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New Single Molecule In Vivo And Ex Vivo Methods To Understand The Physiological Effects Of Nicotine Use, Xu Fu

Theses and Dissertations--Chemistry

The consumption of tobacco products leads to several health risks, including respiratory diseases, cardiovascular diseases, and many different cancers. Nicotine, the primary addictive compound in tobacco, acts on the cholinergic system through nicotinic acetylcholine receptors (nAChRs), which is believed to be an essential component of addiction. However, the lack of a complete understanding of the neurobiological mechanisms of nicotine abuse is one of the primary barriers to finding potent therapeutics for smoking cessation. Single-molecule imaging is an ideal tool to study molecular mechanisms of protein and physiological changes both in vitro and in vivo. In this dissertation, our research …

Deconvolution Tools For Extracting Insight From Challenging Two-Flavin Systems, Dallas Michael Bell

Theses and Dissertations--Chemistry

Flavoproteins have long been explored for their ubiquity among a number of metabolic and energetic reactions. The flavin cofactor has the inherent benefit of distinct spectral changes associated with redox transitions; however, the double-edged sword is incurred as these distinct signatures overlap and take up much of the UV-vis spectral range. Therefore, it is crucial to create a method to demarcate the expressed redox transitions for studying these systems. The first portion of these studies discusses the creation of a program that deduces spectra for redox transitions in a single-flavin containing model protein: flavodoxin. The latter portions discuss the application …

Single-Domain Vhh Antibodies As Protein Stabilization Modules, Nourhan Mohamed Disouky Mostafa

Graduate Research Theses & Dissertations

VHH domains are the antigen-binding units found in heavy-chain-only antibodies from the Camelidae family (e.g., camels and llamas). They are considered a unique minimalist antibody scaffold due to the lack of a variable light chain domain. Despite having half the antigen-binding interface of conventional antibodies, these VHH antibodies bind their target antigens with affinities comparable to conventional antibodies. This project explored how flexible linkers connecting an anti-GFP VHH (αGFP VHH) domain to its antigen (EGFP) enhanced antigen stability and influenced oligomeric assembly. It was hypothesized that linking the anti-GFP VHH antibody to its target antigen (EGFP) will increase the local …

A Bioinformatic Approach To Exploring Land Plant Evolution, Lauren Margaret Orton

Graduate Research Theses & Dissertations

Today, researchers are inundated with an overwhelming amount of data due to advances in DNA sequencing technologies. This has benefited the fields of both bioinformatics and phylogenomics substantially. With the ability to increase sampling at both the taxonomic and molecular levels, we are now able to produce robust phylogenomic reconstructions and better discern relationships between taxa.

The goals of this dissertation are in two distinct parts: 1) to advance the understanding of land plant terrestrialization and evolution through examination of the closest relative to the land plant lineage, a species of green algae; and 2) to explore evolution among a …

Seasonal Variation In Mitochondrial Bioenergetics Of The Bluegill Sunfish, Lepomis Macrochirus, From A Shallow Midwest River, Derick Isaac Lamptey

Masters Theses

As average global temperature increase, the frequency and magnitude of extreme temperatures in shallow aquatic ecosystems are more ubiquitous. In order to understand how these changing thermal regimes affect aquatic ectotherms, it is essential to develop studies evaluating the response of ectotherms to seasonal fluctuating thermal regimes. Previous studies on fluctuating temperature regimes have reported an increased physiological stress leading to morphological, behavioral and biochemical adaptations. From the latter, the adaptive capacity and seasonal performance associated with optimal function of the oxidative phosphorylation system (OXPHOS) are key for species persistence. However, studies on this matter are scarce. This study explores …

Flavodoxin, The Hydrogen Atom Of Flavoproteins: A 19f Nmr Study Of Dynamics And Conformational Changes Utilizing Flavodoxin From Rhodopseudomonas Palustris, Taylor Varner

