archives@tulane.edu / Despite great progress in the treatment viral illnesses, epidemics of emerging infectious diseases often have no specific treatments, leaving most patients to be treated with only supportive care. Broad-spectrum antiviral drugs could help ease this burden especially in situations where diagnostics are limited. Recently, many antiviral peptides have been discovered to inhibit a wide array of viruses using the Wimley-White interfacial hydrophobicity scale to predict inhibitory peptides, some of which were found to possess broad-spectrum activity. These broad-spectrum peptides share some common characteristics: they are moderately hydrophobic and have a propensity to disrupt membranes. We hypothesize that there are general molecular characteristics that these peptides possess, such as a propensity for membrane interaction, that drive broad-spectrum antiviral activity, which we can utilize for the design of novel potent antiviral peptides. To test this hypothesis, we first selected membrane-active peptides with no previously known antiviral activity. We screened these peptides against multiple diverse viruses and found that a majority possess antiviral activity with little cytotoxicity. We next explored the mechanism of action and found that peptides bind virus and cause rapid aggregation. We then used this information to guide the design of novel gain-of-function antiviral peptides. We sequentially designed peptides to test specific hypotheses about sequence motifs that may alter antiviral activity and/or cytotoxicity. Through our screenings we uncovered sequence motifs that led to greater antiviral activity, lower cytotoxicity, and potent broad-spectrum activity. More optimization is needed to turn these peptides into viable drug candidates, but we believe our findings identified important design aspects of antiviral peptides that could lead to the creation of potent broad-spectrum antiviral therapeutics. / 1 / Andrew Hoffmann

This present research explores the impact of Flannery’s (1969) Broad Spectrum Revolution model within the context of the Levantine Epipalaeolithic, specifically the early (Kebaran) Epipalaeolithic. This model attempts to explain changes in subsistence behaviour associated with the end of the Pleistocene leading up to the development of agriculture. The Jordan valley represents an ideal model for Flannery’s marginal habitat because of the range of different flora and fauna required more intensive resource exploitation strategies. The discussion here focuses on the zooarchaeological and taphonomic analysis of two faunal assemblages (representing two sub-horizons) from Urkan e-Rubb IIa, a Kebaran site located in the lower Jordan Valley, and excavated by E. Hovers (Institute of Archaeology, Hebrew University Jerusalem), in 1986 and 1988. This site shows evidence of specialised gazelle hunting and processing. Initial interpretations of the site argued for a multi-purpose residential base camp, supported by lithic tool and shell bead manufacturing. Recent discussions of Epipalaeolithic diet breadth have concentrated on the end of the Pleistocene (Munro 2001, 2003, 2004, 2009; Stiner 2001; Stiner & Munro 2002; Stiner et al. 1999, 2000; Stutz et al. 2009) and have been directed towards explaining changing subsistence patterns towards the end of the Epipalaeolithic (i.e. the Natufian), where an increased use of wild cereals and small game has been well documented. The view that it is not until the Natufian that broad spectrum resource strategies were used is questioned here. Major questions that are addressed with this research include: What do the faunal assemblages from Urkan e-Rubb IIa tell us about Kebaran subisistence behaviour? Do broad spectrum models help to shed light on subsistence adaptations at the end of the Last Glacial Maximum? What can recent analyses of Jordan Valley faunal assemblages contribute to the discussion? These are addressed through a detailed zooarchaeological and taphonomic analysis of the Urkan e-Rubb IIa fauana, followed by synchronic and diachronic analyses of Levantine Epipalaeolithic assemblages, through a combination of taxonomy-based diversity indices and prey-ranking indices.

