Repair of DNA double-strand breaks is critical to the maintenance of genomic stability, and failure to repair these DNA lesions can cause loss of chromosome telomeric regions, complex translocations, or cell death. In humans this can lead to severe developmental abnormalities and cancer. A central pathway for double-strand break repair is homologous recombination (HR), a mechanism that operates during the S and G2 phases of the cell cycle and primarily utilizes the replicated sister chromatid as a template for repair. Most knowledge of HR is derived from work carried out in prokaryotic and eukaryotic model organisms. To probe the HR pathway in human cells, we performed a genome-wide siRNA-based screen; and through this screen, we uncovered cellular functions required for HR and identified proteins that localize to sites of DNA damage. Among positive regulators of HR, we identified networks of pre-mRNA-processing factors and canonical DNA damage response effectors. Within the former, we found RBMX, a heterogeneous nuclear ribonucleoprotein (hnRNP) that associates with the spliceosome, binds RNA, and influences alternative splicing. We found that RBMX is required for cellular resistance to genotoxic stress, accumulates at sites of DNA damage in a poly(ADP-ribose) polymerase 1-dependent manner and through multiple domains, and promotes HR by facilitating proper BRCA2 expression. Screen data also revealed that the mammalian recombinase RAD51 is commonly off-targeted by siRNAs, presenting a cautionary note to those studying HR with RNAi and highlighting the vulnerability of RNAi screens to off-target effects in general. Candidate validation through secondary screening with independent reagents successfully circumvented the effects of off-targeting and set a new standard for reagent redundancy in RNAi screens.

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.

Research was conducted at the Mississippi State University Delta Research and Extension Center from 2016 to 2018 to determine the effects of sub-lethal concentrations of paraquat, glyphosate, saflufenacil, and sodium chlorate exposure to rice at late-season growth stages, determine the effects of exposure to sub-lethal concentrations of glyphosate or paraquat on multiple rice cultivars, and characterize the effects of paraquat exposure and Oebalus pugnax feeding on rice grain quality. In the current research, rough rice grain yields were reduced by exposure to sub-lethal concentrations of soybean desiccants 0 to 28 DAH. Rice injury was determined to not be an accurate predictor of rough rice grain yield loss as injury did not exceed 20% with any desiccant, and no injury was observed from glyphosate applications. Rough rice grain yield reductions were reflected in yield component reductions following desiccant exposure. Applications of sub-lethal concentrations of glyphosate or paraquat to rice at 50% heading caused rough rice grain yield decreases ranging from 0 to 20 and 9 to 21 % respectively. Hybrid cultivars were unaffected following glyphosate exposure at 50% heading. In the current research, observations of paraquat exposure or O. pugnax infestation of rice at the soft dough growth stage suggest rice may exhibit severe sensitivity to both events in the form of reduced kernel weight and reductions in rice milling quality. Rough rice grain yield reductions coupled with milling quality reductions and driven by the proximity of rice to corn, cotton, soybean, and sorghum in Mississippi creates the need to exercise caution when applying desiccants.

Distinguishing 2,4-D and dicamba herbicide formulations in cotton and soybean tissue is challenging in regulation of crop injury from these herbicides. Additionally, stewardship of 2,4-D and dicamba technologies is important to maximize their longevity and efficacy. Research was conducted to (1) characterize cotton and soybean response to various formulations of 2,4-D or dicamba with or without glyphosate, (2) develop a method for classifying these formulations in crop tissue, and (3) optimize use of chloroacetamide herbicides in dicamba systems for mitigation of selection pressure on dicamba. Formulations evaluated include dicamba diglycolamine (DGA), dimethylamine (DMA), N,N-Bis-(3-aminopropyl) methylamine (BAPMA), and DGA plus potassium acetate (KAc); and 2,4-D DMA, acid, isooctyl ester (ESTER), and choline. Weed management by the chloroacetamides s-metolachlor and acetochlor was evaluated with applications preemergence (PRE), early postemergence (EP), late postemergence (LP), PRE followed by (fb) EP, PRE fb LP, and EP fb LP. Cotton and soybean response differed by 2,4-D and dicamba formulation, and glyphosate presence. Cotton yield was reduced by 200 to 500 kg ha-1 following exposure to 2,4-D choline or DMA relative to acid or ESTER. Glyphosate presence led to a reduction in cotton and soybean yield of 377 and 572 kg ha-1, respectively. Exposure to dicamba DMA resulted in a 263 kg ha-1 reduction in soybean yield relative to dicamba DGA, and glyphosate presence reduced yield by 439 and 246 kg ha-1 in cotton and soybeans, respectively. Chemometric analyses generated models capable of up to 85% accuracy in identifying dicamba formulation in cotton and soybean tissue, and up to 80% accuracy in identifying 2,4-D formulation. Split chloroacetamide applications improved cotton yield up to 60%, reduced weed densities up to 90%, and improved control up to 56% relative to single applications. Cotton height was reduced up to 23% if a single chloroacetamide application was made. Soybean yield was maximized following any chloroacetamide application timing except PRE alone, and weed control was reduced up to 31% following single chloroacetamide application relative to split applications. These results will aid regulatory bodies in managing use of new weed control technologies and will assist producers in stewarding these new technologies.

