brown trout sites which overlap with Atlantic salmon or rainbow trout sites are also included within surveillance programmes and that the role that brown trout could play in disseminating infections needs to be factored into contingency/management plans. Throughout the course of the study, a number of parasite samples were sent to the Aquatic Parasitology Laboratory at Stirling for evaluation. Some of these samples represented Gyrodactylus material that were associated with fish mortalities, but the species of Gyrodactylus responsible appeared to be new to science. A further aspect of this study was, therefore, to investigate these Gyrodactylus related mortalities in aquaculture stock and to describe the species found in each case, which may represent emerging pathogens. The two new species, Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 and Gyrodactylus longipes Paladini, Hansen, Fioravanti et Shinn, 2011 on farmed gilthead seabream, Sparus aurata L., were collected from several Mediterranean farms. The finding of G. orecchiae in Albania and Croatia was associated with 2–10% mortality of juvenile stock and represents the first species of Gyrodactylus to be formally described from S. aurata. Subsequently, G. longipes was found in Bosnia-Herzegovina and Italy, and at the Italian farm site, it occurred as a mixed infection with G. orecchiae, but these infections did not appear to result in any loss of stock. Unconfirmed farm reports from this latter site, however, suggest that a 5–10% mortality of juvenile S. aurata was also caused by an infection of Gyrodactylus, which is suspected to be G. longipes. Additional samples of Gyrodactylus from a gilthead seabream farm located in the north of France have been morphologically identified as G. longipes, extending the geographical distribution of this potentially pathogenic species to three countries and three different coasts. In addition to these samples, some specimens of Gyrodactylus from a Mexican population of rainbow trout were sent for evaluation.

We perceive the world through sensory experience. Sensory information is registered and processed by our brain in a modality specific fashion. Interestingly, studies have shown that the visual cortex of early but not late blind subjects is able to respond to touch or sound (Sadato et al., 1996; Buchel et al., 1998; Weeks et al., 2000; Gougoux et al., 2009). Here, we investigated whether sensory parcellation in adult cortex is innate or is acquired during early postnatal life in an experience-dependent manner. Furthermore, we studied the anatomical substrates and molecular pathways possibly involved in cross-modal activation and its plasticity. First, mice were reared from birth in total darkness until adulthood (DR) to replicate the human blind condition. Cross-modal activity and the underlying circuitry were analyzed. We found that DR visual cortex was strongly activated by sound stimulation using functional imaging, single-unit recording, and c-Fos immunohistochemistry. Functional analysis was followed by anatomical tracing studies, which showed ectopic projections from the auditory thalamus and auditory cortex into the secondary visual area in DR animals. The second half of our study looked at how visual experience affects cross-modal plasticity. We found that cross-modal activity and ectopic connectivity is present in normally reared young mice (25 postnatal days: P25). Normal sensory experience through the first two months of postnatal life was sufficient to decrease the number of ectopic inputs. Interestingly, exposing DR mice to visual experience as adults established transient functional sensory specificity in the visual cortex without eliminating the ectopic anatomical inputs. Lastly, we tested several molecular pathways that can potentially regulate cross-modal plasticity. We found that myelin signaling and cholinergic modulation controls the duration of cross-modal plasticity and consolidates sensory modularization. Overall, our work proposes a model of how cross-modal inputs into early sensory areas are pruned or retained depending on early life experience. This study provides insight into how the cortex develops functional specificity, and help approach disorders that exhibit abnormal sensory integration and disrupted neuronal connectivity such as Autism Spectrum Disorder.

Ubiquitination is a post-translational modification that can take several forms. Some proteins are modified with a single ubiquitin molecule, while others are modified with polyubiquitin chains. Each type of ubiquitination is thought to have distinct biological functions. The best-characterized types of ubiquitin modification are K48-linked polyubiquitination, which serves as a signal for proteasomal degradation and K63-linked polyubiquitination, which has non-proteolytic functions such in DNA repair, signaling, and endocytosis. HECT ubiquitin ligases (HECT E3s) form a class of E3s, defined by a C terminal catalytic domain. Several lines of evidence suggested that the HECT E3s assemble a polyubiquitin chain in a sequential manner with one molecule of ubiquitin at a time being conjugated to the distal ubiquitin of the chain. In the process of chain elongation, not all HECT E3s target a common internal lysine of ubiquitin, leading to diversification of chain type specificity in HECT E3s. For example, yeast Rsp5 forms K63 chains, while human E6AP forms K48 chains. Two important mechanistic questions were addressed in my work: 1) what are the determinants of chain type specificity of HECT E3s, and 2) what allows the distal ubiquitin of a chain to be continuously oriented near the active site of the HECT domain in the course of a sequential polyubiquitination reaction? I have determined that the chain type specificity of Rsp5 is a function solely of the HECT domain. Further, through the generation of chimeric HECT E3s, I demonstrated that chain type specificity determinants are located within the last 60 amino acids of the C lobe of the HECT domain. To address the second question, we solved the structure of Rsp5 HECT domain in complex with non-covalently bound ubiquitin in collaboration with Jue Chen’s laboratory (Purdue University). From the structure, we found that the N lobe of the HECT domain binds ubiquitin in a manner distinct from other known ubiquitin binding domains, and I have shown that Rsp5 proteins defective for ubiquitin binding are defective for chain elongation. We hypothesize that the ubiquitin binding site functions in the recruitment of the distal ubiquitin of polyubiquitin chain for efficient polyubiquitination. / text