This present research explores the impact of Flannery’s (1969) Broad Spectrum Revolution model within the context of the Levantine Epipalaeolithic, specifically the early (Kebaran) Epipalaeolithic. This model attempts to explain changes in subsistence behaviour associated with the end of the Pleistocene leading up to the development of agriculture. The Jordan valley represents an ideal model for Flannery’s marginal habitat because of the range of different flora and fauna required more intensive resource exploitation strategies. The discussion here focuses on the zooarchaeological and taphonomic analysis of two faunal assemblages (representing two sub-horizons) from Urkan e-Rubb IIa, a Kebaran site located in the lower Jordan Valley, and excavated by E. Hovers (Institute of Archaeology, Hebrew University Jerusalem), in 1986 and 1988. This site shows evidence of specialised gazelle hunting and processing. Initial interpretations of the site argued for a multi-purpose residential base camp, supported by lithic tool and shell bead manufacturing. Recent discussions of Epipalaeolithic diet breadth have concentrated on the end of the Pleistocene (Munro 2001, 2003, 2004, 2009; Stiner 2001; Stiner & Munro 2002; Stiner et al. 1999, 2000; Stutz et al. 2009) and have been directed towards explaining changing subsistence patterns towards the end of the Epipalaeolithic (i.e. the Natufian), where an increased use of wild cereals and small game has been well documented. The view that it is not until the Natufian that broad spectrum resource strategies were used is questioned here. Major questions that are addressed with this research include: What do the faunal assemblages from Urkan e-Rubb IIa tell us about Kebaran subisistence behaviour? Do broad spectrum models help to shed light on subsistence adaptations at the end of the Last Glacial Maximum? What can recent analyses of Jordan Valley faunal assemblages contribute to the discussion? These are addressed through a detailed zooarchaeological and taphonomic analysis of the Urkan e-Rubb IIa fauana, followed by synchronic and diachronic analyses of Levantine Epipalaeolithic assemblages, through a combination of taxonomy-based diversity indices and prey-ranking indices.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13110号 / 論医博第2128号 / 新制||医||1022(附属図書館) / (主査)教授 髙折 晃史, 教授 佐藤 俊哉, 教授 川上 浩司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tobacco phylloplanins (T-phylloplanin) are a group of closely-related glycoproteins that are formed and disposed at the interface between the plant aerial surface (the phylloplane) and the atmosphere. They are synthesized in short procumbent trichomes and are secreted to aerial surfaces where they are thought to serve the plant as a first line of defense against fungal pathogens. Here it is shown using in vitro and in planta assays that tobacco and sunflower phylloplanins have broad-spectrum antifungal activities against spores - and also hyphae for two species - of several true fungi. Field tests show that T-phylloplanin reduces diseases caused by three important fungal pathogens of turf grasses. Tobacco phylloplanins are distinct proteins but they have properties in common with small, membrane-pore-forming, antimicrobial peptides formed by other organisms. To directly determine if T-phylloplanin has pore-forming activity we monitored conductivity change and specific ion leakage from spores and hyphae in suspension. Results indicate that phylloplanin causes fungal membrane disruption that leads to ion depletion and cell death. Having observed broad efficacy of T-phylloplanin against spores and/or hyphae of several true fungi, but no activity towards hyphae of the oomycetes, Pythium and Peronospora parasitica, we tested for possible effects on zoospores of the latter two pathogens. T-phylloplanin was shown to be effective against their zoospores, extending the efficacy of T-phylloplanin to include water molds. In the course of these experiments we also tested the effects of the diterpene cis-abienol that is secreted from tall trichomes of tobaccos and found this compound impacted zoospores and could prevent black shank disease caused by P. parasitica when applied to soil-grown tobaccos as a root drench. Thus, results of these studies with phylloplanins and cis-abienol, two different tobacco surface accumulated compounds are consistent with their serving the plant as first line of defense systems against a wide array of invading fungal pathogens. Phylloplanins and cis-abienol may be useful for controlling fungal diseases in tobacco float beds. The efficacy shown here for T-phylloplanin control of fungal pathogens of turf grasses in the field suggests that this natural product may find use in IPM of turf and other crops.

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.

No description available.

