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Articles 1 - 30 of 96
Full-Text Articles in Biochemistry
High Resolution Mass Spectrometry As A Platform For The Analysis Of Polyoxometalates, Their Solution Phase Dynamics, And Their Biological Interactions., Daniel T. Favre
High Resolution Mass Spectrometry As A Platform For The Analysis Of Polyoxometalates, Their Solution Phase Dynamics, And Their Biological Interactions., Daniel T. Favre
Doctoral Dissertations
Polyoxometalates (POMs) are a class of inorganic molecule of increasing interest to the inorganic, bioinorganic and catalytic communities among many others. While their prevalence in research has increased, tools and methodologies for the analysis of their fundamental characteristics still need further development. Decavanadate (V10) specifically has been postulated to have several unique properties that have not been confirmed independently. Mass spectrometry (MS) and its ability to determine the composition of solution phase species by both mass and charge is uniquely well suited to the analysis of POMs. In this work we utilized high-resolution mass spectrometry to characterize V10 in aqueous …
The Impact Of A Non-Ionic Adjuvant To The Persistence Of Pesticides On Produce Surfaces, Daniel Barnes
The Impact Of A Non-Ionic Adjuvant To The Persistence Of Pesticides On Produce Surfaces, Daniel Barnes
Masters Theses
Adjuvants can enhance the performance of the pesticide active ingredients in many ways including decreasing surface tension and reducing evaporation. Understanding how adjuvants effect pesticide behavior (e.g., surface persistence) is crucial for developing effective pesticide formulations, as well as facilitating the development of effective approaches to reduce pesticide residues from the surface of fresh produce post-harvest. The objective of this study is to investigate the effect of a non-ionic surfactant, Surf-Ac 910, on the persistence of two model pesticides, thiabendazole and phosmet on apple surfaces. The result shows that the addition of Surf-Ac 910 increased both the maximum wetted area …
Caulobacter Clpxp Adaptor Popa’S Domain Interactions In The Adaptor Hierarchy Of Ctra Degradation, Thomas P. Scudder
Caulobacter Clpxp Adaptor Popa’S Domain Interactions In The Adaptor Hierarchy Of Ctra Degradation, Thomas P. Scudder
Masters Theses
The degradation and recycling of protein is a process essential for the maintenance and regulation of cellular function. More specifically, in Caulobacter crescentus, the ClpXP protease is responsible for driving progression through the cell cycle and protein quality control. This protease utilizes three known adaptors to selectively degrade proteins that initiate different stages of development. This thesis will elaborate on the specific binding interface on one of these adaptors, PopA, with another, RcdA, and focus in on specific residues on PopA and investigate their roles in adaptor binding and delivery of CtrA, the master regulator of Caulobacter. Finally, I …
Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz
Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz
Masters Theses
p53 is a transcription factor and an important tumor suppressor protein that becomes activated due to DNA damage. Because of its role as a tumor suppressor, mutations in the gene that encodes it are found in over 50% of human cancers. The N-terminal transactivation domain (NTAD) of p53 is intrinsically disordered and modulates the function and interactions of p53 in the cell. Its disordered structure allows it to be controlled closely by post-translation modifications that regulate p53’s ability to bind DNA and interact with regulatory binding partners. p53 is an attractive target for developing cancer therapeutics, but its intrinsically disordered …
Modulation Of Plant Immunity During The Establishment Of The Nitrogen-Fixing Symbiosis, Miriam Hernandez-Romero
Modulation Of Plant Immunity During The Establishment Of The Nitrogen-Fixing Symbiosis, Miriam Hernandez-Romero
Doctoral Dissertations
Nitrogen is essential for plant tissue growth but is often a limited resource in soils. Many legumes overcome this limitation by entering a symbiotic association with soil microbes, called rhizobia, which provide nitrogen to the plant while rhizobia receive fixed carbon. To successfully form a symbiosis, the host and symbiont exchange a series of molecular signals. One major obstacle during this interaction is the host's innate immune system, which becomes active upon rhizobial detection. It is therefore the main focus of this thesis to identify the mechanisms that modulate host immunity. In the subsequent chapters, we focus on a rhizobial …
Characterizing The Roles Of The Variable Linker And Hub Domains In Camkii Activation, Noelle Dziedzic
Characterizing The Roles Of The Variable Linker And Hub Domains In Camkii Activation, Noelle Dziedzic
Doctoral Dissertations
Learning and memory formation at the cellular level involves decoding complex electrochemical signals between nerve cells, or neurons. Understanding these processes at the molecular level requires a comprehensive study of calcium-sensitive proteins that serve as signal mediators within cells. More specifically, the protein calcium/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of downstream cellular signaling events in the brain, playing an important role in long term memory formation. CaMKII is encoded in humans on four different genes: alpha, beta, gamma and delta. For added complexity, each of these gene products can be alternatively spliced and translated into multiple protein …