Theses and Dissertations--Chemistry

Flavodoxin is a small, highly stable protein that contain a single FMN cofactor used to transfer single electrons at low potentials. The organism Rhodopseudomonas palustris contains a long-chain flavodoxin. Long-chain flavodoxins are characterized by a 20 amino acid loop that is proposed to allow interactions with partner proteins. We plan to utilize this protein as a model to build our repertoire with protein fluorination and fluorine NMR in flavoproteins. This tool kit will then be applied to the study of a partner protein of interest that is capable of performing electron bifurcation. We have incorporated m-fluoro tyrosine into flavodoxin and …

Hijacking The Fusion Complex Of Human Parainfluenza Virus As An Antiviral Strategy, T. C. Marcink, E. Yariv, K. Rybkina, V. Más, F. T. Bovier, A. Des Georges, A. L. Greninger, C. A. Alabi, M. Porotto, N. Ben-Tal, A. Moscona

Advanced Science Research Center

The receptor binding protein of parainfluenza virus, hemagglutinin- neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein …

Polymer Brush Hypersurface Photolithography, Carlos Carbonell, Daniel J. Valles, Alexa M. Wong, Andrea S. Carlini, Mollie A. Touve, Joanna Korpanty, Nathan C. Gianneschi, Adam B. Braunschweig

Advanced Science Research Center

Polymer brush patterns have a central role in established and emerging research disciplines, from microarrays and smart surfaces to tissue engineering. The properties of these patterned surfaces are dependent on monomer composition, polymer height, and brush distribution across the surface. No current lithographic method, however, is capable of adjusting each of these variables independently and with micrometer-scale resolution. Here we report a technique termed Polymer Brush Hypersurface Photolithography, which produces polymeric pixels by combining a digital micromirror device (DMD), an air-free reaction chamber, and microfluidics to independently control monomer composition and polymer height of each pixel. The printer capabilities are …

A Proteomic Analysis Of Corydoras Sterbai Secretions And Tissues, Erik Powell Wictor

University of the Pacific Theses and Dissertations

Defensive mechanisms vary widely in the animal kingdom ranging from physical defenses like spines to chemical defenses such as toxins. Toxins in these secretions and tissues can fluctuate from enzymes to lipids to uncharacterized chemicals. Next generation -omics technology and mass spectrometry are extremely important in analyzing these samples because of their ability to distinguish minute amounts of toxic substance within a complicated sample. The goal of this experiment was to look at secretions and tissues from Corydoras sterbai. All samples in this study were proteolyzed using a mixture of Trypsin and Lys-C, fractionated, and run through nanoLC-MS/MS analysis using …

Proteomic Analysis Of Fetal Rat Neural Stem Cells After Treatment With Hericium Erinaceus, Bright Adam Test

University of the Pacific Theses and Dissertations

The fungus, Hericium erinaceus, has outstanding chemical properties, displaying health benefits in digestive, hepatic, and nervous tissues. Its ease of accessibility and use makes it one of the most common substances used for treatment in Eastern medicine. More and more recent research is confirming the incredible health benefits of this fungus, especially the impact that is seen on nervous tissue growth and recovery post-treatment. Such neurite outgrowth and myelin sheath regeneration could illustrate the beginning of the cure to lifelong neurodegenerative diseases such as Multiple Sclerosis. In this first-of-its-kind study, we cultured and differentiated fetal rat neural stem cells while …

A Novel Pool Of Microparticle Cholesterol Is Elevatedin Rheumatoid Arthritis But Not In Systemic Lupus Erythematosus Patients, Shuai Shuai Hu, Brenton L. Cavanagh, Robert Harrington, Muddassar Ahmad, Grainne Kearns, Steve Meaney, Claire Wynne

Articles

Microparticles are sub-micron, membrane-bound particles released from virtually allcells and which are present in the circulation. In several autoimmune disorders their amountand composition in the circulation is altered. Microparticle surface protein expression has beenexplored as a differentiating tool in autoimmune disorders where the clinical pictures can overlap.Here, we examine the utility of a novel lipid-based marker—microparticle cholesterol, present in allmicroparticles regardless of cellular origin—to distinguish between rheumatoid arthritis (RA) andsystemic lupus erythematosus (SLE). We first isolated a series of microparticle containing lipoproteindeficient fractions from patient and control plasma. There were no significant differences in thesize, structure or protein content of …

