In recent years the normativity of thought and meaning has been the subject of an extensive debate. What is at issue is whether intentionality has normative features, and if so, whether that constitutes a problem for naturalistic attempts to account for intentional phenomena. The origin of the debate is Saul Kripke’s interpretation of Ludwig Wittgenstein’s later philosophy, published in Wittgenstein on Rules and Private Language. Kripke claimed, on behalf of Wittgenstein, that dispositional accounts of linguistic meaning - accounts, i.e., which attempt to reduce semantic phenomena to facts about how speakers are disposed to employ words - fail to ground the factuality of semantic statements. From this, and other arguments, the far reaching conclusion was drawn by Kripke’s Wittgenstein that there are no semantic facts, that every application of a word is “a leap in the dark”. This position has become known as meaning scepticism. In the present essay, it will be argued that meaning scepticism is incoherent, but that the normativity argument is interesting in its own right. The development of the debate will be traced, primarily through detailed consideration of the writings of Paul Boghossian, who has shifted the focus from the normativity of linguistic meaning to that of belief. It will be contended that even though Boghossian’s attempt to locate a normativity of belief fails, there is a related form of normativity that has to do with the intrinsic badness of false beliefs. Also, suggestions made by Kripke regarding the normativity of intentions will be investigated, and related to contemporary arguments in the philosophy of rationality. The tentative conclusion is that there are some interesting kinds of normativity associated with the intentional, but of a somewhat different variety than those usually discussed.

Ricin and Shiga toxin type 1 are potent cytotoxins known as ribosome inhibition proteins, abbreviated RIPs. Proteins of this family shut down protein synthesis by removing a critical adenine in the conserved stem-loop structure of 28S rRNA. Due to its exquisite cytotoxicity, the plant toxin ricin has been used as a biological warfare agent. Although great achievement has been made on ricin research, including catalytic mechanism and structure analysis, there is still no specific treatment available for ricin exposure. In addition, ricin A chain inhibitors may also be useful against the homologous bacterial proteins shiga toxins, which are responsible for dysentery, and diseases related to food poisoning, including hemolytic uremic syndrome. Previous study on RTA inhibitor search has provided a number of substrate analog inhibitors, all of which, however, are weaker inhibitors. Therefore, the goal of this work is to improve the binding affinity of known inhibitors and to discovery new scaffolds for inhibitor discovery and development. In this work, multiple approaches were employed for this purpose, including optimizing known inhibitors and searching new inhibitors by Virtual Drug Screening (VDS) and High Throughput Screening (HTS). A number of new RTA inhibitors were discovered by these strategies, which provide a variety of pharmacophores for RTA inhibitor design, and also added a new line of evidence for VDS as an advanced technology for drug discovery and development. / text

Ricin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10-4 M range, leaving significant room for improvement. This thesis discusses the development of a subset of inhibitors belonging to the pterin family in which amino acids have been utilized as building blocks. Inhibitors in this family have achieved a significant increase in potency, and have provided valuable structural information for further development. / text

There is limited information currently available regarding the underlying physiological responses and molecular mechanisms of osmoregulation, acetate metabolism [in relation to the synthesis of glycerolipids, polyunsaturated fatty acids (PUFA), and ichthyotoxins], and transport in Prymnesium parvum N. Carter, a microalga that causes devastating harmful algal blooms (HAB) worldwide. This dissertation examines gene expression under environmental conditions that are associated with HAB formation, including phosphate limitation and low salinity, using microarrays and RNA sequencing (RNA-Seq). A comparative fatty acid methyl ester (FAME) analysis at 30 vs. 5 practical salinity units (psu) was performed to gain additional insight into acetate metabolism. The RNA-Seq analysis included a de novo assembly of the P. parvum transcriptome, generating 47,289 transcripts, of which 35.4% were identifiable. This permitted the evaluation of the expression of many more genes compared with the microarray analysis, which examined ~3,500 genes. Relevant candidate genes identified included those whose products are involved in osmolyte production, salinity stress, and ion transport. With respect to the putative synthesis of polyketide ichthyotoxins, 32 different polyketide synthase (PKS) transcripts were identified in the transcriptome assembly, none of which were differentially expressed. Hemolysin and monogalactosyldiacylglycerol synthase were downregulated at 30 vs. 5 psu, suggesting the increased presence of additional ichthyotoxins at the lower salinity. Evidence for several PUFA synthesis pathways was also revealed. Fatty acid compositions were largely similar at the two salinities, containing relatively prominent quantities of the PUFA stearidonic acid, but compositions varied among strains. The transcription of genes whose products are associated with vesicular transport was elevated, and higher levels of extracellular prymnesins were observed in HAB-forming conditions. Thus, with regard to acetate metabolism, I have revealed evidence for the post-transcriptional regulation of the production of prymnesins and the contributory effects of hemolysin, monogalactosyldiacylglycerol, and PUFA towards ichthyotoxicity. Further, I propose that toxin transport is triggered in HAB-forming conditions, in which the toxins are actively being excreted. Collectively, these data shed light on the transcriptional responses that occur following alterations in phosphate availability and salinity, including those associated with the synthesis and delivery of a number of potential ichthyotoxins from P. parvum. / text

