Browsing by Subject "Regeneration"
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Item Chemical biology studies of neuroregenerative small molecules using Caenorhabditis elegans(2015-05) Zlotkowski, Katherine Hannah; Liu, Hung-wen, 1952-; Siegel, Dionicio R.; Pierce-Shimomura, Jon; Keatinge-Clay, Adrian; Dong, GuangbinThe debilitating effects of spinal cord injury can be attributed to a lack of regeneration in the central nervous system. Identification of growth-promoting pathways, particularly ones that can be controlled by small molecules, could provide significant advancements in regenerative science and lead to potential treatments for spinal cord injury. The biological investigations of neuroregenerative small molecules, specifically the natural products clovanemagnolol and vinaxanthone, have been expanded to a whole organism context using the nematode Caenorhabditis elegans (C. elegans) as a tool for these studies. A straightforward assay using C. elegans was developed to screen for compounds that promote neuronal outgrowth in vivo. This outgrowth assay was then used to guide the design of chemically edited analogs of clovanemagnolol that maintained biological activity while possessing structures amenable to further modification for mechanism of action studies. Pull-down experiments using affinity reagents synthesized from a neuroactive structural derivative, clovanebisphenol, and the C. elegans proteome combined with mass spectrometry-based protein identification and genetic recapitulation using mutant C. elegans identified the putative protein target of the small molecule as a kinesin light chain, KLC-1. Furthermore, the small molecule-promoted regeneration of injured neurons in vivo was studied using laser microsurgery to cut specific axons in C. elegans followed by treatment with a library of analogs of the growth-promoting natural product vinaxanthone. Enhanced axonal regeneration was observed following small molecule treatment and the results were used to determine the structure-activity relationship of vinaxanthone, which may guide future development of potential drug candidates for the treatment of spinal cord injury.Item Controlling neural cell behavior with electric field stimulation across a conductive substrate(2012-12) Nguyen, Hieu Trung 1980-; Schmidt, Christine E.Electrical stimulation of tissues induces cell alignment, directed migration, extended processes, differentiation, and proliferation, but the mechanisms involved remain largely unknown. To reveal effects of electric fields (EF) through the media on cell behavior, voltage (7.45 – 22 V), current density (36 – 106 mA/cm2), duration (2 – 24 hrs), and alternating currents (AC, 2 – 1000 Hz) were varied independently when exposed to cell cultures. It was determined that current density and duration are the primary attribute Schwann cells respond to when an EF is applied through the media. This implies that the number of charges moving across the cell surface may play a key role in EF-induced changes in cell behavior. Identical conditions were used to stimulate cells grown on the surface of a conductive substrate to examine if a scaffold can provide structural and EF cues. The effects of an EF through the substrate were examined by placing a protein gel on the surface during stimulation and observing the morphology of subsequent cell cultures and the physical topology of the gel. EFs were shown to create Ca2+ redistribution across gels and subtle changes in collagen I fibril banding. Stimulated gels were able to induce perpendicular Schwann cell alignment on newly seeded cultures days after initial EF exposure, and the cell response decreased when seeded at longer times, indicating the effects of EF on the matrix environment has a relaxation time. These findings were then integrated into a biodegradable, electrically conductive polypyrrole-poly-ε-caprolactone polymer developed by collaborators. Dorsal root ganglia placed in matrix gels on top of conducting polymer exhibited significantly longer axons when stimulated with DC and AC signals. The overall results demonstrate that EFs have a significant effect on the extracellular environment. The broad implication of this data grants researchers with the ability to physically and metabolically control cell behavior with EFs, including improved wound healing or reduced cancer metastasis.Item The development of immunomodulatory approaches to restore skeletal muscle function after injury(2015-05) Rybalko, Viktoriya Yurievna; Farrar, Roger P.; Suggs, Laura J; Brothers, Robert M; Thompson, Wesley J; Adamo, Martin LEfficient restoration of skeletal muscle function after severe injury is a major goal of intervention therapies. Ischemia/reperfusion (I/R) injury to skeletal muscle leads to exaggerated inflammatory response and significant ultrastructural tissue damage slowing restoration of muscular structure and function. Herein, we used animal model of tourniquet-induced ischemia/reperfusion injury (TK-I/R) to test the effects of exogenously delivered growth factors and cells on skeletal muscle regeneration. The delivery of PEGylated fibrin along with stromal cell derived factor-1α and/or insulin-like growth factor-I into acutely injured muscle, differentially affected functional muscle