Innovazioni nel campo dell'ElettroEncefalografia

Il funzionamento del cervello umano, organo responsabile di ogni nostra azione e pensiero, è sempre stato di grande interesse per la ricerca scientifica. Dopo aver compreso lo sviluppo dei potenziali elettrici da parte di nuclei neuronali in risposta a stimoli, si è riusciti a graficare il loro andamento con l'avvento dell'ElettroEncefaloGrafia (EEG). Tale tecnologia è entrata a far parte degli esami di routine per la ricerca di neuropsicologia e di interesse clinico, poiché permette di diagnosticare e discriminare i vari tipi di epilessia, la presenza di traumi cranici e altre patologie del sistema nervoso centrale. Purtroppo presenta svariati difetti: il segnale è affetto da disturbi e richiede un'adeguata elaborazione tramite filtraggio e amplificazione, rimanendo comunque sensibile a disomogeneità dei tessuti biologici e rendendo difficoltoso il riconoscimento delle sorgenti del segnale che si sono attivate durante l'esame (il cosiddetto problema inverso). Negli ultimi decenni la ricerca ha portato allo sviluppo di nuove tecniche d'indagine, di particolare interesse sono la ElettroEncefaloGrafia ad Alta Risoluzione (HREEG) e la MagnetoEncefaloGrafia (MEG). L'HREEG impiega un maggior numero di elettrodi (fino a 256) e l'appoggio di accurati modelli matematici per approssimare la distribuzione di potenziale elettrico sulla cute del soggetto, garantendo una migliore risoluzione spaziale e maggior sicurezza nel riscontro delle sorgenti neuronali. Il progresso nel campo dei superconduttori ha reso possibile lo sviluppo della MEG, che è in grado di registrare i deboli campi magnetici prodotti dai segnali elettrici corticali, dando informazioni immuni dalle disomogeneità dei tessuti e andando ad affiancare l'EEG nella ricerca scientifica. Queste nuove tecnologie hanno aperto nuovi campi di sviluppo, più importante la possibilità di comandare protesi e dispositivi tramite sforzo mentale (Brain Computer Interface). Il futuro lascia ben sperare per ulteriori innovazioni.

SAR ed effetti termici su Cheratinociti sottoposti a Radiofrequenze

Negli ultimi anni, la popolazione è stata esposta a vari tipi di campi elettromagnetici generati da strumentazioni elettroniche e dispositivi di telecomunicazione. In questa tesi, si valutano SAR ed effetti termici prodotti da tre antenne patch a radiofrequenza sia su Cheratinociti (cellule dell'epidermide) in vitro che sull'epidermide umana in vivo caratterizzata in un modello multistrato contenente tessuti biologici. Le antenne progettate hanno frequenze di risonanza di 1.8 e 2.4 GHz, tipiche delle bande utilizzate rispettivamente da LTE (Long Term Evolution, la più recente evoluzione degli standard di telefonia mobile cellulare) e dalle moderne tecnologie Wi-Fi, e di 60 GHz, propria delle cosiddette onde millimetriche. Vengono valutati quindi il SAR (Specific Absorption Rate, grandezza che fornisce una misura dell'assorbimento delle onde da parte dei tessuti biologici) e le variazioni di temperatura prodotte dall'applicazione del campo elettromagnetico: ciò viene realizzato attraverso l'equazione del calore stazionaria e, nel caso dell'epidermide in vivo, con la Bioheat Equation, che contempla anche la circolazione sanguigna ed il calore generato nei processi metabolici che avvengono nell'organismo.

Attivita antimicrobica di oli essenziali incapsulati in chitosano in hamburger di bovino