Extraction Of Protein From Four Different Seaweeds Using Three Different Physical Pre-Treatment Strategies, Jack O'Connor, Steve Meaney, Gwilym A. Williams, Maria Hayes

Articles

Seaweeds are a rich source of protein and can contain up to 47% on the dry weight basis. It ischallenging to extract proteins from the raw biomass of seaweed due to resilient cell-wall complexes.Four species of macroalgae were used in this study-two brown,Fucus vesiculosusandAlaria esculenta,and two red,Palmaria palmataandChondrus crispus. Three treatments were applied individually tothe macroalgal species: (I) high-pressure processing (HPP); (II) laboratory autoclave processing and(III) a classical sonication and salting out method. The protein, ash and lipid contents of the resultingextracts were estimated. Yields of protein recovered ranged from 3.2% forFucus vesiculosuspre-treatedwith high pressure processing to 28.9% protein recovered …

Pt(Iv) Pro-Drugs With An Axial Hdac Inhibitor Demonstrate Multimodal Mechanisms Involving Dna Damage And A Poptosis Independent Of Cisplatin Resistance In A2780/A2780cis Cells, Awatif Rashed Z. Almotairy, Diego Montagner, Liam Morrison, Michael Devereux, Orla Howe, Andrea Erxlebena

Articles

Epigenetic agents such as histone deacetylase (HDAC) inhibitors are widely investigated for use in combined anticancer therapy and the co-administration of Pt drugs with HDAC inhibitors has shown promise for the treatment of resistant cancers. Coordination of an HDAC inhibitor to an axial position of a Pt(IV) derivative of cisplatin allows the combination of the epigenetic drug and the Pt chemotherapeutic into a single molecule. In this work we carry out mechanistic studies on the known Pt(IV) complex cis,cis,trans-[Pt(NH3)2Cl2(PBA)2] (B) with the HDAC inhibitor 4-phenylbutyrate (PBA) and its derivatives cis,cis,trans-[Pt(NH3)2Cl2(PBA)(OH)] (A), cis,cis,trans-[Pt(NH3)2Cl2(PBA)(Bz)] (C), and cis,cis,trans-[Pt(NH3)2Cl2(PBA)(Suc)] (D) (Bz = benzoate, Suc …

Synthesis And Optimisation Of P3 Substituted Vinyl Sulfone-Based Inhibitors As Anti-Trypanosomal Agents, William Doherty, Nikoletta Adler, Andrew Knox, Thomas J. Butler, Paul Evans

Articles

A series of lysine-based vinyl sulfone peptidomimetics were synthesised and evaluated for anti-trypanosomal activity against bloodstream forms of T. brucei. This focused set of compounds, varying in the P3 position, were accessed in a divergent manner from a common intermediate (ammonium salt 8). Several P3 analogues exhibited sub-micromolar EC50 values, with thiourea 14, urea 15 and amide 21 representing the most potent anti-trypanosomal derivatives of the series. In order to establish an in vitro selectivity index the most active anti-trypanosomal compounds were also assessed for their impact on cell viability and cytotoxity effects in mammalian cells. Encouragingly, all compounds only …

Optimisation Of Estrogen Receptor Subtype-Selectivity Of A 4-Aryl-4h-Chromene Scaffold Previously Identified By Virtual Screening, Miriam Carr, Andrew Knox, Daniel Nevin, Niamh O'Boyle, Shu Wang, Billy Egan, Thomas Mccabe, Brendan Twamley, Daniela Zisterer, David Lloyd, Mary Meegan

Articles

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).

Tools To Evaluate Nanodiamond-Mediated Delivery Of Tiopronin For Cataract Prevention, Justin Beltz

Doctoral Dissertations

”There is a growing demand for non-surgical means of cataract treatment. This dissertation presents three bodies of work that reflect the early-stage development of eye drop formulations aimed at delaying cataract progression. These formulations consist of the antioxidant 2-mercaptopropionylglycine (MPG) loaded onto nanodiamond particles.

