Physical Sciences and Mathematics Commons^™

Molecular Mechanism Of Dream Dimerization And Interactions With A Non-Physiological Ligand Zn2+., Maria D. Santiago Estevez

FIU Electronic Theses and Dissertations

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensor proteins and it is involved in several processes in the brain.

Zinc has been shown to bind to recoverin, with submillimolar affinity. Based on the high sequence homology between the NCS family, it is proposed that DREAM can also serve as an intracellular target for Zn²⁺. Fluorescence and CD studies confirm that zinc binds to DREAM with a of K_d = 4 µM, triggering changes in the proteins’ tertiary structure.

The calcium association to DREAM leads to the formation of a Ca^{2+ …}

Consequences Of Non-Physiological Ligands Binding To Dream On Its Secondary And Tertiary Structures And Interactions With Intracellular Partners., Samiol Azam

FIU Electronic Theses and Dissertations

Downstream regulatory antagonist modulator (DREAM) is 29 kDa neuronal calcium sensor (NCS) protein which is expressed in the hippocampus of the brain. DREAM is a multifunctional protein which is involved in a wide spectrum of biological processes, including regulation of potassium current in brain, modulation of pain, presenilin-1 processing, regulation of memory and learning. Exposure to toxic metals such as Pb2+ and Cd2+ have been associated with different types of neurodegenerative diseases. However, how these toxic metals induce neurotoxicity remains an active area of research. In this study, utilizing a combination of spectroscopic and calorimetric techniques, we demonstrated binding of …

Topological Markers For Subcellular Chemical Mapping Using Tof-Sims, Anthony Castellanos

FIU Electronic Theses and Dissertations

Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) is a powerful mass spectrometry imaging technique that is uniquely suited to the study of small and challenging biological samples. Mass spectrometry imaging permits the simultaneous detection of chemical species without a priori knowledge of a sample. In the present work, the use of endogenous chemical species, three-dimensional dual-beam depth profiling, and multi-modal mass spectrometry approaches are utilized to increase the specificity and reliability of sub-cellular chemical assignments. In addition, the use of these complimentary indicators serve as useful quality controls to ensure optimal preparation of samples for a high-vacuum environment. In the study …

Development Of Microfluidic Paper-Based Analytical Devices For The Detection Of Low Explosives, Kathryn R. Chabaud

FIU Electronic Theses and Dissertations

Incidents of terrorism have been on the rise despite increased government regulation of explosives. These regulations and the internet have made the application of improvised explosive devices (IEDs) such as fireworks and smokeless powders more prevalent. These devices contain low explosives which are easier to acquire than high explosives, which are more closely monitored. Pipe bombs typically contain smokeless powders, which are mostly comprised of energetics such as nitroglycerine (NG) and organic additives such as diphenylamine. Another type of easily obtainable material containing low explosives is pyrotechnics. Pyrotechnics are usually made up of a mixture of inorganic chemical oxidizers and …

Novel Mips-Parabens Based Spe Stationary Phases Characterization And Application, Angela Tartaglia, Abuzar Kbir, Songhul Ulusoy, Halil Ibrahim Ulusoy, Giuseppe Maria Merone, Fabio Savini, Cristian D'Ovidio, Ugo De Grazia, Serena Gabrielli, Fabio Maroni, Pantaleone Bruni, Fausto Croce, Dora Melucci, Kenneth G. Furton, Marcello Locatelli

Department of Chemistry and Biochemistry

In this work, the synthesis, characterization, and application of novel parabens imprinted polymers as highly selective solid-phase extraction (SPE) sorbents have been reported. The imprinted polymers were created using sol–gel molecular imprinting process. All the seven parabens were considered herein in order to check the phase selectivity. By means of a validated HPLC-photodiode array detector (PDA) method all seven parabens were resolved in a single chromatographic run of 25 min. These SPE sorbents, in-house packed in SPE empty cartridges, were first characterized in terms of extraction capability, breakthrough volume, retention volume, hold-up volume, number of theoretical plates, and retention factor. …