Off-target herbicide movement onto rice is an annual problem in rice-producing areas within Mississippi. In Mississippi, rice is routinely drill‐seeded in April to early May. Because these dates often coincide with preplant and/or preemergence (PRE) herbicide applications to corn (Zea mays L.), cotton (Gossypium hirsutum L.), and soybean [Glycine max (L.) Merr.], drift onto neighboring rice crops is likely to occur. Although the effects of off-target movement of paraquat on rice may not be visibly apparent, the potential effect on rough rice yield could be detrimental. Field research was conducted at the Delta Research and Extension Center in Stoneville, MS, in 2019, 2020, and 2021 to characterize rice response to exposure to a range of sub-lethal concentrations of paraquat during the vegetative and reproductive growth phases. Other field experiments characterized rice response to exposure to a sub-lethal concentration of paraquat at different stages of reproductive growth. A final field experiment evaluated rice response and barnyardgrass control with labeled herbicides after exposure to a sub-lethal concentration of paraquat.

Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.

Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors. Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity. Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively. Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione. The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.

La détection de potentielles cibles secondaires ou off-targets d’un ligand donné requiert la détermination de son site d’interaction et la recherche de sites d’interaction similaires sur d’autres protéines. Dans le but de mener à bien cette étude, nous avons développé PatchSearch : cet outil compare un patch requête, correspondant à un site d’interaction, avec la surface d’une cible potentielle. L’algorithme employé s’appuie sur une méthode originale de recherche de quasi-cliques dans un graphe produit : cette approche identifie des groupes d’atomes du patch appariés avec ceux de la surface ciblée avec des propriétés physico-chimiques conservées et dans des configurations proches. Nous montrons que PatchSearch trouve des patches qui correspondent à ceux qui sont connus sur les surfaces ciblées. De plus, les résultats de l’application de PatchSearch sur des protéines flexibles indiquent que l’approche des quasi-cliques permet de retrouver à la fois les parties rigides et flexibles des patches,contrairement à la recherche classique de cliques. Enfin, les performances de Patch-Search sont équivalentes à celles des autres outils de comparaison de sites de liaison.Nous avons également appliqué PatchSearch sur des off-targets de médicaments impliqués dans le traitement de cancers. Nos expériences suggèrent l’utilisation de PatchSearch dans la recherche des éventuelles off-targets d’un médicament. / Detection of putative off-targets for a ligand requires to search for some similarbinding sites onto other proteins surface. In order to achieve this goal, we developeda tool named PatchSearch. This program compares a query patch, whichcontains the binding site, with the surface a potentially targeted protein. Patch-Search’s algorithm is based on an original method searching for some quasi-cliquesin a graph product, which identifies some atoms both in the patch and in the surfacewith conserved physicochemical properties and in similar configurations. Weshow that PatchSearch efficiently finds known patches on protein surfaces. Moreover,application of PatchSearch on flexible proteins shows that, unlike the classiccliques approach, quasi-cliques method allows to find both rigid and flexible partsof the patches. PatchSearch gets similar results compared to the other binding sitecomparison tools. We also applied PatchSearch to find patches binding polypharmacologicaldrugs involved in cancer treatment, in order to identify them on knownoff targets. Our experiments suggest to employ PatchSearch in off-targets detectionprocess.

Research was conducted at the Mississippi State University Delta Research and Extension Center from 2015 to 2018 to (1) determine the effects of sub-lethal concentrations of paraquat, metribuzin, fomesafen, and cloransulam-methyl applied at different rice growth stages, determine the effects on rice growth of simulated off-target paraquat applications at varying concentration based on a proportionally decreased carrier volume characterize rice response to a sub-lethal concentration of paraquat in combination with common POST and residual herbicides, assess whether starter N fertilizer or different N fertilizer management strategies can aide in rice recover after exposure to a sub-lethal concentration of paraquat, and define a maximum soil concentration of S-metolachlor that will allow rice to germinate and emerge. Rice yield was negatively affected following exposure to paraquat applied any time after rice emergence. Paraquat applications to rice in early reproductive growth reduced rough rice yield and seed germination the greatest. Paraquat plus metribuzin injured rice 68 to 69% 14 and 28 d after treatment (DAT), which was 10 to 13% greater than following paraquat alone or paraquat plus fomesafen. Pooled across metribuzin and fomesafen treatments, paraquat reduced rough rice yields 23%. Paraquat plus 10 different residual herbicides injured rice ≥51% 28 DAT and reduced rough rice yields ≥21%. In spite of starter N fertilizer applications, paraquat injured rice ≥41%, reduced height 57%, reduced dry weight prior to flooding 77%, delayed maturity 10 d, reduced dry weight at maturity 33%, and reduced rough rice yield 35%. Similar results were observed in the N Fertilizer Timing Study. Soil concentrations of s-metolachlor 28 DAT were 30, 31, 32, 36, 61, and 488 ppm following exposure to s-metolachlor applied at 0, 1/64, 1/32, 1/16, 1/4, and 1X concentration. A soil analysis would be the best option to determine levels of s-metolachlor prior to planting rice if an off-target herbicide movement containing s-metolachlor occurred. These data indicate that paraquat can have negative impact on rice growth and development. Therefore, it is crucial that if environmental conditions are conducive for off-target herbicide movement extreme caution should be exercised when applying paraquat adjacent to fields devoted to rice production.

This study was conducted to determine whether soybean productivity is affected by multiple, sublethal herbicide exposures. The effects of dicamba and 2,4-D on soybean (Glycine max) productivity was investigated at 17 site-years. Relative to a single exposure of dicamba at R1, an additional exposure at either V3 or R3 reduced yield up to 23%. Three or more applications did not further decrease yields relative to an R1&R3 exposure. For 2,4-D, a single application to V3, R1, R3, or R5 soybean did not affect grain yield. However, two exposures of 2,4-D occurring from V3 through R3 reduced yield 5 to 7%. Three or more applications of 2,4-D had no effect on yield relative to exposing soybean to 2,4-D twice between V3 and R3. Exposing soybean to multiple, sublethal rates of auxin herbicides can reduce yield relative to a single exposure and may be most deleterious from flowering to initial pod set.