Characterization Of The Poly (Adp-Ribose) Polymerase Family In The Fusarium Oxysporum Species Complex, Daniel Norment
Characterization Of The Poly (Adp-Ribose) Polymerase Family In The Fusarium Oxysporum Species Complex, Daniel Norment
Masters Theses
Fusarium oxysporum is a filamentous fungus that is known to invade over a hundred different hosts and poses a major threat to the economy and food supply world-wide. Poly (Adenosine diphosphate-Ribose) Polymerase (PARP) is a family of regulatory proteins that affect change in the cell through transfer of ADP-Ribose moieties onto target molecules. The most well-studied PARP protein is the human PARP1, a PARylating nuclear protein that serves as our model PARP protein. F. oxysporum was found to contain a large expansion of PARP catalytic-domain-containing proteins compared to other filamentous fungi. We utilized in silico multiple sequence alignments and domain …
Investigation Of Kinase Conformational Dynamics And Analytes Detection With Protein Nanopore, Fanjun Li
Investigation Of Kinase Conformational Dynamics And Analytes Detection With Protein Nanopore, Fanjun Li
Doctoral Dissertations
Protein nanopores are pore-forming proteins which have been developed as single-molecule biosensors. Due to the high sensitivity, selectivity, label-free and real-time detection methodology, protein nanopores have been used for a wide variety of applications. In this dissertation, we use ClyA nanopore to investigate kinase conformational dynamics and develop a kinase/nanopore system for the specific detection of kinase allosteric inhibitors. Besides, we engineer OmpG nanopore to be a sensor for nucleic acid detection. Protein kinases play essential roles in cellular regulation by catalyzing the phosphorylation of target proteins and are promising drug targets. The conformational dynamics are critical for kinase functions. …
Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward
Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward
Doctoral Dissertations
Molecular motors, such as myosin, have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction in these motors is a root cause of many pathologies necessitating the application of intrinsic control over molecular motor function. We hypothesized that altering the myosin’s energy substrate via minor positional changes to the triphosphate portion of the molecule will allow us to control the protein and affect its in vitro function. We utilized positional isomers of a synthetic non-nucleoside triphosphate, azobenzene triphosphate, and assessed whether myosin’s force- and motion-generating capacity could …
Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni
Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni
Doctoral Dissertations
Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …
Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage
Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage
Masters Theses
Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a …
Deciphering Protein Higher-Order Structure And Interactions Via Diethylpyrocarbonate Labeling-Mass Spectrometry, Xiao Pan
Doctoral Dissertations
The study of protein higher-order structures is vital because it is closely related to the investigation of protein folding, aggregation, interaction and protein therapeutics. Consequently, numerous biochemical and biophysical tools have been developed to study protein higher-order structures in many different situations. The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a powerful tool for studying protein structures and offers many advantages over other traditional techniques, such as better structural coverage, high throughput, high sensitivity, and the ability to study proteins in mixtures. This dissertation focuses on diethylpyrocarbonate (DEPC) as an effective CL reagent that can …
Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri
Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri
Doctoral Dissertations
Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA …
Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo
Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo
Doctoral Dissertations
Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …
Mechanistic Insights Into Diverse Protease Adaptor Functions, Nathan J. Kuhlmann
Mechanistic Insights Into Diverse Protease Adaptor Functions, Nathan J. Kuhlmann
Doctoral Dissertations
Protein degradation is an essential cellular process that helps maintain proper homeostasis. The ClpXP protease broadly regulates bacterial development and quality control during the cell cycle. The range and order of substrates that ClpXP degrades during the cell cycle is dictated by 3 accessory proteins, which are known as adaptors. This thesis will elaborate on how dimerization tightly regulates the stability and activity of the adaptor protein at the center of this hierarchy, RcdA, and show how this affects normal cellular processes in Caulobacter crescentus. I will discuss the mechanism by which dimerization limits RcdA activity and how the dimerization …
Hsp70 Phosphorylation: A Case Study Of Serine Residues 385 And 400, Sashrika Saini
Hsp70 Phosphorylation: A Case Study Of Serine Residues 385 And 400, Sashrika Saini
Masters Theses
Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, …