Molecular Mass Growth Through Ring Expansion In Polycyclic Aromatic Hydrocarbons Via Radical–Radical Reactions, Long Zhao, Ralf I. Kaiser, Wenchao Lu, Bo Xu, Musahid Ahmed, Alexander N. Morozov, Alexander Mebel, A. Hasan Howlader, Stanislaw F. Wnuk

Department of Chemistry and Biochemistry

Polycyclic aromatic hydrocarbons (PAHs) represent key molecular building blocks leading to carbonaceous nanoparticles identified in combustion systems and extraterrestrial environments. However, the understanding of their formation and growth in these high temperature environments has remained elusive. We present a mechanism through laboratory experiments and computations revealing how the prototype PAH—naphthalene—can be efficiently formed via a rapid 1-indenyl radical—methyl radical reaction. This versatile route converts five- to six-membered rings and provides a detailed view of high temperature mass growth processes that can eventually lead to graphene-type PAHs and two-dimensional nanostructures providing a radical new view about the transformations of carbon in …

Development Of Dna Methylation Markers To Infer Age, Smoking Status And Body Fluid Types For Forensic Application, Hussain J.H. Alghanim

FIU Electronic Theses and Dissertations

In forensic investigations, biological evidences have great potential to place a suspect at the scene of a crime. However, in many cases suspect(s) usually can’t be identified with any local or national databases. In such challenging cases, police need a tool that can provide investigative leads. Thus, different genetic biomarkers including DNA methylation markers have been developed for different phenotypic traits to serve as source of intelligence information to investigators in cases of unknown DNA profiles. In this project, novel sets of DNA methylation markers were developed for the forensic estimation of human age, determining the smoking status and body …

Functionalized Aptamers For Detection Of Small-Molecule Targets, Haixiang Yu

FIU Electronic Theses and Dissertations

Aptamers have recently gained considerable attention for small-molecule detection in diverse applications such as drug identification, medical diagnostics, and environmental monitoring. However, the performance of aptamer-based sensors has been greatly limited by the low target affinity and responsiveness of small-molecule binding aptamers. This dissertation describes several novel aptamer engineering and isolation strategies to remedy this problem. Specifically, we first develop a generally applicable strategy to engineer split aptamers containing two binding domains termed cooperative-binding split aptamers (CBSAs). CBSAs exhibit higher target binding affinity and are far more responsive in terms of target-induced split aptamer assembly compared to single-domain parent split …

A Randomized Cross-Over Trial Comparing The Effect Of Intramuscular Versus Intranasal Naloxone Reversal Of Intravenous Fentanyl On Odor Detection In Working Dogs, Jennifer L. Essler, Paige G. Smith, Danielle Berger, Elizabeth Gregorio, M. Ross Pennington, Amanda Mcguire, Kenneth G. Furton, Cynthia M. Otto

Department of Chemistry and Biochemistry

Fentanyl is a potent opioid used clinically as a pain medication and anesthetic but has recently seen a sharp rise as an illicit street drug. The potency of fentanyl means mucous membrane exposure to a small amount of the drug can expose first responders, including working canines, to accidental overdose. Naloxone, a fast-acting opioid antagonist administered intranasally (IN) or intramuscularly (IM) is currently carried by emergency personnel in the case of accidental exposure in both humans and canines. Despite the fact that law enforcement relies heavily on the olfactory abilities of canine officers, the effects of fentanyl exposure and subsequent …

Frailty, Cognitive Decline, Neurodegenerative Diseases And Nutrition Interventions, Maria Elena Gomez-Gomez, Sara C. Zapico

Department of Chemistry and Biochemistry

Currently the human population is aging faster. This leads to higher dependency rates and the transformation of health and social care to adapt to this aged population. Among the changes developed by this population is frailty. It is defined as a clinically detectable syndrome, related to the aging of multiple physiological systems, which prompts a situation of vulnerability. The etiology of frailty seems to be multifactorial and its pathophysiology is influenced by the interaction of numerous factors. Morley et al. propose four main mechanisms triggering the frailty: atherosclerosis, sarcopenia, cognitive deterioration and malnutrition, with their respective metabolic alterations. Malnutrition is …