The threat from infectious diseases dates as far back as prehistoric times. Pathogens continue to pose serious challenges to human health. The emergence and spread of diseases such as HIV/AIDS, Severe Acute Respiratory Syndrome (SARS), avian influenza, and the threats of bioterrorism have made infectious diseases major public health concerns. Despite many successes in the discovery of anti-infective medications, the treatment of infectious diseases faces serious challenges, which include (i) the emergence and reemergence of infectious pathogens, (ii) the ability of pathogens to adapt and develop resistance to drugs, and (ii) a shortage in the development and discovery of new anti-infective drugs. Host-Oriented Broad-Spectrum (HOBS) treatments have the promising potential to alleviate these problems. The HOBS treatment paradigm focuses on finding drug targets in human host that are simultaneously effective against a wide variety of infectious agents and toxins. In this dissertation, we present a computational approach to predict HOBS treatments by integrative analysis of three types of data, namely, (a) gene expression data representing host responses upon infection by a pathogen, (b) annotations of genes to pre-defined biological pathways and processes, and (iii) genes that are targets of known drugs. Our methods combine gene set-level enrichment with biclustering. We applied our approach to a compendium of gene expression data sets derived from host cells exposed to bacterial or to fungal pathogens, to functional annotation data from multiple databases, and to drug targets from DrugBank. We present putative host drug targets and drugs with extensive support in the literature for their potential to treat multiple bacterial and fungal infections. These results showcase the potential of our computational approach to predict HOBS drug targets that may be effective against two or more pathogens. Our study takes a clean-slate approach that promises to yield unsuspected or unknown associations between pathogens and biological processes, and thus discern candidate gene/proteins to be further probed as HOBS targets. Furthermore, by focusing on host responses to pathogens as captured by transcriptional data, our proposed approach stimulates host-oriented drug target identification, which has potential to alleviate the problem of drug resistance. / Ph. D.