regeneration. These data suggest that local balance and release kinetics of growth factors in the tissue microenvironment can significantly impact the success of skeletal muscle repair. Cell-mediated treatment of I/R-injured muscle demonstrated significant tissue regeneration using adoptively transferred and in vitro polarized macrophages. Functional activation status of transplanted macrophage populations impacted the outcome of muscle repair. We showed that increasing macrophage populations at the site of injury in temporally regulated manner is beneficial for efficient recovery of muscle force and function.Item The effect of peroneal nerve relocation on skeletal muscle regeneration within an extracellular matrix seeded with mesenchymal stem cell populations derived from bone marrow and adipose tissue(2009-08) Tierney, Matthew Timothy; Farrar, Roger P.; Suggs, Laura J.Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials are capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation, reinnervation and revascularization of the injured area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1.0 x 1.0 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle-derived extracellular matrix (ECM). Constructs were seeded with bone marrow-derived (BMSCs) or adipose-derived stem cells (ADSCs) and the peroneal nerve was relocated over the implanted ECM. Creation of the defect resulted in a functional impairment of the LGAS, only capable of producing 85.1 ± 4.1% of the force generated in the contralateral LGAS following ECM implantation. A significant increase in specific tension (SPo) was seen in all groups following the nerve relocation procedure when compared to their corresponding cellular treatment without nerve relocation (p < 0.05). Histological quantification revealed significant increases in cellular content and blood vessel density in the top and bottom regions of ECM implants seeded with BMSCs (p < 0.05). The nerve relocation procedure significantly increased these same variables within the middle region of the ECM when compared to all groups lacking this treatment (p < 0.05). The presence of regenerating myofibers was immunofluorescently confirmed using antibodies against desmin, myosin heavy chain and laminin, while their developmental state was substantiated by the presence of central nuclei. These data corroborate a therapeutic effect of BMSCs on skeletal muscle regeneration within the ECM implant that was not seen following ADSC injection. Furthermore, the nerve relocation procedure stimulated an increased cellular and vascular growth within the middle region of the construct, likely the cause of improved functional output.Item Heart Regeneration: An Evolutionary Tale(2013-01-17) Mahmoud, Ahmed Ibrahim; Sadek,Hesham A., M.D., Ph.D.Lower vertebrates like urodele amphibians and teleost fish retain a robust cardiac regenerative capacity throughout their life, a phenomenon that is mediated through the proliferation of pre-existing cardiomyocytes. The adult mammalian heart lacks any meaningful endogenous regenerative response following injury. However, embryonic mammalian cardiomyocytes are proliferative and exit the cell cycle shortly after birth. The question of whether the mammalian heart lacks this regenerative potential or is lost early after birth was not clear. We were able to show that the hearts of 1-day-old mice regenerated following partial surgical resection of the neonatal heart, a phenomenon that is lost within a week after birth. Thus, for a brief period after birth, the mammalian heart appears to have the capacity to regenerate due to the proliferative competency of cardiomyocytes. However, one major unresolved question was whether the neonatal mouse heart could also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. To examine this question, we induced myocardial infarction (MI) in 1-day-old mice by ligating the left anterior descending coronary artery, and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal mouse heart mounted a robust regenerative response, through proliferation of pre-existing cardiomyocytes, which resulted in full functional recovery within 21 days. Moreover, we were able to demonstrate that the neonatal heart is capable of regeneration following mild, but not severe cryoinjury. Therefore, our work identifies a short period of time after birth where the mammalian heart is capable of regeneration following various types of injury. To unravel the molecular mechanisms that regulate the regenerative capacity of the neonatal mammalian heart, we determined that the miR-15 family regulates neonatal heart regeneration through inducing post-natal cardiomyocyte cell cycle arrest. Moreover, inhibition of the miR-15 family at an early post-natal age until adulthood induces cardiomyocyte proliferation in the adult heart and improves left ventricular systolic function following MI. In conclusion, our findings indicate that the mammalian heart harbors a robust regenerative capacity for a short period of time after birth, mediated by proliferation of pre-existing cardiomyocytes, and that the miR-15 family is an important regulator of post-natal cardiomyocytes cell cycle arrest.Item The Identification and Characterization of MKRP, a Novel Kelch Related Protein(2007-12-17) Embree, Laurence