Cataractogenesis is linked to oxidative damage to lens proteins. To investigate the potential of MPG for protection against oxidative damage, A549 cells were incubated in 0.6 mM tert-butylhydroperoxide (tBHP). Cells exposed to tBHP without MPG exhibited elevated levels of reactive oxygen species, which led to the depletion of the vital antioxidant glutathione and, ultimately, apoptosis. Co-administration …

Selectivity For Allosteric Effectors Of Bacterial Adp-Glucose Pyrophosphorylase: Structural And Functional Studies, Mashael Alghamdi

Dissertations

The biosynthesis of the intracellular polysaccharide in bacteria and plants, glycogen and starch, respectively, controlled by the key regulatory step that catalyzed by ADP-Glucose Pyrophosphorylase (ADP-Glc PPase). ADP-Glc PPase is an allosteric enzyme regulated by metabolites produced of the principle carbon assimilation pathway in each organism. Agrobacterium tumefaciens enzyme activated by fructose 6-phosphate (Fru6P) and pyruvate, whereas Escherichia coli enzyme activated by fructose 1,6-bisphosphate (FBP), and both enzymes inhibited by AMP. Here, we targeted the allosteric regulation of the A. tumefaciens enzyme, examined some residues that may impact the regulation mechanism (Ser72, His71, Arg75, Ser351, Ser334, Arg368, Asn350, and Asp291), …

Human Isocitrate Dehydrogenase Enzymes, Joseph Valentein Roman

Dissertations

Mammals have three forms of isocitrate dehydrogenase (ICDH) that each catalyze the oxidative decarboxylation of isocitrate forming alpha-ketoglutarate (AKG), carbon dioxide and reduced pyridine nucleotide. The intra-mitochondrial, citric acid cycle enzyme is ICDH III that exists as a hetero tetramer and uses NAD+ as a substrate. ICDH I and ICDH II are both dimeric enzymes that reside in the cytosol and use NADP+. While, the precise function of the cytosolic ICDHs is not known, active site variants of both ICDH I and II cause a variety of cancers. The R132H variant of ICDH1 is one of the most common alterations. …

Controlled Release Of Fibroin Repeat-Tagged Proteins From Silk Hydrogel, Jaturong Promsuk

Chulalongkorn University Theses and Dissertations (Chula ETD)

Protein-based biologic drugs such as growth factors are playing important roles in modern medicine. These proteins can be used to stimulate local tissue repair. Growth factors are often incorporated into a scaffold, such as a hydrogel, and then put into the desired area of the body to achieve therapeutic effects. However, diffusion of these growth factors outside of the designated area or inappropriate release time can have deleterious consequences, such as inducing tumors in other areas of the body or uncontrolled inflammation. Therefore, this project aims to create a model to study diffusion of protein from hydrogel by using green …

Roles Of Bacterial Toxin Receptor And Piwi-Interacting Rna Of Pacific White Shrimp Penaeus Vannamei In Response To Vibrio Parahaemolyticus-Causing Acute Hepatopancreatic Necrosis Disease Infection, Waruntorn Luangtrakul

Chulalongkorn University Theses and Dissertations (Chula ETD)

Acute hepatopancreatic necrosis disease (AHPND) or Early mortality syndrome (EMS) caused by virulent strains of Vibrio parahaemolyticus AHPND (VPAHPND) has been a leading cause of significant losses of shrimp production. In this study, we aim to explore the response and the role of immune-related gene/protein and small RNA in the VPAHPND-infected shrimp. This research was divided into 2 parts. In the first part, we identified candidate genes of VPAHPND toxin receptor and further confirmed its function as a VPAHPND toxin receptor. APN has been reported as a Cry toxin from Bacillus thuringiensis, whose structure is similar to VPAHPND toxin. Therefore, …

Metabolite Profiling And Gene Expression Analysis In Starch And Sugar Metabolisms Of Ripening Durian Durio Zibethinus Pulp, Lalida Sangpong

Chulalongkorn University Theses and Dissertations (Chula ETD)