Development Of Improved Extraction/Purification Methods And Comprehensive Screening/Confirmation By Lc-Qqq-Ms Analysis For Novel Psychoactive Substances, Ashley N. Kimble

FIU Electronic Theses and Dissertations

The presence of novel psychoactive substances (NPS) in forensic casework poses major difficulties for detection, since there are many structural variations of NPS circulating in the street market. There are currently no comprehensive screening/confirmatory/quantitation methods available that encompass the majority of NPS encountered in forensic toxicology. A major issue faced with developing such a method is that full validation is extremely time consuming. The use of a liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) method makes the detection of a large number of NPS possible because of high selectivity and sensitivity.

This research included four main tasks: 1) development …

Comprehensive Analysis Of Synthetic Cannabinoids And Metabolites In Oral Fluid By Liquid Chromatography Triple Quadrupole Mass Spectrometry, Carmen T. Mulet

FIU Electronic Theses and Dissertations

According to NIDA, synthetic cannabinoids (SC) include man-made, mind-altering chemicals. Their typically rapid metabolism and instability in biological matrices requires fast analytical methods for detection and confirmation; therefore, the use of oral fluid (OF) matrix is advantageous because collection is fast and non-invasive. The use of liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) and online solid phase extraction (SPE) for sample pretreatment minimizes bias and facilitates low level detection.

This study demonstrates the first use of online SPE for sample pretreatment in the comprehensive analysis of 100 SC and metabolites in OF by LC-QqQ-MS. The method was validated according the …

Uv And Visible Light-Driven Production Of Hydroxyl Radicals By Reduced Forms Of N, F, And P Codoped Titanium Dioxide, A. M. Abdullah, Miguel A. Gracia-Pinilla, Suresh C. Pillai, Kevin E. O'Shea

Department of Chemistry and Biochemistry

The photocatalytic activities of reduced titanium dioxide (TiO₂) materials have been investigated by measuring their ability to produce hydroxyl radicals under UV and visible light irradiation. Degussa P25 TiO₂ was doped with nitrogen (N), fluorine (F), and/or phosphorus (P) and then subjected to surface modification employing a thermo-physicochemical process in the presence of reducing agent sodium borohydride (NaBH₄). The reduced TiO₂ materials were characterized by a number of X-ray, spectroscopic and imaging methods. Surface doping of TiO₂ was employed to modulate the band gap energies into the visible wavelength region for better overlap …

Uv And Visible Light-Driven Production Of Hydroxyl Radicals By Reduced Forms Of N, F, And P Codoped Titanium Dioxide, A. M. Abdullah, Miguel A. Gracia-Pinella, Suresh C. Pillai, Kevin E. O'Shea

Department of Chemistry and Biochemistry

The photocatalytic activities of reduced titanium dioxide (TiO₂) materials have been investigated by measuring their ability to produce hydroxyl radicals under UV and visible light irradiation. Degussa P25 TiO₂ was doped with nitrogen (N), fluorine (F), and/or phosphorus (P) and then subjected to surface modification employing a thermo-physicochemical process in the presence of reducing agent sodium borohydride (NaBH₄). The reduced TiO₂ materials were characterized by a number of X-ray, spectroscopic and imaging methods. Surface doping of TiO₂ was employed to modulate the band gap energies into the visible wavelength region for better overlap …

Label-Free, Visual Detection Of Small Molecules Using Highly Target-Responsive Multimodule Split Aptamer Constructs, Yingping Luo, Haixiang Yu, Obtin Alkhamis, Yingzhu Liu, Xinhui Lou, Boyang Yu, Yi Xiao

Department of Chemistry and Biochemistry

Colorimetric aptamer-based sensors offer a simple means of on-site or point-of-care analyte detection. However, these sensors are largely incapable of achieving naked-eye detection, because of the poor performance of the target-recognition and signal-reporting elements employed. To address this problem, we report a generalizable strategy for engineering novel multimodule split DNA constructs termed "CBSAzymes" that utilize a cooperative binding split aptamer (CBSA) as a highly target-responsive bioreceptor and a new, highly active split DNAzyme as an efficient signal reporter. CBSAzymes consist of two fragments that remain separate in the absence of target, but effectively assemble in the presence of the target …