Bacterial toxins have co-opted host cell machinery in order to enter cells and exert their deleterious effects. Anthrax toxin is composed of the receptor binding protein protective antigen (PA), and the enzymatic subunits lethal factor (LF) and edema factor (EF), which form the binary toxin complexes lethal toxin, LeTx (PA + LF), and edema toxin, EdTx (PA + EF). PA binds to receptors on the surface of host cells and shuttles LF and EF into cells through the endocytic pathway. Upon endosome acidification, PA oligomers insert into the endosomal membrane and form functional pores that deliver LF and EF into the cytoplasm. Translocation of the N-terminal domain of LF, \(LF_N\), through PA pores formed in lipid bilayers in vitro does not require host machinery. However, translocation of the related fusion protein \(LF_N\)-DTA across the membrane of toxin-loaded endosomes in vitro requires the addition of cytosolic translocation factors that include the COPI coatamer complex. We performed high-throughput small molecule and RNAi screens to identify host factors required for LF translocation, using LeTx-induced cell death as a phenotype. We describe the characterization of small molecule inhibitors of LeTx-induced cell death that inhibit toxin entry. Further, we describe the role of the endosomal chaperone GRP78 and the cytoplasmic CCT chaperonin in toxin translocation. RNAi knockdown of GRP78 and CCT subunits inhibited LeTx and EdTx delivered through the endocytic pathway. CCT knockdown additionally inhibited translocation of LF through PA pores formed directly in the plasma membrane, while GRP78 had no effect. Furthermore, we show that the role of GRP78 in toxin translocation is specific to translocation from the early endosome. Together with biochemical data, we propose that GRP78 facilitates translocation by unfolding LF and EF at near-neutral pH. In addition, we show that in CCT-knockdown cells, lethal levels of toxin reach the endosome, suggesting that CCT has a role in translocation and/or refolding of LF and EF. These studies highlight previously unidentified strategies used by anthrax toxin to hijack host cellular machinery in order to gain access to the cytosol.

Disease caused by Bordetella pertussis persists with rates increasing over the past decade in industrialized countries. A hindrance to vaccine development has been the lack of a clear serological correlate of protective immunity. Pertussis toxin (PTx), an AB-type toxin, is one of the bacteria’s major virulence factors and among the lead candidates for potential correlates. Of the numerous monoclonal antibodies (mAbs) binding PTx, the murine IgG2a mAb 1B7 is potently neutralizing in all in vitro assays and in vivo murine models of infection. 1B7 binds an epitope on the enzymatic S1-subunit of PTx with some linear elements but previous work was unable to more precisely define the epitope or determine its exact mechanism of protection. We characterize the epitope bound by 1B7 on PTx-S1 in molecular detail and define energetically important interactions between residues at the interface including six residues on PTx-S1 and six residues on 1B7. Using this information, a model of the 1B7-S1 interaction was developed, indicating a predominantly conformational epitope located on the base of S1 near S4. The location of this epitope is consistent with previous data and is shown to be conserved across several naturally occurring strain variants including PTx-S1A, B, D, and E in addition to the catalytically inactive 9K/129G variant. Using immunofluorescent microscopy, it was determined that 1B7’s unique mode of action lies in its ability to bind to the toxin and co-traffic into target cells. Upon endocytosis, 1B7 protects from PTx intoxication by redirecting its intracellular retrograde trafficking. In order to determine whether antibody responses are differently induced by infection or acellular vaccination, we analyzed sera from 30 adults with confirmed exposure to pertussis and 30 recent vaccinees. Natural infection resulted in significantly higher titers of anti-PTx-S1, 1B7-like, and 11E6-like antibodies, while overall anti-PTx titers were similar to vaccinated samples. We also observed a direct correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Thus, natural infection elicits higher titers of protective antibodies indicating that the use of detoxified PTx in current acellular vaccines although highly immunogenic results in the elicitation of predominantly non-neutralizing antibodies. / text