Jonathan; Gurry, Daniel J.The cells of adult myofibers in mammals are terminally differentiated and are incapable of division and self-renewal. Regeneration of damaged skeletal muscle tissue is facilitated through the proliferation and differentiation of resident stem cells known as satellite cells . These satellite cells remain quiescent in uninjured tissue, occupying a sublaminar position between the sarcolemma and basal lamina of adult myofibers. In response to trauma, these cells become activated, and proliferate. The activated cells will reestablish the pool of reside quiescent satellite cells, while others will proliferate, migrate by chemotaxis to the area of injury, withdraw from the cell cycle, and differentiate into new myoblasts. These myoblasts will fuse with and repair the injured fibers, or align with each other and fuse to form new fibers. Genes involved in this process should exhibit an altered pattern of expression in skeletal muscle in response to injury by cardiotoxin, and the changes in expression level can be quantified though the measurement of mRNA levels at specified time points following injury. Utilizing transcriptome analysis, we identified a completely novel transcript that is induced in the myogenic progenitor cells following cardiotoxin injury. The novel transcript contained an open reading frame that coded for a protein belonging to the Kelch superfamily. Expression of the transcript was restricted to skeletal muscle lineages during development, and to myogenic progenitor cells and immature myotubes during injury regeneration. Because of its structural identity and restriction to skeletal muscle, the novel transcript was named the myogenic Kelch related protein (MKRP). Knockdown of MKRP expression using siRNA in C2C12 cells revealed an inhibition of both migration and differentiation in myogenic progenitor cells. A yeast two-hybrid screen identified calsarcin-2 as a potential interacting protein, indicating a possible role for MKRP in the calcineurin pathway during myogenic differentiation.Item Influence of insulin-like growth factor-I on skeletal muscle regeneration(2012-12) Hammers, David Wayne; Farrar, Roger P; Suggs, Laura J; Adamo, Martin L; Sweeney, H. Lee; Thompson, Wesley J; Ivy, John LSkeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury.Item Lean NOx trap regeneration control strategy on a 1.9L turbocharged diesel(2009-12) Munguia, Nicole; Ezekoye, D K; Matthews, Ronald D.; Justh, David; Longoria, RaulVarious diesel NOx control technologies are being explored today as solutions for minimizing NOx emissions. The Lean NOx Trap is one of two products that offer high NOx conversion efficiency, but the LNT requires a regeneration strategy. The Lean NOx Trap control strategy offers a systematic approach in which engine control parameters are fully utilized to create an exhaust “rich” environment necessary for LNT catalyst regeneration. In the present study EGR throttling, inlet air-throttling and late-injections were the three control parameters tuned for LNT regeneration. The strategy was tested in-house and by GM engineers on a 1.9 L diesel engine coupled to a Diesel Oxidation Catalyst, Diesel Particulate Filter and a Lean NOx Trap. Siemens NOx sensors were used to evaluate the NOx conversion efficiency. It was found that the catalyst did reduce NOx output but was neither efficient at low-loads nor fully-effective throughout the drive-cycle as the DPF did clog as a result of too low a CO/HC ratio. The regeneration mode is open loop and should be closed loop to minimize the additional fueling necessary for creating a rich environment for LNT regeneration. There was no evidence that late injections contributed to regeneration. Instead of late-injections, a more robust, closed-loop control strategy should be explored that uses the air/fuel ratio from the exhaust manifold as the control parameter for maximizing NOx conversion efficiency while minimizing the fuel penalty.Item Mechanical signals for compensatory lung growth assessed by high resolution computed tomography(2008-09-18) Ravikumar, Priya; Hsia, Connie C.W.This dissertation involves the use of high resolution computed tomography (HRCT) to understand the role of intra-thoracic mechanical force and its distribution in regenerative growth in dogs i.e. to quantify lobar lung volumes and density gradients in normal and post-pneumonectomy (following lung resection) lungs. HRCT was used to quantitatively assess regional distribution of lung volume and density gradients among lobes of the lung in order to follow the expansion of remaining lobes following lung resection with a high degree of anatomical precision, and to determine the relationships between lung expansion and alveolar tissue growth. I also extended this work by relating regional lung expansion and growth assessed by radiology to regional alveolar tissue growth assessed by detailed quantitative histology under light and electron microscopy. This study illustrates for the first time a powerful and novel use of in vivo imaging to quantify regional lung distortion and changes in local volume, lung compliance as well as soft tissue density. These changes can be followed non-invasively and serially in a wide range of clinical and investigational applications, such