Durian (Durio zibethinus L.), an economically important fruit of Southeast Asian countries, expresses unique flavor at the edible ripe stage, resulting from metabolic changes during the ripening process. Nevertheless, the study in such modulations is still limited. In the first manuscript, we profiled metabolites from unripe and ripe pulps of durian cv. Monthong using capillary electrophoresis- (CE) and gas chromatography- (GC) time-of-flight mass spectrometry (TOF/MS) and obtained the total 94 ripening-related metabolites from a combination of the two datasets, of which we focused on flavor-related metabolites. For sugar, sucrose is the most abundant sugar that increased during ripening, and the …

Protective Effects Of Tiger Milk Mushroom (Lignosus Rhinocerus) Extracts On Oxidative Stress-Induced Neurotoxicity And Aging In Ht22 Cells And C. Elegans, Parinee Kittimongkolsuk

Chulalongkorn University Theses and Dissertations (Chula ETD)

Lignosus rhinocerus (LR) or Tiger Milk Mushroom, a fork medicinal mushroom, has been reported for several pharmacological effects including asthma treatment, anti-inflammatory, anti-proliferative, immuno-modulating effects, promote neurite outgrowth in PC-12 cells, anti-HIV-1 activity, and antioxidants properties. However, the antioxidant properties have only focus on in vitro and no or few studies have reported their protective effects in mouse hippocampal (HT22) cells and Caenorhabditis elegans (C. elegans). This study aims to investigate the neuroprotective effect of three extracts of LR against oxidative stress in both HT22 cells and. C. elegans as well as longevity in C. elegans. In HT22 cells, we …

Generation Of Neural Stem Cells (Nscs) From Human Fibroblasts Using Qq-Modified Sox2 And Neurod1 Proteins, Abdullah Ibrahim Alhomoudi

Wayne State University Theses

The generation of induced neural stem cells (iNSCs) and induced neuronal cells (iNCs) from somatic cells provides new avenues for basic research and potential transplantation therapies for neurological diseases. However, clinical applications must consider the tumor formation capabilities of the implanted cells, the inability of iNCs to self-renew in culture, and reprogramming methods that use retroviral transduction which permanently alter genetic network of the cells. Here we report the generation of protein-induced neural stem cells (piNSCs) from human dermal fibroblasts using QQ-SON pluripotent reprogramming as a tool to quickly reset the time clock of the human somatic fibroblasts to a …

Can The Carbon And Nitrogen Isotope Values Of Offspring Be Used As A Proxy For Their Mother's Diet? Using Foetal Physiology To Interpret Bulk Tissue And Amino Acid Δ15N Values, Nico Lübcker, John P. Whiteman, Seth D. Newsome, Robert P. Millar, P.J. Nico De Bruyn

Biological Sciences Faculty Publications

The measurement of bulk tissue nitrogen (δ¹⁵N) and carbon isotope values (δ¹³C) chronologically along biologically inert tissues sampled from offspring can provide a longitudinal record of their mothers' foraging habits. This study tested the important assumption that mother-offspring stable isotope values are positively and linearly correlated. In addition, any change in the mother-offspring bulk tissues and individual amino acids that occurred during gestation was investigated. Whiskers sampled from southern elephant seal pups (Mirounga leonina) and temporally overlapping whiskers from their mothers were analyzed. This included n = 1895 chronologically subsampled whisker segments for bulk …

Characterization Of The Tsc/Dyrk1a Interaction, Supriya Joshi

Theses and Dissertations

The Tuberous sclerosis complex (TSC) includes TSC1, TSC2 and the TBC1D7 subunits that together function as a principal inhibitor of the mTOR protein kinase complex 1 (mTORC1). mTORC1 is a master regulator of cell growth and proliferation that responds to signaling cues such as growth factors and nutrient availability. Proteomic studies in our lab revealed an interaction between the TSC subunits and DYRK1A, a ubiquitous protein kinase encoded by a gene located in the Down syndrome (DS) region on human chr21. In this study, we sought to validate the interaction of the TSC components with DYRK1A and to determine the …

Characterizing Glycosaminoglycan And Glycosaminoglycan-Oligo Interactions With Cancer Stem Cells And Their Regulators, Rahaman Navaz Gangji

Theses and Dissertations

Tumor metastasis and recurrence account for more than 90% of cancer-associated deaths and are thought to occur due to a small subpopulation of neoplastic cells known as cancer stem cells (CSCs). Because CSCs are resistant to conventional chemotherapy, there is a critical need to develop CSC-targeting agents. Microenvironmental components, especially glycosaminoglycans (GAGs), play a key role in regulating CSC phenotype. GAGs such as heparan sulfate (HS) interact with hundreds of proteins and influence a variety of biological processes including tumor proliferation, angiogenesis, metastasis and stem cell biology.