No description available.

Ramularia collo cygni (Rcc), der Erreger der Sprenkelkrankheit an Gerste, gewinnt zunehmend an Bedeutung. Das pilzliche Pathogen wird den Deuteromyceten zugeordnet. Typisch für die Krankheit ist ein auffallend spätes Auftreten von Symptomen in der Vegetationsperiode. Nach Abschluss der Blüte nimmt die Befallsstärke stark zu, was sich in einer Verbräunung und Absterben der Blätter innerhalb von 12 Tagen zeigt. Klassische Untersuchungsmethoden der Rcc-Gerste-Interaktion werden kontinuierlich durch molekulare Untersuchungen zur Befallsdynamik unterstützt. So stehen seit kurzem PCR-basierte Verfahren zum Nachweis von Rcc in Pflanzengewebe zur Verfügung. Ziel dieses Projektes waren Untersuchungen zur Epidemiologie und Pathogenität von Rcc, weiterhin wurde die Herkunft des Inokulums und die Verbreitung des Pathogens untersucht. Ein weiterer Schwerpunkt war die Entwicklung von robusten Phänotypisierungs-Assays zur Bewertung der Resistenz unterschiedlicher Gersten-Genotypen sowohl unter Gewächshaus- als auch unter Feldbedingungen. Zunächst wurde eine auf SYBR-Green basierende quantitative Real-Time PCR (qPCR)-Methode zum Nachweis von pilzlicher DNA in Pflanzengewebe entwickelt. Die Nachweisgrenze der pilzlichen DNA lag hier bei 0,1 pg in Flüssigkultur, sowie in Pflanzengewebe und in Samenmaterial; auch in Regenwasser und Schnee konnte diese DNA-Konzentration noch nachgewiesen werden. Mittels qualitativer PCR konnte eine Übertragung des Pathogens von Samen auf Keimlinge gezeigt werden. Die Untersuchungen bestätigten auch, dass die weitere Ausbreitung des Pathogens in älteren Pflanzen ohne Ausbildung von Symptomen erfolgte. Unter Gewächshausbedingungen traten in infizierten Pflanzen erste Symptome erst zur Abreife und Kornentwicklung auf. Eine Desinfektion der Samen mit heißem Wasser führte nicht zur Abtötung von Rcc und somit zur Verbreitung des Pathogens in der Pflanze. In weiteren Untersuchungen wurde die Wirkung von Saatgutbehandlung und Blattfungiziden zu unterschiedlichen Entwicklungsstadien der infizierten Pflanze getestet. Die Beizmittel Zardex G (Cyproconazol und Imazalil) und das systemische Blattfungizid Proline (Prothioconazole) wurden zu unterschiedlichen Wachstumsphasen der Pflanze appliziert und die Verbreitung von Rcc mittels Real-Time PCR analysiert. Dabei konnte gezeigt werden, dass die Fungizide bei separater Anwendung keine Wirkung hatten, eine Kombination der beiden Pflanzenschutzmittel jedoch Effekte zeigte. So konnte nach Beizung mit Zardex G und anschließender Anwendung von Proline im Wachstumsstadium 39-41 ein starker inhibitorischer Effekt auf das pilzliche Wachstum demonstriert werden. In einer weiteren Studie wurde die Verbreitung und Mobilität von Rcc Inokulum über die Luft mittels Sporenfallen untersucht, die entweder in der Nähe eines Gerstenfeldes oder weiter enfernt aufgestellt wurden. Im späten Herbst und in den Wintermonaten wurden Sporen von Rcc in größerer Entfernung von Gerstenfeldern und in höheren Lagen nachgewiesen. Rcc Inokulum ist demnach weit verbreitet und kann auch über größere Distanzen durch die Luft, im Regenwasser oder Schnee transportiert werden. Interessanterweise ist Inokulum auch auch in kühleren Jahreszeiten nachweisbar. Zur Identifizierung resistenter Genotypen erfolgten Phänotypisierungen von Blattsegmenten in der Klimakammer und parallel dazu wurden Screenings unter Feldbedingungen im Reifestadium 73-75 durchgeführt. Es konnten signifikant unterschiedliche Anfälligkeiten gegenüber Rcc sowohl im Feld, als auch unter kontrollierten Bedingungen im Gewächshaus und der Klimakammer gezeigt werden. So konnten einige Genotypen identifiziert werden, die besonders gute Resistenzen aufwiesen wie z. B. die Sorte IPZ 24727. Eine signifikante Korrelation konnte zwischen den Gewächshausdaten (Inokulation ganzer Pflanzen) und den Felddaten 2009 (p=0,005, rs=0,483) und 2010 (p=0,03, rs=0,384) nachgewiesen werden. Ein Vergleich der Daten des Blattsegment-Assays und der ermittelten Befallsstärke im Gewächshausversuch zeigte eine signifikante Korrelation (p=0,0002, rs=0,592). Darüber hinaus korrelierten die Werte des Assays mit den Felddaten aus dem Jahr 2009 (p=0,0005, rs=0,576) und 2010 (p=0,002, rs=0,513). Eine signifikante Korrelation wurde auch zwischen den Daten der Feldversuche der beiden untersuchten Jahren gezeigt (p=0,04, rs= 0,419). Mittels qPCR wurde Rcc in allen getesteten Genotypen nachgewiesen, d.h., dass keiner der Genotypen komplett resistent war. Die Ergebnisse zeigen, dass ein Nachweis des Pathogens in frühen Wachstumsstadien bereits vor der Symptomausbildung mittels PCR möglich ist. QPCR Analysen zeigten eine starke Korrelation (p=0,00179, rs=0,851) zwischen den visuellen Boniturdaten und der pilzlichen DNA-Konzentration im F-1Blatt. Eine Anwendung der qPCR zur Selektion resistenter Gerste Genotypen ist daher möglich. Weiterhin wurde mittels HPLC eine neue Detektionsmethode für das Rcc Phototoxin Rubellin entwickelt. Mit dieser sensitiven Methode konnten Konzentrationen des Toxins bereits vor der Symptomausbildung in frühen Wachstumsstadien der Pflanze nachgewiesen werden. Die gemessenen Toxinwerte in infiziertem Blattgewebe korrelierten stark mit den Werten der Sichtbonitur (p=0,00005, rs=0,966657). Diese Methode ist daher zur Identifizierung von resistenten Gerste-Genotypen unter kontrollierten Bedingungen geeignet.