Exploring Knockdown Phenotypes And Interactions Between Atad3 Proteins In Arabidopsis Thaliana, Eli S. Gordon
Exploring Knockdown Phenotypes And Interactions Between Atad3 Proteins In Arabidopsis Thaliana, Eli S. Gordon
Masters Theses
Mitochondria are required for a diverse array of cellular functions and processes. ATAD3 (ATPase family AAA domain containing protein 3) proteins are newly discovered mitochondrial membrane proteins in Arabidopsis thaliana. Homologous to ATAD3A in metazoans, Co-Immunoprecipitation/Mass spectrometry and genomic analysis identified a four ATAD3A homologues in A. thaliana. The four A. thaliana proteins are referred to as ATAD3A1 (At3g03060), ATAD3A2 (At5g16930), ATAD3B1 (At2g18330), and ATAD3B2 (At4g36580). Studies in metazoans indicate that ATAD3A localizes to Mitochondria-ER contact sites and is involved in a variety of processes required for proper mitochondrial function, but ATAD3A proteins are poorly defined in plants. …
Amyloidogenesis Of Β-2-Microglobulin Studied By Mass Spectrometry And Covalent Labeling, Blaise G. Arden
Amyloidogenesis Of Β-2-Microglobulin Studied By Mass Spectrometry And Covalent Labeling, Blaise G. Arden
Doctoral Dissertations
Amyloid-forming proteins are implicated in a number of debilitating diseases. While many amyloid-forming proteins are well studied, the early stages of amyloidosis are still not well understood on a molecular level. Covalent labeling, combined with mass spectrometry (CL-MS), is uniquely well suited to provide molecular-level insight into the factors governing the early stages of amyloidosis. This dissertation leverages CL-MS techniques to examine the early stages of β-2-microglobulin (β2m) amyloidosis. β2m is the protein that forms amyloids in the condition known as dialysis-related amyloidosis. An automated CL-MS technique that uses dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide as a labeling reagent was developed and used …
Primary Kinetic Isotope Effect For Substrate Hydroxylation And Iron Binding Regulation In Factor Inhibiting Hif-1, Michael A. Mingroni
Primary Kinetic Isotope Effect For Substrate Hydroxylation And Iron Binding Regulation In Factor Inhibiting Hif-1, Michael A. Mingroni
Doctoral Dissertations
The hypoxic response is a vast and complex system, delicately designed through evolution to allow our tissues to rapidly adapt and survive fluctuations in pO2. HIF1α, the master regulator of oxygen, is tightly controlled through oxygen-dependent, post-translational hydroxylation via PHD2 and FIH1.The aberrant stabilization of HIF1α is an adverse consequence of disease states, but can also occur under normoxia in the presence of ROS or in iron depletion. The extensive transcriptional network regulated by HIF1α makes the HIF pathway an attractive therapeutic target, particularly through the inhibition of the hydroxylases, PHD2 and FIH, however the active site chemistry is mechanistically …
Link Between Muscle And Whole-Body Energetic Responses To Exercise, Christopher M.T. Hayden
Link Between Muscle And Whole-Body Energetic Responses To Exercise, Christopher M.T. Hayden
Masters Theses
Substantial evidence exists regarding how skeletal muscles use energy and how this affects muscular performance. What remains unclear is how characteristics of muscle energetics affect whole-body energetics during daily living, and what effects this may have on mobility. The goal of this study was to determine the associations between muscle and whole-body energetics including the relationships between: 1) muscle PCr depletion (∆PCr) in response to light intensity isotonic contractions and the oxygen deficit at the onset of a 30-min treadmill walk (30MTW), and, 2) muscle oxidative capacity and excess post-exercise oxygen consumption (EPOC; 30MTW), respiratory exchange ratio (RER; 30MTW), and …
Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac
Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac
Doctoral Dissertations
High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes …
Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu
Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu
Doctoral Dissertations
Human lysosomal neuraminidase 1 (hNEU1) is an exo-a-sialidase which cleaves a(2-3) and a(2-6) linked sialic acids on glycoproteins in the lysosome. Deficiency of hNEU1 in the lysosome results in sialidosis, a lysosomal storage disease. Currently there is no effective treatment for sialidosis, which leads to a rising interest in discovering potential therapies. Here we presented a small molecule, α-D-N-acetylneuraminic acid (NANA), increases the protein amount and activity of both wild-type hNEU1 and three different hNEU1 mutations found in sialidosis patients in our mammalian cell system, suggesting that NANA works as a potential pharmacological chaperone for hNEU1 and provides …
Investigating Structural Proteins By Light Scattering, Uma Nudurupati
Investigating Structural Proteins By Light Scattering, Uma Nudurupati
Masters Theses
This thesis evaluates the organization of the structural proteins, Human Gamma D crystallin and Collagen type II, into higher-order structures using light scattering. Specifically, it evaluates the natures of incipient aggregation in Human Gamma D crystallin and the nature of its interactions with CAPEGn, an electrostatic blocker. Additionally, this thesis evaluates whether Collagen type II growth kinetics follows Classical nucleation theory.