La sperimentazione aveva il fine di valutare l’attività antimicrobica di oli essenziali di timo e cannella in hamburger di bovino. In primo luogo è stata fatta una caratterizzazione di questi oli mediante analisi GC-MS-SPME, da cui è emerso che le molecole a maggiore attività antimicrobica (rispettivamente timolo/carvacrolo ed aldeide cinnamica) erano presenti a concentrazioni piuttosto basse. Successivamente sono state valutate MIC e MBC nei confronti di microrganismi patogeni di interesse alimentare. Infine, tali oli sono stati incapsulati in chitosano per valutare un eventuale effetto sinergico e favorire un rilascio graduale degli oli. Il processo di incapsulazione ha mostrato una efficienza piuttosto bassa (solo il 25% dell’olio aggiunto veniva effettivamente incapsulato), per cui non è stato possibile utilizzare nanoparticelle “caricate” con quantità di olio tali da inibire significativamente lo sviluppo microbico in hamburger di carne bovina. Infatti, l’effetto sinergico chitosano/olio essenziale è stato in grado di ridurre solo lievemente il carico di enterobatteri durante la conservazione refrigerata, soprattutto nei campioni confezionati in atmosfera modificata. Una prova effettuata utilizzando i due oli in forma libera ad elevata concentrazione (1000 mg/kg), la cannella si è dimostrata molto più efficace del timo, riducendo il carico cellulare di enterobatteri a fine shelf-life, con effetto più evidente nei campioni di hamburger confezionati in atmosfera modificata (riduzione di un ciclo logaritmico). Inoltre, essa è risultata in grado di ridurre lievemente lo sviluppo di lieviti e funghi. Concludendo, gli oli essenziali usati in questa sperimentazione, poiché caratterizzati da una bassa concentrazione di molecole ad elevata attività antimicrobica (timolo/carvacrolo per il timo, aldeide cinnamica per la cannella), non sono stati in grado di prolungare in modo significativo la shelf-life di hamburger di bovino, nemmeno se incapsulati in chitosano.

Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling

There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here, we report the findings of a plasma metabolomic investigation of HCC patients by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm, and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate, and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also conducted by ultraperformance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). By this method, we found that HCC was also associated with reduced levels of lysophosphocholines and in 4 of 20 patients with increased levels of lysophosphatidic acid [LPA(16:0)], where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GC-MS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GC-MS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.

Characterization of Halyomorpha halys (brown marmorated stink bug) biogenic volatile organic compound emissions and their role in secondary organic aerosol formation.

The formation of aerosols is a key component in understanding cloud formation in the context of radiative forcings and global climate modeling. Biogenic volatile organic compounds (BVOCs) are a significant source of aerosols, yet there is still much to be learned about their structures, sources, and interactions. The aims of this project were to identify the BVOCs found in the defense chemicals of the brown marmorated stink bug Halymorpha halys and quantify them using gas chromatography-mass spectrometry (GC/MS) and test whether oxidation of these compounds by ozone-promoted aerosol and cloud seed formation. The bugs were tested under two conditions: agitation by asphyxiation and direct glandular exposure. Tridecane, 2(5H)-furanone 5-ethyl, and (E)-2-decenal were identified as the three most abundant compounds. H. halys were also tested in the agitated condition in a smog chamber. It was found that in the presence of 100-180 ppm ozone, secondary aerosols do form. A scanning mobility particle sizer (SMPS) and a cloud condensation nuclei counter (CCNC) were used to characterize the secondary aerosols that formed. This reaction resulted in 0.23 microg/ bug of particulate mass. It was also found that these secondary organic aerosol particles could act as cloud condensation nuclei. At a supersaturation of 1%, we found a kappa value of 0.09. Once regional populations of these stink bugs stabilize and the populations estimates can be made, the additional impacts of their contribution to regional air quality can be calculated.

Characterization of Halyomorpha Halys (Brown Marmorated Stink Bug) Biogenic Volatile Organic Compound Emissions and Their Role in Secondary Organic Aerosol Formation

The formation of aerosols is a key component in understanding cloud formation in the context of radiative forcings and global climate modeling. Biogenic volatile organic compounds (BVOCs) are a significant source of aerosols, yet there is still much to be learned about their structures, sources, and interactions. The aims of this project were to identify the BVOCs found in the defense chemicals of the brown marmorated stink bug Halymorpha halys and quantify them using gas chromatography-mass spectrometry (GC/MS) and test whether oxidation of these compounds by ozone-promoted aerosol and cloud seed formation. The bugs were tested under two conditions: agitation by asphyxiation and direct glandular exposure. Tridecane, 2(5H)-furanone 5-ethyl, and (E)-2-decenal were identified as the three most abundant compounds. H. halys were also tested in the agitated condition in a smog chamber. It was found that in the presence of 100-180 ppm ozone, secondary aerosols do form. A scanning mobility particle sizer (SMPS) and a cloud condensation nuclei counter (CCNC) were used to characterize the secondary aerosols that formed. This reaction resulted in 0.23 mu g/bug of particulate mass. It was also found that these secondary organic aerosol particles could act as cloud condensation nuclei. At a supersaturation of 1%, we found a kappa value of 0.09. Once regional populations of these stink bugs stablilize and the populations estimates can be made, the additional impacts of their contribution to regional air quality can be calculated. Implications: Halymorpha halys (brown marmorated stink bugs) are a relatively new invasive species introduced in the United States near Allentown, Pennsylvania. The authors chemically speciated the bugs' defense pheromones and found that tridecane, 5-ethyl-2(5H)-furanone, and (E)-2-decenal dominated their emissions. Their defense emissions were reacted with atmospherically relevant concentrations of ozone and resulted in 0.23 g of particulate matter per emission per bug. Due to the large population of these bugs in some regions, these emissions could contribute appreciably to a region's PM2.5 (particulate matter with an aerodynamic diameter 2.5 m) levels.

