No / The naturally occurring bitter principle quassin (1) was converted chemically into the gamma-lactone quassilactone (13) in an attempt to enhance its antiplasmodial activity. The in vitro antiplasmodial activity of 13 against Plasmodium falciparum (K1) (IC(50) = 23 microM) was 40-fold greater than that of 1. However, one of the intermediates, compound 8, the 15beta-hydroxy,16-O-m-chlorobenzoyl analogue of 1, was 506-fold more active than 1 against P. falciparum (IC(50) = 1.8 microM) and only 3-fold less potent than chloroquine. In addition, 8 displayed the best cytotoxic/antiplasmodial ratio (112) of all of the compounds tested. In the course of this work a dimer, neoquassin ether (6), linked at C-16 was also prepared; 6 was found to have weak antiplasmodial activity (IC(50) = 9.7 microM).

In a series of related experiment: some aspects of the histology and physiology of the disorder bitter pit in apple (Malus domestica Borkh.) were studied. A two year field study was conducted to induce consistent bitter pit development in type ’Delicious’ (D) and ’Golden Delicious’ (GD) apple fruit. Multiple spray treatments of CaCl₂ and MgCl₂, combined with paper bag fruit covers, were applied and subsequent bitter pit development examined. The main effect of bags in both cultivars was increased pit development, decreased Ca in fruit and increased fruit K. CaCl₂ sprays resulted in less pit development, increased Ca in fruit, and less fruit Mg. MgCl₂ sprays resulted in increased bitter pit, decreased fruit Ca, and increased fruit Mg. Overall, field treatments provided a wide range of bitter pit incidence after storage, from 100% (bag and MgCl₂ spray combined) to 3% (CaCl₂ spray alone) in both D and GD. The cellular morphology of pitted apple fruit from field trees was examined using transmission and scanning electron microscopy. The overall tissue morphology of both cultivars was similar, but in pitted tissues differences were observed in tannin localization, starch hydrolysis, and cell wall morphology. Cation levels in the tissues were examined using X-ray microanalysis. High Mg levels were localized in pit cells, while K levels were similar in both healthy and pitted cells. Ca levels in both tissue types were too low to be detected by this method. Using ’Golden Delicious’ fruit from the field study, the relationship between pyruvate kinase activity, fruit cation concentration and bitter pit was investigated. Pyruvate kinase activity during early fruit growth was higher in fruit which developed 100% bitter pit after storage (MgCl, spray + bag), than in fruit that developed 3% bitter pit (CaCl₂ spray). Fruit with a high bitter pit incidence had a lower Ca: Mg + K ratio than fruit with a low level of the disorder. There was a strong positive correlation between enzyme activity early in the season and bitter pit incidence alter storage. An assay for pyruvate kinase may be valuable for early prediction of postharvest bitter pit development. Finally, the qualitative electrophoretic patterns of soluble fruit proteins from each treatment were examined starting early in the season and continuing until termination of fruit storage. Patterns from all treatments were almost identical throughout the season. / Ph. D.

Menschen nehmen Tausende von Stoffen als bitter wahr. Die chemische Struktur der verschiedenen Bitterstoffe ist sehr vielfältig: Sie reicht von kleinen Molekülen wie Kaliumchlorid oder Harnstoff, bis zu sehr komplexen organischen Verbindungen. Die Größe der einzigen bekannten menschlichen Familie von Bitterrezeptoren (TAS2Rs) wurde auf nur ca. 80-120 Mitglieder geschätzt. In Anbetracht der hohen Zahl und Komplexität der Bitterstoffe erscheint die Zahl von Rezeptoren als sehr gering. Dies führt natürlich zu einer Reihe von Fragen: Wie viele Mitglieder hat die menschliche TAS2R-Genfamilie? Wie viele verschiedene Substanzen können denselben Rezeptor aktivieren? Scheint die Zahl der TAS2R-Rezeptoren ausreichend, alle Bitterstoffe wahrnehmen zu können oder muss es noch andere Bitterrezeptorfamilien geben? Diese Fragen zu beantworten, ist das Ziel der vorliegenden Arbeit. <br /> <br /> Hier durchgeführte Analysen des menschlichen Genomprojektes zeigen, dass Menschen ca. 25 TAS2R-Rezeptoren