Investigation Of Shot1-Binding Atpases In Arabidopsis Thaliana, Sam Zelman
Investigation Of Shot1-Binding Atpases In Arabidopsis Thaliana, Sam Zelman
Masters Theses
Mitochondria play critical roles not only in primary metabolism as a central organelle for ATP generation, but also in responding to abiotic stresses. We identified a mutation in the MTERF18 (Mitochondrial Transcription Termination factor)/SHOT1 (Suppressor of hot1-4 1) gene in Arabidopsis thaliana that enables plants to better tolerate heat and oxidative stresses, presumably due to reduced oxidative damage, but the exact molecular mechanism of the heat tolerance is unknown. In order to reveal the stress tolerance mechanisms of mterf18/shot1 mutations, it is critical to understand the molecular defects of the mutant and to identify …
Assessing Stress Tolerance Of Organelle Small Heat Shock Protein Mutants In Arabidopsis Thaliana, Parth Patel
Assessing Stress Tolerance Of Organelle Small Heat Shock Protein Mutants In Arabidopsis Thaliana, Parth Patel
Masters Theses
Molecular chaperones are proteins found in virtually every organism and are essential to cell survival. When plants are heat stressed, they upregulate and downregulate multiple genes, many of which are associated with the heat shock response. Small heat shock proteins (sHSPs) are one class of molecular chaperones that are upregulated during heat shock. They are proposed to act as the first line of defense by binding to heat sensitive proteins and preventing their irreversible aggregation. However, many details of sHSP function remain to be discovered and exactly what proteins they protect is unresolved. In addition to cytosolic sHSPs found in …
A Rational Design Approach To Developing Second Generation Fabry Disease Treatments, Matthew Metcalf
A Rational Design Approach To Developing Second Generation Fabry Disease Treatments, Matthew Metcalf
Doctoral Dissertations
Fabry disease is an X-linked lysosomal storage disorder that affects approximately 1 in 40,000 males in its classical form and as many as 1:4,600 in its late-onset form [1]. The disease is caused by mutations in the gene encoding α- galactosidase (α-GAL), which results in deficient levels of α-GAL activity in the lysosomes of patients [2, 3]. This lack of enzymatic activity causes macromolecular substrates to accumulate in tissues, and can result in a wide range of symptoms such as impaired renal and cardiac function [4]. The severity of disease is linked to the amount of residual …
Investigating The Accumulation, Sub-Organ Distribution, And Biochemical Effects Of Nanomaterials Using Mass Spectrometry, Kristen Nicole Sikora
Investigating The Accumulation, Sub-Organ Distribution, And Biochemical Effects Of Nanomaterials Using Mass Spectrometry, Kristen Nicole Sikora
Doctoral Dissertations
Gold nanoparticles (AuNPs) are attractive materials for use in various biomedical applications, such as therapeutic delivery, due to their unique chemical properties and modular tunability. Mass spectrometry methods, including laser desorption/ionization mass spectrometry (LDI-MS) and inductively coupled plasma mass spectrometry (ICP-MS) have been successfully used to evaluate the distribution of AuNPs in complex biological systems. As new AuNP-based materials are developed for applications in therapeutic delivery, it is essential to simultaneously develop analytical techniques that can comprehensively assess their behavior in vivo. In this dissertation, novel mass spectrometric methods have been developed and utilized to evaluate the uptake, distribution, …
Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma
Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma
Doctoral Dissertations
The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material …
Study Of The Role Of Biologically-Relevant, Labile Nickel Pools In The Maturation Of Nickel-Dependent Enzymes, Priyanka Basak
Study Of The Role Of Biologically-Relevant, Labile Nickel Pools In The Maturation Of Nickel-Dependent Enzymes, Priyanka Basak
Doctoral Dissertations
Cellular nickel pools, comprised of static and labile pools of nickel complexes, play important roles in maintaining nickel homeostasis in various organisms (microbes, fungi, and plants), which utilize it as a cofactor of one or more nickel enzymes that catalyze specific reactions and are essential for their proper growth and survival in various ecological niches. Like other metals, tight regulation of cellular nickel levels is critical to prevent toxic effects of nickel deprivation, nickel overload, and ‘free’ nickel. While more static nickel pools include nickel tightly bound to nickel-dependent enzymes, nickel in the labile pool is exchangeable and weakly bound …
Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther
Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther
Doctoral Dissertations
Many legumes form a symbiosis with nitrogen-fixing bacteria found in the soil. This relationship is beneficial to both the plant and the bacteria; the plant receives nitrogen that is otherwise limited, and the bacteria receive fixed carbon. Upon sensing the bacteria, the plant forms a new organ (the nodule) where the bacteria are housed within the cells. Many genes are required for the proper formation and function of nodules; this dissertation is broadly focused on how genes required for nitrogen-fixing symbiosis are co-opted from other cellular processes and how they are specialized for symbiosis. Protein trafficking from the plant to …