Many functional RNAs are required to fold into specific three-dimensional structures. A fundamental property of RNA is that its secondary structure and even some tertiary contacts are highly stable, which gives rise to independent modular RNA motifs and makes RNAs prone to adopting misfolded intermediates. Consequently, in addition to stabilizing the native structure relative to the unfolded species (defined here as stability), RNAs are faced with the challenge of stabilizing the native structure relative to alternative structures (defined as structural specificity). How RNAs have evolved to overcome these challenges is incompletely understood. Self-splicing group I introns have been used to study RNA structure and folding for decades. Among them, the Tetrahymena intron was the first discovered and has been studied extensively. In this work, we found that a version of the intron that was generated by in vitro selection for enhanced stability also displayed enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. Further dissection suggests that the increased specificity against misfolding arises from two point mutations, which strengthen a local tertiary contact network that apparently cannot form in the misfolded conformation. Our results suggest that the structural rigidity and intricate networks of contacts inherent to structured RNAs can allow them to evolve exquisite structural specificity without explicit negative selection, even against closely-related alternative structures. To explore further how RNAs gain stability from intricate architectures, we examined a novel group I intron from red algae (Bangia). Biochemical methods and computational modeling suggest that this intron possesses general motifs of group IC1 introns but also forms an atypical tertiary contact, which has been reported previously in other subgroups and helps position the reactive helix at the active site. In the Bangia intron, the partners have been swapped relative to known group I RNAs that include this contact. This result underscores the modular nature of RNA motifs and provides insight into how structured RNAs can arrange helices and contacts in multiple ways to achieve and stabilize functional structures. / text

Most amino acid sequences are predicted to specify a single three-dimensional protein structure. However, the identification of "metamorphic" proteins, which can adopt two folds from a single amino acid sequence, has challenged the one sequence/one structure paradigm. Polar-to-hydrophobic substitutions have been suggested computationally as one mechanism to decrease structural specificity, allowing the population of novel folds. Here, we experimentally investigate the role of polar-to-hydrophobic substitutions on structural specificity in the homodimeric ribbon-helix-helix protein Arc repressor. Previous work showed that a single polar-to-hydrophobic surface substitution in the strand region of Arc repressor (Arc-N11L) populates the wild-type fold and a novel dimeric "switch" fold. In this work, we investigate an Arc repressor variant with the N11L substitution plus two additional polar-to-hydrophobic surface substitutions (Arc-S-VLV). We determine that this sequence folds into at least three structures: both dimer forms present in Arc-N11L, and a novel octamer structure containing higher stability and less helicity than the dimer folds. We are able to isolate and stabilize a core of the S-VLV octamer by limited trypsinolysis and deletion mutagenesis (Arc-VLV 4-44). The shortened construct contains only the octameric structure by removing disordered C-terminal segments nonessential for this fold. A two-dimensional NMR spectrum of VLV 4-44 and subsequent trypsinolysis of this construct suggests that at least two types of subunits comprise the S-VLV octamer: subunits structured from residues 4 to 44 and subunits structured from residues 4 to 31. Crystal trials of trypsinolyzed Arc-VLV 4-44 yielded several leads, suggesting that obtaining a high resolution structure of the S-VLV octamer is possible. Relatedly, we determine that the proline residues flanking the Arc repressor strand act in concert as "gatekeepers" to prevent aggregation in the S-VLV sequence. We also find that three highly hydrophobic surface substitutions in the Arc repressor strand region are necessary and sufficient to promote higher-order oligomer formation. In summation, this work reveals in an experimental context that progressive increases in polar-to-hydrophobic surface substitutions populate increasingly diverse, structurally degenerate folds. These results suggest that "metamorphic" as well as "polymetamorphic" proteins, which adopt numerous folds, are possible outcomes for a single protein sequence.