besitzt, die eine sehr divergente Aminosäurestruktur aufweisen. Diese Rezeptoren wurden in eine neu entwickelte Expressionskassette kloniert, die den Transport des Rezeptors an die Zelloberfläche ermöglicht. Um Liganden für die menschliche TAS2R-Rezeptoren zu identifizieren, wurden die Rezeptoren in HEK293 Zellen exprimiert und mit verschiedenen Bitterstoffen stimuliert. Der Nachweis der Rezeptoraktivierung erfolgte durch Calcium-Imaging. Es konnte gezeigt werden, dass hTAS2R16 der menschliche Rezeptor zur Wahrnehmung von Salicin und verwandten bitteren Pyranosiden ist. So wird hTAS2R16 in HEK293 Zellen durch Salicin und chemisch verwandte Substanzen aktiviert. Ein Vergleich der in diesem Messsystem erhaltenen Daten mit psychophysikalisch ermittelten Geschmackswahrnehmungen beim Menschen, ergab eine hohe Übereinstimmung. Die Ergebnisse deuten auch darauf hin, dass die Desensitiverung einzelner Rezeptoren die Ursache für die Adaption des Bittergeschmacks ist. Der Nachweis der Expression des Rezeptors in menschlichen Geschmackspapillen, sowie die festgestellte Assoziation des G/A Polymorpphismus an Position 665 des hTAS2R16 Gens mit einer reduzierten Salicinwahrnehmung, sind weitere unabhängige Beweise für diese These. Ein anderer menschlicher Rezeptor, hTAS2R10, wird durch die Bitterstoffe Strychnin, Brucin und Denatonium aktiviert. Dies sowie die Tatsache, dass die zur Aktivierung benutzten Konzentrationen eine sinnvolle Korrelation zu dem menschlichen Geschmacksschwellwert von Strychnin zeigen, sind starke Hinweise, dass hTAS2R10 der menschliche Rezeptor zur Wahrnehmung von Strychnin und verwandten Substanzen ist. <br /> <br /> Die vorliegenden Daten zeigen eindeutig, dass die TAS2R-Rezeptoren auch beim Menschen Bitterrezeptoren darstellen. Sowohl hTAS2R16, als auch hTAS2R10 werden durch ein Spektrum strukturell sehr unterschiedlicher Bitterstoffe aktiviert. Falls die anderen Mitglieder der TAS2R-Familie ebenfalls dieses Verhalten zeigen, wäre es möglich, dass die nur ca. 25 Mitglieder umfassende TAS2R-Rezeptorfamilie des Menschen tatsächlich zur Wahrnehmung aller Bitterstoffe ausreicht. / Bitter taste generally is aversive and protects vertebrates from ingestion of toxic substances, but bitter tastants also contribute to the enjoyment and palatability of food influencing human nutritional habits. Although bitter taste has been extensively studied, receptor mechanisms are still poorly understood. Anatomic, functional and genetic evidence from rodents suggested, however, that the T2R genes encode a family of bitter receptors while definite proof is missing in humans 6. We here report that the hT2R16 receptor is present in vallate papilla taste buds of the human tongue and activated by bitter b-glucopyranosides. These phytonutrients did not activate any other human T2R and showed similar concentration-response relations and cross-desensitization in hT2R16 expressing cells and psychobiological experiments. hT2R16 is broadly tuned. It binds bitter compounds that share only a b-glycosidic bond and a glucose residue. The broad tuning of T2Rs could explain how a limited number of receptors permits the perception and coding of numerous and different bitter substances. Together our data identify the hT2R16 as a broadly tuned, genuine human bitter receptor for b-glucopyranosides.

Fairy tales as a form of social acculturation can subvert and/or perpetuate potentially harmful social norms. In this essay, Chris Anne Wolfe’s lesbian romance novel Bitter Thorns (1994) and the film Shrek (2001) are analysed as adaptations of the fairy tale Beauty and the Beast, with a focus on the extent to which they challenge and/or reinforce three fairy tale norms: women as tradeable objects, heteronormativity and idealised beauty. Both these texts can be seen as subversive, Bitter Thorns in how it challenges heteronormativity and Shrek in how it challenges the norm of idealised beauty. This subversion, however, is limited, as both texts do more to perpetuate fairy tale norms than to challenge them. They both reinforce the idea of women as objects for trade, Bitter Thorns perpetuates the norm of idealised beauty, and Shrek advocates heteronormative relationships and the dominance of heterosexual masculinity.