Endocannabinoid content in fetal bovine sera - unexpected effects on mononuclear cells and osteoclastogenesis

The major endocannabinoids (ECs) arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) and related N-ethanolamines act as full and partial agonists at CB(1), CB(2), GPR55, PPAR and TRPV1 receptors to various degrees. These receptors are also expressed in immune cells like monocytes/macrophages where they regulate different cellular processes. In this study, potentially bioactive lipids in fetal bovine sera (FBS) were quantified by GC/MS. We found that several commercial FBS contain ECs and bioactive amounts of 2-AG (250-700 nM). We show that residual 2-AG from FBS can activate primary macrophages and increase migration and RANKL-stimulated osteoclastogenesis. Furthermore, 2-AG high-content sera specifically upregulated LPS-stimulated IL-6 expression in U937 cells. Polymyxin B beads may be used to selectively and efficiently remove 2-AG from sera, but not arachidonic acid and N-ethanolamines. In conclusion, 2-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual 2-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Primary male osteoporosis is associated with enhanced glucocorticoid availability

OBJECTIVE: While systemic glucocorticoids compromise bone metabolism, altered intracellular cortisol availability may also contribute to the pathogenesis of primary male osteoporosis (MO). The objective of this study was to assess whether intracellular cortisol availability is increased in MO due to a distorted local cortisol metabolism. METHODS: Forty-one patients with MO were compared with age- and BMI-matched non-osteoporotic subjects after excluding overt systemic hypercortisolism (N = 41). Cortisol, cortisone and the respective tetrahydro-, 5α-tetrahydro- and total cortisol metabolites were analysed by GC-MS in 24 h urine. Apparent 11β-hydroxysteroid dehydrogenase (11β-HSD) enzyme activities, excretion of cortisol metabolites and calcium, and fractional urinary calcium excretion were assessed and related to BMD. RESULTS: Fractional and total urinary calcium excretion negatively correlated with BMD at all (P < 0.05) and at three of five (P < 0.05) measurement sites, respectively. While systemic cortisol was unchanged, apparent 11β-HSD enzyme activity in MO patients (P < 0.01) suggested increased intracellular cortisol availability. Total and fractional urinary calcium excretion was higher, with apparent 11β-HSD enzyme activities consistent with an enhanced intracellular cortisol availability (P < 0.05). CONCLUSION: Apparent 11β-HSD enzyme activities consistent with increased intracellular cortisol availability correlated with urinary calcium loss and reduced bone mineral density in MO. The changes in 11β-HSD activity were associated with both the fractional calcium excretion, suggesting altered renal calcium handling, and the absolute urinary calcium excretion. Both mechanisms could result in a marked bone calcium deficiency if insufficiently compensated for by intestinal calcium uptake.

Urinary metabolites and antioxidant products of exogenous melatonin in the mouse

Exogenous melatonin is widely used for sleep disorders and has potential value in neuroprotection, cardioprotection and as an antioxidant. Here, a novel method is described for the determination of melatonin and six metabolites in mouse urine by use of LC-MS/MS and GC-MS. LC-MS/MS is used for the measurement of melatonin, N1-acetyl-5-methoxykynuramine (AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 6-hydroxymelatonin (6-HMEL), while GC/MS is used for the determination of N-[2-(5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (2-OMEL) and cyclic 3-hydroxymelatonin (3-HMEL) with detection limits on column of 0.02-0.5 pmol, depending on the metabolite. Following oral administration of melatonin to mice, a 0-24 hr urine collection revealed the presence of melatonin (0.2% dose), 6-HMEL (37.1%) and NAS (3.1%) comprising >90% of the total metabolites; AMK and AFMK were also detected at 0.01% each; 2-OMEL was found at 2.2% of the dose, which is >100 times more than the AMK/AFMK pathway, and comprises >5% of the melatonin-related material detected in mouse urine. 3-HMEL was largely found as a sulfate conjugate. These studies establish sensitive assays for determination of six melatonin metabolites in mouse urine and confirm the potential for antioxidant activity of melatonin through the identification in vivo of AMK and AFMK, ring-opened metabolites with a high capacity for scavenging reactive oxygen species.