as a) assessing the extent and progression of regional heterogeneity in lung disease or injury; b) assessing local response to treatment or surgical intervention; or c) assessing normal or abnormal patterns of lung growth.Item Removal of Chloride from Wastewater by Advanced Softening Process Using Electrochemically Generated Aluminum Hydroxide(2014-07-23) Mustafa, Syed FaisalWastewater recycle and reuse is limited in many cases by high concentrations of dissolved solids. The majority of dissolved solids can be removed by precipitation. However, chloride is among ionic species that is difficult to remove due to its high solubility. Chloride can be removed from water and wastewater by precipitation as calcium chloroaluminate using advanced softening process. This research was conducted to evaluate chloride removal using electrochemically generated aluminum hydroxide and lime. Kinetics of chloride removal was investigated and the system reached equilibrium within two hours of reaction time indicating that removal kinetics is suitable for practical application of the process. Equilibrium characteristics of chloride removal were characterized. Good chloride removal was obtained at reasonable ranges of lime and aluminum doses. However, the stoichiometry of chloride removal deviated from the theoretical stoichiometry of calcium chloroaluminate precipitation. Analysis of experimental data indicated that this deviation was due to the formation of other solid phases such as tricalcium hydroxyaluminate and tetracalcium hydroxyaluminate. This research obtained the optimum doses of electrochemically produced aluminum hydroxide and lime which achieved maximum chloride removal. An attempt to regenerate and recycles precipitated solids was also investigated.Item Resistance training as a modality to enhance muscle regeneration in a rat skeletal muscle defect(2009-12) Taylor, Daniel Ryan; Farrar, Roger P.; Suggs, LauraTraumatic skeletal muscle injuries that include loss of large amounts of muscle mass are becoming more common in today’s warfare. Traditional treatments often do not prevent long term functional impairments. Using a decellularized extracellular matrix (ECM) as scaffolding to replace lost muscle tissue allows for transmission of force through the injury site, and provides a suitable microenvironment receptive to myofiber growth. Seeding the ECM with progenitor cells improves cellular content in the defect area. Exercise exposes the muscle to improved blood flow as well as higher than normal loading. This results in increased blood vessel density as well as higher levels of cellular content, and near complete restoration of function.Item Skeletal muscle repair following Plantar nerve relocation on an extracellular matrix seeded with mesenchymal stem cells in PEGylated fibrin gel as a treatment model for volumetric muscle loss.(2014-08) Da Costa, Adriana Jocelyn; Farrar, Roger P.The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs.Item Stripper modeling for CO₂ removal using monoethanolamine and piperazine solvents(2011-08) Van Wagener, David Hamilton; Gary, Rochelle T.; Seibert, Frank; Edgar, Thomas F.; Truskett, Thomas M.; Schubert, Craig N.This dissertation seeks to reduce the energy consumption of steam stripping to regenerate aqueous amine used for CO₂ capture from coal-fired power plants. Rigorous rate-based models in Aspen Plus® were developed, and rate-based simulations were used for packed vapor/liquid separation units. Five main configurations with varying levels of complexity were evaluated with the two solvents. 8 m piperazine (PZ) always performed better than 9 m monoethanolamine (MEA). More complex flowsheets stripped CO₂ with higher efficiency due to the more reversible separation. Multi-stage flash configurations were competitive at their optimal lean loadings, but they had poor efficiency at low lean loading. The most efficient configuration was an interheated column, with more effective and distributed heat exchange. It had a secondary benefit of a cooler overhead temperature, so less water vapor exited with the CO₂. Using a rich loading of 0.40 mol CO₂/mol alkalinity in 8 m PZ, the optimal lean loading was 0.28 and the energy requirement was 30.9 kJ/mol CO₂. Case studies were also performed on cold rich bypass and the use of geothermal heat. When cold rich bypass is used with the 2-stage flash and 8 m PZ, it reduces equivalent work by 11% to 30.7 kJ/mol CO₂. PZ benefited the most from cold rich bypass because it had a higher water concentration in the overhead vapor than with MEA. In an advanced 2-stage flash with 8 m PZ, geothermal heat available from 150 down to 100 °C requires 35.5 kJ work/mol CO₂. The heat duty and equivalent work was higher than other optimized configurations, but it would be a valid option if separating the heat source from the steam cycle of a coal-fired power plant was highly valued. Pilot plant campaigns were simulated with the available thermodynamic models. Two campaigns with 8 m PZ were simulated within small deviation from the measured values. The average absolute errors in these campaigns were 2.5 and 2.7%. A campaign with 9 m MEA in a simple stripper demonstrated that the MEA model did not predict the solvent properties well enough to appropriately represent the pilot