A mandioca (Manihot esculenta Crantz) é uma das principais culturas do mundo, havendo grande variabilidade genética. As variedades são classificadas com base na palatabilidade e toxicidade das raízes, em mansas ou doces e bravas ou amargas. Apesar da importância, o potencial da mandioca é pouco explorado, não sendo conhecidos, em nível molecular, os elementos determinantes para as suas características. Assim, pretendeu-se identificar, empregando a 2D-PAGE, proteínas que possam estar associadas com as diferenças físico-químicas das raízes tuberosas de variedades de mesa (IAC 576-70 e IAC 06-01), indústria (Cigana Preta, IAC 12 e IAC 90) e de uso misto (Vassourinha Paulista). Após extração de proteínas e separação por 2D-PAGE, as imagens dos géis foram analisadas no programa Delta2D (DECODON), sendo realizada análise estatística utilizando-se ANOVA (p<0,01), Heat Map e Análises de Componentes Principais (ACP) e de Agrupamentos. Os 146 spots de interesse foram removidos dos géis e suas proteínas digeridas e sequenciadas por espectrometria de massas. Algumas proteínas refletiram as características fenotípicas das variedades em estudo, especialmente entre as de mesa e indústria. Pela ACP, foram explicados 54,54% da variabilidade entre as amostras. A primeira componente separou as variedades exclusivamente de mesa de todas as demais, enquanto a segunda separou a IAC 90 de todas as outras, sendo esta caracterizada por um perfil proteico diferente das demais amostras de uso industrial. A IAC 576-70 e a IAC 12 apresentaram alta correlação positiva, assim como, a Vassourinha e a Cigana. A Análise de Agrupamentos corroborou as informações da ACP, revelando que o proteoma das raízes tuberosas refletiu diferenças fenotípicas entre as variedades. / Cassava (Manihot esculenta Crantz) is a main crop with large genetic variability. The varieties are classified according palatability and toxicity of the roots as sweet or bitter cassavas. Despite its importance, little is known about the molecular basis of phenotypic characteristics. Therefore, this study aimed to identify proteins associated to the differences between the sweet (\'IAC 576-70\' e \'IAC 06-01\'), bitter (\'Cigana Preta\', \'IAC 12\' e \'IAC 90\') and the mixed-use (\'Vassourinha Paulista\') varieties by 2D-PAGE. After the protein extraction and separation by 2D-PAGE, the gel images were analyzed through the software Delta 2D (DECODON), and the statistical analysis were performed with ANOVA (p<0,01), Heat Map, Principal Component Analysis (PCA) and Cluster Analysis. The 146 significant spots were removed from the gels, digested and sequenced by mass spectrometry. Some proteins were related to the physico-chemical characteristics of the varieties, especially between the sweet and the bitter. Variability of the samples was explained at the level of 54,54% by the PCA. The first component separated the sweet varieties from all others while the second one separated the \'IAC 90\' from all others. This variety was characterized by a different protein profile among the bitter cassavas. The \'IAC 576-70\' and the \'IAC 12\' were positively correlated, as well as, \'Vassourinha\' and the \'Cigana\'. Cluster Analysis agreed the PCA information, revealing that the proteomes of the tuberous roots reflected phenotypic differences among the varieties.