5′,8-Cyclopurine Lesions In Dna Damage: Chemical, Analytical, Biological, And Diagnostic Significance, Chryssostomos Chatgilialoglu, Carla Ferreri, Nicholas E. Geacintov, Marios G. Krokidis, Yuan Liu, Annalisa Masi, Vladimir Shafirovich, Michael A. Terzidis, Pawlos S. Tsegay

Department of Chemistry and Biochemistry

Purine 5′,8-cyclo-2′-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5′R and 5′S, for each 2′-deoxyadenosine and 2′-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2′-deoxyribose units generating C5′ radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic …

Towards Trigonal Prismatic Hexanuclear Copper Complexes For Catalytic Water Oxidation, David I. Kreiger

FIU Electronic Theses and Dissertations

Our planet is running out of energy resources and traditional renewables are not easily transported nor will be sufficient to cover the void left by fossil fuels. Therefore, a new energy storage system needs to be adopted. The medium with highest energy density that is readily available is hydrogen from water. However, to access hydrogen, water needs to be oxidized and water oxidation catalysts (WOCs) will be required for widespread adoption. In pursuit of WOCs, several hexauclear copper complexes were synthesized and characterized, including the isolation and characterization of a mixed-valent Cu₆ (formally, Cu^II₅Cu^III) …

Phenolate Derivatives Of Octanuclear Iron-Oxo-Pyrazolato Complexes; Enantiomeric Resolution Of T-Symmetry Species, Konstantinos Lazarou

FIU Electronic Theses and Dissertations

Octanuclear ferric complexes involving achiral phenolates of the general formula [Fe₈(μ-O)₄(μ-pz)₁₂(R-C₆H₄O)₄], (R = 4-NO₂, 4-CN, 4-F, 4-Cl, 4-I, 4-CH₃O, and 3-NO₃), where synthesized and characterized by single crystal X-ray crystallography, elemental analysis, ¹H-NMR, UV-Vis, IR, ⁵⁷Fe Mössbauer spectroscopy and electrochemical methods (cyclic and differential pulse voltammetry). Results indicate that the nature of the phenolate substituents allows the fine tuning of the spectroscopic data of the compounds.

Subsequently, this project focused on the resolution of the racemic …

Oil Hydrocarbon Degradation By Caspian Sea Microbial Communities, John I. Miller, Stephen Techtmann, Julian Fortney, Nagissa Mahmoudi, Dominique Joyner, Jiang Liu, Scott Olesen, Eric Alm, Adolfo Fernandez, Piero Gardinali, Nargiz Garajayeva, Faig S. Askerov, Terry C. Hazen

Department of Chemistry and Biochemistry

The Caspian Sea, which is the largest landlocked body of water on the planet, receives substantial annual hydrocarbon input from anthropogenic sources (e.g., industry, agriculture, oil exploration, and extraction) and natural sources (e.g., mud volcanoes and oil seeps). The Caspian Sea also receives substantial amounts of runoff from agricultural and municipal sources, containing nutrients that have caused eutrophication and subsequent hypoxia in the deep, cold waters. The effect of decreasing oxygen saturation and cold temperatures on oil hydrocarbon biodegradation by a microbial community is not well characterized. The purpose of this study was to investigate the effect of oxic and …

Oil Hydrocarbon Degradation By Caspian Sea Microbial Communities, John I. Miller, Stephen Techtmann, Julian Fortney, Nagissa Mahmoudi, Dominique Joyner, Jiang Liu, Scott Olesen, Eric Alm, Adolfo Fernandez, Piero Gardinali, Nargiz Garajayeva, Faig S. Askerov, Terry C. Hazen

