Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

The performativity of nonviolent protest in South Asia (1918 - 1948)

This study seeks to address a gap in the study of nonviolent action. The gap relates to the question of how nonviolence is performed, as opposed to the meaning or impact of nonviolent politics. The dissertation approaches the history of nonviolent protest in South Asia through the lens of performance studies. Such a shift allows for concepts such as performativity and theatricality to be tested in terms of their applicability and relevance to contemporary political and philosophical questions. It also allows for a different perspective on the historiography of nonviolent protest. Using concepts, modes of analysis and tropes of thinking from the emerging field of performance studies, the dissertation analyses two different cases of nonviolent protest, asking how politics is performatively constituted. The first two sections of this study set out the parameters of the key terms of the dissertation: nonviolence and performativity, by tracing their genealogies and legacies as terms. These histories are then located as an intersection in the founding of the nonviolent. The case studies at the analytical core of the dissertation are: fasting as a method in Gandhi's political arsenal, and the army of nonviolent soldiers in the North-West Frontier Province, known as the Khudai Khidmatgar. The study begins with an overview of current theorisations of nonviolence. The approach to the subject is through an investigation of commonly held misconceptions about nonviolent action, such as its supposed passivity, the absence of violence, its ineffectiveness and its spiritual basis. This section addresses the lacunae within existing theories of nonviolence and points to possible fertile spaces for further exploration. Section 3 offers an overview of the different shades of the concept of performativity, asking how it is used in various contexts and how these different nuances can be viewed in relation to each other. The dissertation explores how a theory of performativity may be correlated to the theorisation of nonviolence. The correlations are established in four boundary areas: action/inaction, violence/absence of violence, the actor/opponent and the body/spirit. These boundary areas allow for a theorising of nonviolent action as a performative process. The first case study is Gandhi's use of the fast as a method of nonviolent protest. Using a close reading of his own writings, speeches and letters, as well as a reading of responses to his fast in British newspapers and within India, the dissertation asks what made fasting into Gandhi's most favoured mode of protest and political action. The study reconstructs his unique praxis of the fast from a performative perspective, demonstrating how display and ostentation are vital to the political economy of the fast. It also unveils the cultural context and historical reservoir of body practices, which Gandhi drew from and adapted into 'weapons' of political action. The relationship of Gandhian nonviolence to the body forms a crucial part of the analysis. The second case study is the nonviolent army of the Pashtuns, Khudai Khidmatgar (KK), literally Servants of God. This anti-imperialist movement in the North-West Frontier Province of what is today the border between Pakistan and Afghanistan existed between 1929 and 1948. The movement adopted the organisational form of an army. It conducted protest activities against colonial rule, as well as social reform activities for the Pashtuns. This group was connected to the Congress party of Gandhi, but the dissertation argues that their conceptualisation and praxis of nonviolence emerged from a very different tradition and worldview. Following a brief introduction to the socio-political background of this Pashtun movement, the dissertation explores the activities that this nonviolent army engaged in, looking at their unique understanding of the militancy of an unarmed force, and their mode of combat and confrontation. Of particular interest to the analysis is the way the KK re-combined and mixed what appear to be contradictory ideologies and acts. In doing so, they reframed cultural and historical stereotypes of the Pashtuns as a martial race, juxtaposing the institutional form of the army with a nonviolent praxis based on Islamic principles and social reform. The example of the Khudai Khidmatgar is used to explore the idea that nonviolence is not the opposite of violent conflict, but in fact a dialectical engagement and response to violence. Section 5, in conclusion, returns to the boundary areas of nonviolence: action, violence, the opponent and the body, and re-visits these areas on a comparative note, bringing together elements from Gandhi's fasts and the practices of the KK. The similarities and differences in the two examples are assessed and contextualised in relation to the guiding question of this study, namely the question of the performativity of nonviolent action.