Made available in DSpace on 2017-07-10T17:37:18Z (GMT). No. of bitstreams: 1 Silvio Douglas Ferreira.pdf: 1370632 bytes, checksum: 66a09d5927779ac975b32a3541a035ad (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study aimed to evaluate the biological cycle of susceptible biotypes of Digitaria insularis and resistant to glyphosate. The seeds of the biotypes of D. insularis were collected in populations from geographically distant agricultural areas belonging to producers Brejo Paraibano no history of using herbicides, so susceptible's supposed to, and west of Paraná, with a history of resistance. To carry out the evaluations of the growth curves of both experiments the same methodologies were used. The experimental design was a randomized block with four replications. The treatments consisted of plant collections seasons, carried out at regular intervals: 14; 21; 28; 35; 42; 49; 56; 63; 70; 77; 84; 91; 98; 105; 112 and 119 days after emergence (DAE), corresponding to periods of 16 samplings in each period. Biotypes were characterized as follows: AG biotype was considered susceptible, while the CVEL biotypes, ERO and GR were considered resistant and showed FR50 2,7; 3,4 and 7,7 respectively. Between the summer / fall (average temperature of 24,6 °C), the S biotype began issuing tillers and flowering an average of 14 and 21 DAE, respectively, before the resistant biotypes. However, in autumn / Winter (average temperature 19 °C) only biotype R1 was able to develop and finalize the biological cycle. In the summer / autumn period, the S biotype showed the highest absolute growth rate (TCA) at 84 DAE, being 82,3% higher than the resistant biotypes. The higher ratio of leaf area (RAF) was obtained by biotype R3 at 77 DAE, however, could not be verified differences in the relative growth rate (RGR) between biotypes. Biotypes evaluated showed differences during development especially among the phenological phases, so that the susceptible biotype was the earliest and with high reproductive potential / Objetivou-se avaliar o ciclo biológico de biótipos de Digitaria insularis suscetível e resistente ao glyphosate. As sementes dos biótipos de D. insularis, foram coletadas em populações provenientes de áreas agrícolas geograficamente distantes, pertencentes a produtores do Brejo Paraibano sem histórico de uso de herbicidas, logo, suspostamente suscetível, e da região oeste do Paraná, com histórico de resistência. Para realizar as avaliações das curvas de crescimento dos dois experimentos foram utilizadas as mesmas metodologias. O delineamento experimental foi em blocos casualizado com quatro repetições. Os tratamentos foram constituídos por épocas de coletas das plantas, realizadas em intervalos regulares de: 14; 21; 28; 35; 42; 49; 56; 63; 70; 77; 84; 91; 98; 105; 112 e 119 dias após a emergência (DAE), correspondendo a períodos de 16 coletas em cada período. Os biótipos foram caracterizados da seguinte maneira: biótipo AG foi considerado suscetível, enquanto que, os biótipos CVEL, ERO e GR foram considerados resistentes e apresentaram FR50 de 2,7; 3,4 e 7,7 respectivamente. No período o verão/outono (Temperatura média de 24,6°C), o biótipo S iniciou a emissão de perfilhos e o florescimento em média 14 e 21 DAE, respectivamente, antes que os biótipos resistentes. Entretanto, no período outono/inverno (Temperatura média de 19°C) apenas o biótipo R1 conseguiu se desenvolver e finalizar o ciclo biológico. No período verão/outono, o biótipo S apresentou maior Taxa de crescimento absoluto (TCA) aos 84 DAE, sendo 82,3% superior aos biótipos resistentes. A maior Razão da área foliar (RAF) foi obtida pelo biótipo R3 aos 77 DAE, porém, não foi possível verificar diferenças na Taxa de crescimento relativo (TCR) entre os biótipos. Os biótipos avaliados apresentaram diferenças durante o desenvolvimento principalmente entre as fases fenológicas, de modo que, o biótipo suscetível foi o mais precoce e com elevado potencial reprodutivo

Les Saramaka du Suriname, descendants d'esclaves échappés des plantations au XVIIème siècle, ont une pharmacopée traditionnelle encore vive. Le Bitter-cup, ou " gobelet amer ", en est un exemple puisqu'il garde encore sa place sur les étals des marchés de Paramaribo. Nous avons mené une enquête ethnopharmacologique afin de détailler ses usages : mis à macérer pendant une nuit avec du rhum ou de l'eau, le contenu est bu d'un trait au matin comme tonique amer, stomachique, fébrifuge ou encore antimalarique. Pour mettre fin aux controverses que suscite sa provenance botanique, nous avons procédé à une étude anatomique du bois et des gobelets provenant du marché, ainsi qu'à l'établissement de signatures chimiques par Chromatographie Liquide Haute Performance (CLHP). Ces deux méthodes complémentaires nous ont permis d'identifier Quassia amara L. comme source des gobelets. Des tests in vitro sur Plasmodium falciparum n'ont pas confirmé l'activité antipaludique, mais après un fractionnement bioguidé par CLHP semi-préparative, certaines fractions se montrent très performantes contre les parasites.