Recently, the Desai and Patel laboratories have demonstrated that a hexasaccharide form of …

Regulation Of Vascular And Lymphatic Endothelial Barrier Function By Sphingosine-1-Phosphate, Jacqueline Bellaci

Theses and Dissertations

Endothelial cells play an important role in maintaining homeostasis, and disruption of the endothelial barrier have been linked to a number of different pathologies, including cancer metastasis. The vascular endothelial barrier is known to be regulated by a variety of factors, including sphingosine-1-phosphate (S1P), a bioactive lipid metabolite, which has been shown to improve barrier function. However, most studies to date have been focused on the vascular endothelium, and the regulation of the lymphatic endothelial barrier has not been thoroughly investigated. We found that prior to any stimulation the lymphatic endothelial cells (LECs) had a lower trans-endothelial resistance (TER) in …

Structural Features Contributing To Methylenedioxypyrovalerone (Mdpv) Analog Inhibition Of The Human Dopamine Transporter, Zachary R. Spires

Theses and Dissertations

Monoamine transporters (MATs) are transmembrane neurotransmitter sodium symporters consisting of the dopamine, norepinephrine, and serotonin transporters: DAT, NET, and SERT, respectively. Following an action potential and neurotransmitter vesicular release, MAT-mediated reuptake of these neurotransmitters in the presynaptic membrane contributes to the termination of monoaminergic neurotransmission. MATs are the targets of many drugs providing therapeutic benefits or taken recreationally. One of these drugs is methylenedioxypyrovalerone (MDPV), an abused synthetic cathinone that is a high potency uptake inhibitor of the human DAT (hDAT). In contrast, MDPV is a low potency uptake inhibitor of the Drosophila melanogaster dopamine transporter (dDAT). The reasons for …

The Impact Of Aging And Mechanical Injury On Alveolar Epithelial And Macrophage Responses In Acute Lung Injury And Inflammation, Michael S. Valentine

Theses and Dissertations

Patients with severe lung pathologies, such as Acute Respiratory Distress Syndrome (ARDS), often require mechanical ventilation as a clinical intervention; however, this procedure frequently exacerbates the original pulmonary issue and produces an exaggerated inflammatory response that potentially leads to sepsis, multisystem organ failure, and mortality. This acute lung injury (ALI) condition has been termed Ventilator-Induced Lung Injury (VILI). Alveolar overdistension, cyclic atelectasis, and biotrauma are the primary injury mechanisms in VILI that lead to the loss of alveolar barrier integrity and pulmonary inflammation. Stress and strains during mechanical ventilation are believed to initiate alveolar epithelial mechanotransduction signaling mechanisms that contribute …

Structural And Functional Characterization Of Cazyme And Cazyme-Related Proteins From: Bacteroides Thetaiotaomicron And Porphyromonas Gingivalis: Two Abundant Colonizers Of The Human Microbiome, James Stevenson

Theses and Dissertations (Comprehensive)

The human body consists of approximately 30 trillion cells, while non-human microbes that reside on and within the body outnumber human somatic cells by a factor of 1.3 – 2.3. The interplay between our cells and those of the colonizing microorganisms affect physiology in a multitude of ways, both beneficial and detrimental. Microbes found in the oral cavity, such as the Red Complex member Porphyromonas gingivalis, are associated with pathology, namely periodontal diseases including gum deterioration, tooth decay, and loss of underlying alveolar bone. At the other end of the gastrointestinal tract, microbes such as Bacteroides thetaiotaomicron are found …