plant operation.Item Total synthesis and chemical modification of small molecules: a study of axonal regeneration and aryl oxidation(2015-05) Eliasen, Anders Mikal; Willson, C. Grant, 1939-; Siegel, Dionicio R.; Anslyn, Eric; Keatinge-Clay, Adrian; Liu, Hung-Wen; Sessler, JonathanInjuries to the central nervous system are irreversible and debilitating due to the limited regrowth of damaged or severed neurons. Two small molecules, xanthofulvin and vinaxanthone, isolated from P. vinaceum and P. glabrum promote spinal cord regeneration in animal models. It is speculated that these natural products inhibit semaphorin 3A, a chemorepellent that mitigates axonal growth-cone formation. In addition to promoting axonal growth, rats treated with vinaxanthone and xanthofulvin following complete spinal cord transection experienced greater remyelination, increased angiogenesis, attenuated apoptosis, and depressed scaring of the lesion site. The only prior synthesis of vinaxanthone speculated that the xanthone core is constructed via enzyme-catalyzed intermolecular Diels-Alder reaction. We have demonstrated, however, that warming a functionalized acetoacetyl chromone in water, furnishes vinaxanthone in good yield, providing an alternative biosynthetic pathway. With a robust syntheses of both natural products, we determined the protein target of the observed regeneration: succinate receptor 1, providing a new therapeutic target to promote neuronal regeneration. Among the various methods of incorporating oxygen into aryl rings, the direct conversion of a C-H bond into a C-OH bond is ideal. The metal-free hydroxylation of arenes developed in our laboratory, utilizing phthaloyl peroxide, marks the first disclosure of this transformation using mild conditions. Computational and experimental evidence obtained thus far has supported a mechanism involving a diradical intermediate. The reactivity of phthaloyl peroxide was increased by the incorporation of two chlorine atoms onto the ring. To minimize the accumulation of large quantities of peroxide, the optimization of the preparation of the peroxide in flow has been developed. This protocol immediately consumes the peroxide as it is generated. Finally, a new dearomatization reaction has been optimized. This reaction forms two carbon-oxygen bonds and dearomatizes the ring system.Item Tree ferns of Central Veracruz : harvest and conservation implications(2011-08) Vázquez-Domínguez, Othoniel; Doolittle, William Emery; Knapp, Gregory W.; Miller, Jennifer A.Tree ferns are listed as endangered species under Mexican law and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Despite this status, tree ferns are currently being harvested by rural communities, and sold in the form of handicrafts, traditional medicines, and household ornaments In the state of Veracruz, some authors argue that the harvesting of tree fern caudexes (trunks) to obtain a material made out of the fern’s adventitious roots called maquique poses a major threat to tree fern conservation. This thesis systematically explores the effect of harvesting activities on the distribution of tree fern species in the tropical montane cloud forest’s fragmented landscape using vegetative regeneration as a proxy for maquique harvesting. The study was conducted in El Zapotal in the municipality of Acajete and El Riscal in the municipality of Coatepec, two small communities with different land use histories. A census was performed at each site to georeference and document all tree fern individuals, including information on diameter, height and presence/absence of vegetative regenerations due to maquique harvesting per individual tree fern. Four species were present in the study: Alsophila firma, A. tryoniana, Cyathea bicrenata, and C. fulva. ArcGIS Desktop was used to calculate distances from individual tree ferns to trails and rivers, which were regarded as points of access for maquique harvesters. These data were used to infer how and whether maquique affects the distribution and abundance of tree fern species at the two studied sites. This study reports for the first time different forms of vegetative regenerations in Mexican tree fern species such as the resprouting of multiple branches from a single tree fern trunk and also documents different forms of harvesting like the “C cut”. Contrary to common conservation arguments, the study shows that tree ferns can continue to thrive even after a severe environmental modification, such as forest clearing and the establishment of tree plantations. Other results suggest that maquique harvesters operating clandestinely are more likely to target tree ferns with maquique closer to points of access (trails or rivers) rather than according to size. In the long run, this pattern of tree fern harvesting could modify the distribution of tree ferns as they are displaced from areas closer to human access, despite the ability of some tree fern species to regenerate in highly disturbed environments. The discovery of tree fern regenerative properties offers potential for the management of certain tree fern species as umbrella species for conservation in central Veracruz. It suggests that maquique harvesting might be sustainable given careful management and government regulation based on scientific data.