Die Bittergeschmacksrezeptoren stellen in der Superfamilie der G-Protein-gekoppelten Rezeptoren eine besondere Gruppe dar. Im Menschen können die 25 Rezeptoren eine große Anzahl unterschiedlichster Bittergeschmacksstoffe detektieren. Diese Substanzen können sowohl schädlich, wie etwa Strychnin, als auch der Gesundheit förderliche Arzneistoffe, wie etwa Chloramphenicol sein. Unter den Bittergeschmacksrezeptoren des Menschen gibt es eine Gruppe von drei Rezeptoren, die besonders viele Bitterstoffe detektieren können. Einer von ihnen ist der Rezeptor hTAS2R10. In dieser Arbeit konnte sowohl experimentell als auch durch computergestützte Modellierung gezeigt werden, dass der hTAS2R10 nur eine Bindungstasche besitzt. Das stimmt mit den bisher ausführlich experimentell und in silico untersuchten Rezeptoren hTAS2R1, -R16, -R38 und -R46 überein. Die für die Agonisteninteraktionen nachweislich wichtigen Transmembrandomänen sind in den bisher untersuchten Bittergeschmacksrezeptoren, wie auch im hTAS2R10, die Transmembrandomänen 3, 5, 6 und 7. Die Untersuchungen zeigten, dass die Bindungstasche des hTAS2R10 in der oberen Hälfte des zum extrazellulären Raum gerichteten Bereichs lokalisiert ist. Insbesondere konnte für die untersuchten Agonisten Strychnin, Parthenolid und Denatoniumbenzoat gezeigt werden, dass die Seitenketten der Aminosäuren in Position 3.29 und 5.40 ausgeprägte agonistenselektive Wechselwirkungen eingehen. Weitere Untersuchungen haben ergeben, dass das weitgefächerte Agonistenspektrum des hTAS2R10 zu Lasten der Sensitivität für einzelne Bitterstoffe geht. Der Vergleich wichtiger Positionen im hTAS2R10, hTAS2R46 und mTas2r105 hat deutlich gemacht, dass sich die Bindungsmodi zwischen diesen Rezeptoren unterscheiden. Dies deutet auf eine getrennte evolutionäre Entwicklung der Bindungseigenschaften dieser Rezeptoren hin. Gleichfalls zeigten die Untersuchungen, dass einige Positionen wie z.B. 7.39 die Funktion aller untersuchten Bittergeschmacksrezeptoren prägen, sich jedoch die genaue Bedeutung im jeweiligen Rezeptor unterscheiden kann. Einzelne dieser Positionen konnten auch bei der Agonisteninteraktion des Rhodopsins und des β2-adrenergen Rezeptors beobachtet werden. Die Ergebnisse dieser Arbeit helfen dabei die Wechselwirkungen zwischen Bitterstoffen und den Bittergeschmacksrezeptoren zu verstehen und geben erste Einblicke in die Entwicklung der Rezeptoren in Hinblick auf ihren Funktionsmechanismus. Diese Erkenntnisse können genutzt werden, um Inhibitoren zu entwickeln, die sowohl ein wichtiges Werkzeug in der Rezeptoranalytik wären, als auch dazu genutzt werden könnten, den unerwünschten bitteren Geschmack von Medikamenten oder gesundheitsfördernden sekundären Pflanzenstoffen zu mindern. Damit könnte ein Beitrag zur Gesundheit der Menschen geleistet werden. / In the Superfamily of G protein-coupled receptors the bitter taste receptors form a notable group. The 25 human receptors are able to detect a large group of structurally diverse bitter compounds. These compounds can be toxic – like strychnine – or have beneficial effects on health – like the pharmacological agent chloramphenicol. Three of these bitter taste receptors show a strikingly broad agonist spectrum. One of them is the hTAS2R10. It was shown empirically and by computational modelling that the hTAS2R10 has only one binding pocket. This agrees with the findings of studies on the bitter taste receptors hTAS2R1, -R16, -R38 and -R46. The domains important for agonist interaction in these receptors, as well as in the hTAS2R10, are the transmembrane domains 3, 5, 6 and 7. The results of this thesis show that the binding pocket of the hTAS210 is located in the upper part of the receptor which points into the direction of the extracellular area. Interestingly, it has been shown for the amino acid side chains in the positions 3.29 and 5.40, that they can interact with the analysed agonists strychnine, parthenolide and denatonium benzoate in an agonist-selective way. Further analyses showed that the broad tuning of the hTAS2R10 goes at the expense of the sensitivity to single agonists. The comparison of crucial positions in the hTAS2R10, hTAS2R46 and the mTas2r105 reveal that these receptors differ in their binding mode. These could be evidence that the binding abilities of these receptors evolved independently. However, the results show that some positions, e.g. 7.39, influence the receptor activity in all analysed receptors, but the function of these positions in the receptors could be different. Some of these positions also have an influence on the agonist-receptor interaction of Rhodopsin and the β2-adrenergic receptor. The findings in this thesis contribute to the knowledge about interaction between bitter receptors and bitter compounds. The results also provide insight into the evolvement of receptor functions. These outcomes can be of use for the development of inhibitors which could serve as analytical tools in taste research. Furthermore, such inhibitors could be used to reduce the bitter taste of medicine and healthy plant compounds and thus increase palatability. This could contribute to improve human well-being.