Department of Chemistry and Biochemistry

The Caspian Sea, which is the largest landlocked body of water on the planet, receives substantial annual hydrocarbon input from anthropogenic sources (e.g., industry, agriculture, oil exploration, and extraction) and natural sources (e.g., mud volcanoes and oil seeps). The Caspian Sea also receives substantial amounts of runoff from agricultural and municipal sources, containing nutrients that have caused eutrophication and subsequent hypoxia in the deep, cold waters. The effect of decreasing oxygen saturation and cold temperatures on oil hydrocarbon biodegradation by a microbial community is not well characterized. The purpose of this study was to investigate the effect of oxic and …

Amyz1: A Novel Α-Amylase From Marine Bacterium Pontibacillus Sp. Zy With High Activity Toward Raw Starches, Wei Fang, Saisai Xue, Pengjun Deng, Xuecheng Zhang, Xiaotang Wang, Yazhong Xiao, Zemin Fang

Department of Chemistry and Biochemistry

No abstract provided.

Amyz1: A Novel Α-Amylase From Marine Bacterium Pontibacillus Sp. Zy With High Activity Toward Raw Starches, Wei Fang, Saisai Xue, Pengjun Deng, Xuecheng Zhang, Xiaotang Wang, Yazhong Xiao, Zemin Fang

Department of Chemistry and Biochemistry

Background

Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch.

Results

A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli …

Investigating The Structural Complexity Of The Uch Knot Protein Using Trapped Ion Mobility Spectrometry – Mass Spectrometry, Shirley Hernandez

Undergraduate Research at FIU (URFIU) Conference

Knot proteins are rare proteins whose backbones adopt knotted configurations with a wide range of topologically interesting features [Lim, 2015]. They were regarded as structural impossibilities, until they were observed embedded in naturally-occurring proteins in the early 2000s [Taylor, 2000; Lim, 2003]. Ubiquitin carboxyl-terminal hydrolase, or UCH, is one of the most complex knot proteins studied to-date. UCH contains a knot structure composed of 5 crossings around the polypeptide chain (52 knot). Humans have four UCHs: UCH-L1, UCH-L3, UCH-L5 and BAP1. The UCH-L1 in particular is found in abundance in the nerve cells of the brain and is thought to …

Accessibility Of The Heme-Pocket In Hexa-Coordinate Globins, Maria Jose Santiago

Undergraduate Research at FIU (URFIU) Conference

Studies of fluorescence analogues of neuroglobin and cytoglobin to characterize conformational changes upon ligand binding Neuroglobin (Ngb) and cytoglobin (Cygb) are newly discovered hexacoordinate members of the globin family. Ngb is expressed in nerve cells and the retina and its main role is to protect cells under hypoxic–ischemic conditions. Cygb is expressed almost in all tissues in our body and it have tumor suppression role. Here we report a preparation of fluorescent analogues of Ngb and Cygb by replacing the non-fluorescent Fe protoporphyrin IX with a fluorescent probe, Zn protoporphyrin IX (ZnPPIX). The accessibility of the heme binding pocket in …

Novel Synthesis Of The Homologs Of Natural Nucleosides, Erika Lozano

Undergraduate Research at FIU (URFIU) Conference

Homologated nucleoside analogs are frequently studied for their antiviral activity. They are effective for the treatment of infectious species like the herpes virus, varicella-zoster viruses and retroviruses like HIV (Human Immunodeficiency Virus). Successful synthesis of these organic compounds has been achieved in the past through the transformation of the alcohol functionality into a carbonyl, often followed by Wittig reaction or Ardnt-Eistert homologation. Additionally, others have generated the homologated sugar moiety and then proceeded with the coupling to nucleobases. Researchers from Palacky University recently developed novel methodology for the homologation of primary and secondary alcohols. They generated the homologated esters of …

Synthesis Of (E)-2- Chlorhomovinyl Ribose To Examine The Mechanism Of 6-Chlorhomovinyl Adenosine, Mattew Pasteris