Raktiniai žodžiai: spazminė diplegija, spazmiškumas, botulino toksinas, kineziterapija. Tyrimo objektas: botulino toksino ir kineziterapijos dažnumo efektyvumas 4-7 metų vaikams, sergantiems cerebriniu paralyžiumi. Tyrimo problema: Cerebrinis paralyžius – dažniausia vaikų judesių raidos problema, kuri sutrikdo individo santykį su aplinka ir apriboja jo dalyvumą. Apie 70-80% vaikų, sergančių cerebriniu paralyžiu, pasitaiko spazminės formos. Pastaruoju metu pasaulyje botulino toksinas plačiausiai taikomas spazmiškumui slopinti. Nors Lietuvoje šis preparatas taikomas jau šešeri metai, tačiau nėra nustatyta, kaip kineziterapijos dažnumas įtakoja vaikų su spazmine diplegija ir hemiplegija motorinių funkcijų pokyčius po botulino toksino panaudojimo. Darbo tikslas: Nustatyti kineziterapijos ir Botulino toksino (BTX-A) poveikį 4-7 metų vaikams, sergantiems cerebriniu paralyžiumi. Tiriamieji klausimai: Darbe palyginami vaikų su spazmine diplegija ir hemiplegija pasyvios dorzalinės fleksijos, selektyvių p�����dos judesių skalės, pusiausvyros skalės ir bendrosios motorikos funkcijų vertinimo skalės pokyčiai po botulino toksino panaudojimo, kineziterapiją taikant 5 kartus ir 2 kartus per savaitę. Darbe pateikiama gautų rezultatų analizė ir interpretacija. Išvados: 1. Nustatyta, kad čiurnos sąnario pasyvios dorzalinės fleksijos amplitudės padidėjo po BTX-A ir dažnos (5 kartai per savaitę) bei nedažnos (2 kartai per savaitę) kineziterapijos taikymo, tačiau skirtumas... [toliau žr. visą tekstą] / Keywords: spastic diplegia, spasticity, botulinum toxin, physiotherapy. Object: the effectiveness of intensive (5 times/week) and regular (2 times/week) physiotherapy for children with cerebral palsy after the use of botulinum toxin. Problem: Cerebral palsy is a frequent cause of children‘s motor disorder. It affects person‘s relationship with environment and limits his participation. Spasticity predominate for 70-80% of children with cerebral palsy. Botulinum toxin type A (BTX-A) is a relatively new and widely used method of spasticity management in children with cerebral palsy. Despite, that in Lithuania BTX-A has been used for 6 years for spasticity management, there is no evidence how intensivity of physiotherapy influence motor functions for children with cerebral palsy after botulinum toxin A injections. Purpose: to assess the effectiveness of intensive (5 times/week) and regular (2 times/week) physiotherapy for children with cerebral palsy after botulinum toxin A injections. Tasks: to evaluate the effectiveness of intensive and regular physiotherapy for passive ankle range of motions, for selective ankle movements, for balance and gross motor functions for children with cerebral palsy after botulinum toxin A injections. Investigative questions: the purpose of this study to compare the effifacy of botulinum toxin and intensive and regular physiotherapy by assessing changes in passive range of motions, selective movement scale, pediatric balance scale and gross motor... [to full text]