Der Mensch besitzt ~25 funktionelle Bitterrezeptoren (TAS2R), die für die Wahrnehmung potenziell toxischer Substanzen in der Nahrung verantwortlich sind. Aufgrund der großen genetischen Variabilität der TAS2R-Gene könnte es eine Vielzahl funktionell unterschiedlicher TAS2R-Haplotypen geben, die zu Unterschieden der Bitterwahrnehmung führen. Dies konnte bereits in funktionellen Analysen und sensorischen Studien für einzelne Bitterrezeptoren gezeigt werden. In dieser Arbeit wurden die häufigsten Haplotypen aller 25 Bitterrezeptoren verschiedener Ethnien funktionell charakterisiert. Das Ziel war eine umfassende Aussage über die funktionelle Diversität der TAS2Rs, die die molekulare Grundlage für individuelle Bitterwahrnehmung bildet, treffen zu können. Fehlende Varianten wurden aus genomischer DNA kloniert oder durch gezielte Mutagenese bereits vorhandener TAS2R-Konstrukte generiert. Die funktionelle Analyse erfolgte mittels Expression der TAS2R-Haplotypen in HEK293TG16gust44 Zellen und anschließenden Calcium-Imaging-Experimenten mit zwei bekannten Agonisten. Die Haplotypen der fünf orphanen TAS2Rs wurden mit über hundert Bitterstoffen stimuliert. Durch die gelungene Deorphanisierung des TAS2R41 in dieser Arbeit, wurden für die 21 aktivierbaren TAS2Rs 36 funktionell-unterschiedliche Haplotypen identifiziert. Die tatsächliche funktionelle Vielfalt blieb jedoch deutlich hinter der genetischen Variabilität der TAS2Rs zurück. Neun Bitterrezeptoren wiesen funktionell homogene Haplotypen auf oder besaßen nur eine weltweit vorherrschende Variante. Funktionell heterogene Haplotypen wurden für zwölf TAS2Rs identifiziert. Inaktive Varianten der Rezeptoren TAS2R9, TAS2R38 und TAS2R46 sollten die Wahrnehmung von Bitterstoffen wie Ofloxacin, Cnicin, Hydrocortison, Limonin, Parthenolid oder Strychnin beeinflussen. Unterschiedlich sensitive Varianten, besonders der Rezeptoren TAS2R47 und TAS2R49, sollten für Agonisten wie Absinthin, Amarogentin oder Cromolyn ebenfalls zu phänotypischen Unterschieden führen. Wie für den TAS2R16 bereits gezeigt, traten Haplotypen des funktionell heterogenen TAS2R7 und TAS2R41 ethnien-spezifisch auf, was auf lokale Anpassung und verschiedene Phänotypen hinweisen könnte. Weiterführend muss nun eine Analyse der funktionell-variablen TAS2Rs in sensorischen Tests erfolgen, um ihre phänotypische Relevanz zu prüfen. Die Analyse der funktionsmodulierenden Aminosäurepositionen, z.Bsp. des TAS2R44, TAS2R47 oder TAS2R49, könnte weiterführend zum besseren Verständnis der Rezeptor-Ligand- und Rezeptor-G-Protein-Interaktion beitragen. / Bitter taste perception varies markedly from person to person, due to a high number of polymorphisms present in the 25 known functional bitter receptors (TAS2Rs). These polymorphisms lead to a number of haplotypes for each receptor, which are common in different populations, but vary in frequency. The individual combination of receptor variants seems to determine the person’s sensitivity of bitter perception, as could already be shown for single TAS2Rs. Bitter is an aversive taste quality, indicating the ingestion of harmful substances. Different sensitivity could have an impact on food choice. In order to characterize functional consequences of the genetic diversity, we performed calcium imaging experiments with all main haplotypes for the 25 bitter receptors. The obtained information about receptor properties enables us on the one hand to analyze structure-function relationships and on the other hand gives us the functional diverse candidates to focus on in psychophysical studies. The overall aim is to show genotype-phenotype correlation for bitter taste perception and their impact on food choice and therefore diet and health. Our first aim was to identify agonists for the 5 receptors, which could not be deorphaned in previous screens. We challenged all main haplotypes of these TAS2Rs with 106 bitter compounds and could identify the antibiotic chloramphenicol as agonist for bitter receptor TAS2R41. In total we identified 36 functionally different receptor variants of the 21 deorphaned TAS2Rs. Main haplotypes of nine TAS2Rs were functionally homogeneous while twelve TAS2Rs possessed between two and three functionally heterogeneous receptor variants. In summary the observed functional diversity is not as big as expected. Based on our in vitro findings the shown functional diversity of these twelve bitter receptors might be the molecular basis for individual differences in bitter taste perception and will be further analyzed in psychophysical studies.