Undergraduate Research at FIU (URFIU) Conference

Nucleosides function as important agents in fundamental human metabolism and provide a broad range of activity in resulting modification of these nucleosides as functional prodrugs. Various modified nucleosides exhibit inhibitory activity towards enzymes and carry the possibility of containing anti-viral and anti-cancer properties. These properties are often exhibited by modifying the nucleobase moiety or the sugar moiety as synthesized novel nucleosides. In understanding my project aim, the sugar moiety of 6-chlorohomovinyl adenosine will be synthesized as the novel nucleoside (E)-2-chlorovinylribose to examine the nucleoside as the active component of 6-chlorohomovinyl adenosine. 6-chlorohomovinyl adenosine 1 demonstrates an active component …

Synthesis, Structure And Properties Of Two Symmetrical Oxorhenium(V) Dimers Derived From 4-Chloro Pyrazole, Juliana Cazzaniga

Undergraduate Research at FIU (URFIU) Conference

Oxorhenium(V) complexes are of considerable interest due to their multifaceted applications in the field of catalysis and in radiopharmaceuticals. The properties of these oxo-complexes can be “finetuned” through appropriate ligand choices. Our lab has been engaged in the development of coordination chemistry with pyrazoles due to their versatile binding modes. Pyrazole can bind a metal center in either monodentate or bridging bidentate fashion. Moreover, the 3, 4, and 5- positions on pyrazole ring offer the opportunity for suitable functionalization for desired applications. Although there has been a plethora of research work on transition metal coordination complexes involving pyrazole(s), there is …

The Application Of Ion Mobility Spectrometry And Ultrahigh Resolution Spectrometry For Analysis Of Anti- Microbial Peptides From Frog Skin Secretions, Benjamin Bokor

Undergraduate Research at FIU (URFIU) Conference

Frog skin secretions of antimicrobial peptides (AMPs) are of increasing interest due to the protection they impart against microbes. Such peptides offer the prospect of new and efficient sources of bacterial, fungal, and viral treatments. Recently, an epizootic of chytridiomycosis from the fungal pathogen Batrachochytrium dendrobatidis (Bd) caused the death of many amphibian species in the Andes. However, certain species of frogs such as Hypsiboas gladiator were able to survive. It is speculated that the survival of Hypsiboas gladiator is due to AMPs secreted from their skin.

The identification and characterization of these peptides is necessary for understanding the antimicrobial …

Gas Phase Synthesis Of [4]-Helicene, Long Zhao, Ralf I. Kaiser, Bo Xu, Utuq Ablikim, Wenchao Lu, Musahid Ahmed, Mikhail M. Evseev, Eugene K. Bashkirov, Valeriy N. Azyazov, Marsel V. Zagidullin, Alexander N. Morozov, A. Hasan Howlader, Stanislaw F. Wnuk, Alexander M. Mebel, Dharati Joshi, Gregory Veber, Felix R. Fischer

Department of Chemistry and Biochemistry

A synthetic route to racemic helicenes via a vinylacetylene mediated gas phase chemistry involving elementary reactions with aryl radicals is presented. In contrast to traditional synthetic routes involving solution chemistry and ionic reaction intermediates, the gas phase synthesis involves a targeted ring annulation involving free radical intermediates. Exploiting the simplest helicene as a benchmark, we show that the gas phase reaction of the 4-phenanthrenyl radical ([C14H9]•) with vinylacetylene (C4H4) yields [4]-helicene (C18H12) along with atomic hydrogen via a low-barrier mechanism through a resonance-stabilized free radical intermediate (C18H13). This pathway may represent a versatile mechanism to build up even more complex …

The Ability Of Narcotic Detection Canines To Detect Illegal Synthetic Cathinones (Bath Salts), Vanquilla Shellman Francis, Howard K. Holness, Kenneth G. Furton

Department of Chemistry and Biochemistry

Twelve certified narcotic detection canines were tested for their ability to detect confiscated illegal synthetic cathinones (bath salts). These canine teams were randomly assigned to two different groups and each group imprinted on one of two types of bath salts, ethylone and alpha-pyrrolidinovalerophenone (α-PVP), over the period of 1 month; while simultaneously documenting the imprinting procedure. The newly imprinted canines were validated by field testing and found to not only detect the imprinted bath salt to which they were trained, but they were able to detect other bath salts. The imprinting procedure and results are the first scientifically validated studies …