Pathogenic bacteria use an arsenal of toxic protein virulence factors to cause disease in host cells. The mono-ADP-ribosyltransferase (mART) toxins are a family of exotoxins produced by pathogens which contribute to a wide range of diseases including cholera, diphtheria and whooping cough. Specifically, mART toxins act by transferring ADP-ribose from NAD+ to target proteins in host cells, altering or inhibiting target activity with deleterious downstream effects. Recently, in silico analyses have revealed two novel mARTs, Photox and Certhrax, from pathogenic organisms. Photox, from Photorhabdus luminescens was successfully expressed and purified from E. coli and was shown to target actin, specifically at Arg177. This covalent modification inhibits actin polymerization and leads to observed cytotoxicity in yeast cells. Photox has 35% identity with SpvB from Salmonella enterica, which allowed for a structural model to be built, showing the location of all characteristic mART active site components, and the binding site for potential inhibitors. Certhrax originates from Bacillus cereus G9241, implicated in a number of severe pneumonia cases. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we demonstrated that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. In vivo tests employing toxin gene expression in yeast, and receptor-mediated infection of mammalian, cells showed the extreme cytotoxicity of Certhrax (LD50 = 100 pg/mL against mouse macrophage cells), making it 60 times more toxic than its infamous counterpart, anthrax lethal factor. In vitro analysis indicated that Certhrax possesses NAD+ glycohydrolase activity, characteristic of many mART toxins, but we continue to search for the natural host protein target of this toxic enzyme. We determined the crystal structure of Certhrax to 2.2 Å, which illustrates a close structural similarity with anthrax lethal factor. Furthermore, we identified several small molecule inhibitors which show protection against Certhrax both in vitro and in vivo. We determined a 1.9 Å crystal structure of one inhibitor in complex with Certhrax. Through identification and characterization of novel mART enzymes, we seek to better understand this family of toxic enzymes to aid in the discovery and development of more potent therapeutics. / National Sciences and Engineering Research Council, Canadian Institutes of Health Research