Multiple species of Colletotrichum can cause bitter rot disease of apple, but the identities and relative representation of the species causing the disease in Kentucky are unknown. A total of 475 Colletotrichum isolates were collected from diseased apples in 25 counties and characterized both morphologically and by using various molecular approaches. Four morphotypes corresponded to reported descriptions of bitter rot species. Morphotype 1, distinguished by the production of a pink color on potato dextrose agar (PDA), orange conidial masses, and fusiform spores, was consistent with C. acutatum. Morphotype 2, which produced gray or white mycelial colonies with orange conidial masses and fusiform spores, was also similar to C. acutatum. Morphotype 3 had abundant gray mycelium and rounded spores and was identical to C. gloeosporioides. Morphotype 4 produced ascospores and resembled Glomerella cingulata. Species-specific polymerase chain reaction (PCR) indicated that both Morphotype 1 and Morphotype 2 belonged to the C. acutatum species complex, whereas Morphotype 3 and Morphotype 4 corresponded to the C. gloeosporioides complex. Multigene sequence analyses revealed that sample isolates belonged to several newly erected species within these species complexes. Morphotype 1 was identified as C. fioriniae, which resides within the C. acutatum species complex. Morphotype 2 was identified as C. nymphaeae, which is also a species within the C. acutatum species complex. Some isolates of Morphotype 3 were identified as C. siamense and some as C. theobromicola; both species are grouped within the C. gloeosporioides species complex. Morphotype 4 was identified as C. fructicola, which is also placed within the C. gloeosporioides species complex. C. fioriniae was the most common species causing bitter rot in Kentucky, comprising more than 70% of the isolates. Molecular fingerprinting using random amplified polymorphic DNA (RAPD) suggested that isolates within C. fioriniae belonged to a relatively homogeneous population, while isolates within C. siamense, C. theobromicola and C. fructicola were more diverse. Infectivity tests on detached fruit showed that C. gloeosporioides species-complex isolates were more aggressive than isolates in the C. acutatum species complex. However, isolates within the C. acutatum species complex produced more spores on lesions compared to isolates within the C. gloeosporioides species complex. Aggressiveness varied among individual species within a species complex. C. siamense was the most aggressive species identified in this study. Within the C. acutatum species complex, C. fioriniae was more aggressive than C. nymphaeae, causing larger, deeper lesions. Apple cultivar did not have significant effect on lesion development. However, Colletotrichum species produced more spores on Red Stayman Winesap than on Golden Delicious. Fungicide sensitivity tests revealed that the C. acutatum species complex was more tolerant to thiophanate-methyl, myclobutanil, trifloxystrobin, and captan compared to the C. gloeosporioides species complex. The study also revealed that mycelial growth of C. siamense was more sensitive to tested fungicides compared to C. fructicola and C. theobromicola. These research findings emphasize the importance of accurate identification of Colletotrichum species within each species complex, since they exhibit differences in pathogenicity and fungicide sensitivity.