Les dernières décennies ont été marquées par l’émergence ou la réémergence d’un nombre croissant de virus pathogènes. Malheureusement, les antiviraux actuellement sur le marché ciblent un nombre restreint de virus ; il y a donc un besoin urgent de développer des antiviraux à large spectre. Les cyclophilines sont des protéines cellulaires impliquées dans un grand nombre de processus biologiques, qui possèdent une activité enzymatique peptidyl-prolyl cis-trans isomérase (PPIase). Elles sont également impliquées dans la réplication de virus appartenant à des familles éloignées et constituent donc une cible de choix pour le développement d'antiviraux à large spectre. Toutefois, les inhibiteurs de cyclophilines disponibles possèdent de nombreux inconvénients qui rendent leur utilisation clinique difficile.Par une stratégie de "fragment-based drug design", nous avons généré une nouvelle famille d'inhibiteurs de cyclophilines, les SMCypI ("Small-Molecule Cyclophilin Inhibitors"), complètement différents de tous les inhibiteurs de cyclophilines existants. La cristallographie de ces composés a montré qu'ils se fixaient dans les deux poches voisines du site actif des cyclophilines et qu'ils inhibaient leur activité PPIase. Ces composés n’étaient pas immunosuppressifs et bloquaient in vitro l'infection par le VIH, le VHC et les Coronavirus.L'activité anti-VHC du C31, composé le plus actif sur l'activité PPIase des cyclophilines, a été caractérisée. Le C31 était un inhibiteur pan-génotypique du VHC, doté d’une haute barrière contre la résistance et présentant une activité additive avec les inhibiteurs du VHC approuvés. Nous avons montré que le C31 bloquait l'infection par le VHC en rompant l'interaction entre la protéine virale NS5A et la cyclophiline A de façon PPIase-dépendante. Enfin, le C31 était actif sur la réplication des virus zika, de la dengue, de la fièvre jaune et du Nil Occidental.L'activité des SMCypI a été caractérisée sur l'infection par le Coronavirus 229E. De manière intéressante, l’inhibition de l’activité PPIase était nécessaire, mais pas suffisante pour l’activité antivirale. Une étude de la relation structure-activité des composés a révélé qu'un groupement chimique situé à l'interface entre les deux poches du site actif des cyclophilines jouait un rôle clé dans l'effet anti-coronavirus. Le F836 a été identifié comme le composé le plus actif, qui bloquait l'effet cytopathique et la quantité d'ARN du HCoV-229E avec la même efficacité que l'alisporivir, sans toxicité associée. Ce composé bloquait l'entrée du HCoV-229E après l'attachement du virus à la surface cellulaire, et était également actif sur l'entrée des HCoV-OC43 et du MERS-CoV. Nous avons par la suite démontré l’association de la cyclophiline A avec les particules virales. Par l'utilisation de la technologie CRISPR-Cas9, des cellules invalidées pour la cyclophiline A ont été générées. La cyclophiline A apparaissait nécessaire pour l'infection par HCoV-229E et la cible de l'effet antiviral du F836.Les SMCypI constituent un outil pour la compréhension des mécanismes par lesquels les cyclophilines modulent les infections virales et représentent des candidats crédibles pour le développement futur d'antiviraux à large spectre. / Over the past decades, an increasing number of viruses has emerged or re-emerged in humans. Unfortunately, currently approved antiviral drugs target a small set of viruses. Thus, there is an urgent need for the development of broad-spectrum antiviral drugs.Cyclophilins are cellular proteins involved in a large number of biological processes, and in different viral lifecycles from unrelated families. They appear as a potential target for the development of broad-spectrum antiviral approaches. However, currently available cyclophilin inhibitors have drawbacks which limit their clinical use.By means of "fragment-based drug design", we generated a new class of small-molecule cyclophilin inhibitors (SMCypI), unrelated with those already available. Cristallographic studies revealed that the SMCypIs bind to two close pockets of the active site and inhibit cyclophilin PPIase activity. These compounds do not bear immunosuppressive properties and inhibit the replication of HIV, HCV and coronaviruses in vitro.We characterized the anti-HCV activity of C31, the most potent inhibitor of cyclophilin PPIase activity. C31 had pan-genotypic HCV inhibitor properties, with a high barrier to resistance and additive effects with currently approved anti-HCV agents. C31 blocked HCV replication by disrupting the interaction between the nonstructural viral protein NS5A and cyclophilin A in a PPIase-dependent manner. Finally, C31 was active on zika, yellow fever, dengue and West-Nile virus infections.The antiviral activity of the SMCypIs has then been characterized on HCoV-229E infection. Interestingly, PPIase inhibition was necessary, but not sufficient for antiviral effect. A structure-activity relationship study identified a key moiety in the SMCypIs at the interface between the two cyclophilin pockets. F836 has been identified as the most potent compound which inhibited both the cytopathic effect and the intracellular RNA of HCoV-229E without associated cytotoxicity and as potently as alisporivir. This compound targeted HCoV-229E entry at a post-attachment step and was also active on HCoV-OC43 and MERS-CoV strains. We then demonstrated that cyclophilin A was associated with viral particles. By means of CRISPR-Cas9, cell lines depleted for cyclophilin A were generated. Cyclophilin A was identified as a proviral factor for HCoV-229E and was partially involved in F836 antiviral effect. Cyclophilin A expression level was drastically decreased by infection.SMCypIs represent a unique tool to decipher the cellular and molecular mechanisms by which cyclophilins interfere with viral lifecycles, as well as drugable compounds that could find an indication as broad-spectrum antiviral drugs.

The theoretical part of this master's thesis is focused on research of confocal microscopy and FLIM method. There are a principles and types of confocal microscopy and the use of broad-spectrum laser as a basic light source of these microscopes. It gives what the FLIM method and its use not only in cell biology. The practical part thesis includes the acquisition of three sets of fluorescence intensity images with use of applications tunable pulsed laser, function TimeGate and detection of hybrid detectors. For practical elaboration of this thesis is in the software Fiji created a plugin, which is the source code in the Java programming language. The types of plugins and their uses are described in the third chapter of the thesis. This plugin including the graphical user interface in the form of the dialog box, proceses the fluorescence intensity images and creates a graphical representation of data showing the fluorescence lifetime, so called pseudocolor map.