The inhibition of bone resorption in rats treated with (-)-menthol is due to its metabolites

(-)-Menthol, a monoterpene from Mentha species (Lamiaceae), has been shown to inhibit bone resorption in vivo by an unknown mechanism. In the present study, plasma and urine profiling in rats determined by GC/MS demonstrate that (-)-menthol is extensively metabolized, mainly by hydroxylation and carboxylation, and excreted in the urine, in part as glucuronides. In plasma, very low concentrations of (-)-menthol metabolites were detected after a single dose of (-)-menthol, whereas after repeated treatment, several times higher concentrations and long residence times were measured. In contrast, the elimination of unchanged (-)-menthol was increased by repeated treatment. (-)-Menthol, at concentrations found in plasma, did not inhibit bone resorption in cultured mouse calvaria (skull). However, the neutral metabolites of (-)-menthol, extracted from urine of rats fed with (-)-menthol, inhibited bone resorption in vitro, the concentrations being at plasma level or higher. These results suggest that not (-)-menthol itself, but one or several of its neutral metabolites inhibit the bone resorbing cells in vivo.

Application of headspace solid phase microextraction to qualitative and quantitative analysis of tobacco additives in cigarettes

Cigarettes may contain up to 10% by weight additives which are intended to make them more attractive. A fast and rugged method for a cigarette-screening for additives with medium volatility was developed using automatic headspace solid phase microextraction (HS-SPME) with a 65 microm carbowax-divinylbenzene fiber and gas chromatography-mass spectrometry (GC-MS) with standard electron impact ionisation. In three runs, each cigarette sample was extracted in closed headspace vials using basic, acidic and neutral medium containing 0.5 g NaCl or Na2SO4. Furthermore, the method was optimized for quantitative determination of 17 frequently occurring additives. The practical applicability of the method was demonstrated for cigarettes from 32 brands.

CHARACTERIZATION OF ASPHALT MATERIALS CONTAINING BIO OIL FROM MICHIGAN WOOD

The objective of this research is to develop sustainable wood-blend bioasphalt and characterize the atomic, molecular and bulk-scale behavior necessary to produce advanced asphalt paving mixtures. Bioasphalt was manufactured from Aspen, Basswood, Red Maple, Balsam, Maple, Pine, Beech and Magnolia wood via a 25 KWt fast-pyrolysis plant at 500 °C and refined into two distinct end forms - non-treated (5.54% moisture) and treated bioasphalt (1% moisture). Michigan petroleum-based asphalt, Performance Grade (PG) 58-28 was modified with 2, 5 and 10% of the bioasphalt by weight of base asphalt and characterized with the gas chromatography-mass spectroscopy (GC-MS), Fourier Transform Infra-red (FTIR) spectroscopy and the automated flocculation titrimetry techniques. The GC-MS method was used to characterize the Carbon-Hydrogen-Nitrogen (CHN) elemental ratio whiles the FTIR and the AFT were used to characterize the oxidative aging performance and the solubility parameters, respectively. For rheological characterization, the rotational viscosity, dynamic shear modulus and flexural bending methods are used in evaluating the low, intermediate and high temperature performance of the bio-modified asphalt materials. 54 5E3 (maximum of 3 million expected equivalent standard axle traffic loads) asphalt paving mixes were then prepared and characterized to investigate their laboratory permanent deformation, dynamic mix stiffness, moisture susceptibility, workability and constructability performance. From the research investigations, it was concluded that: 1) levo, 2, 6 dimethoxyphenol, 2 methoxy 4 vinylphenol, 2 methyl 1-2 cyclopentandione and 4-allyl-2, 6 dimetoxyphenol are the dominant chemical functional groups; 2) bioasphalt increases the viscosity and dynamic shear modulus of traditional asphalt binders; 3) Bio-modified petroleum asphalt can provide low-temperature cracking resistance benefits at -18 °C but is susceptible to cracking at -24 °C; 3) Carbonyl and sulphoxide oxidation in petroleum-based asphalt increases with increasing bioasphalt modifiers; 4) bioasphalt causes the asphaltene fractions in petroleum-based asphalt to precipitate out of the solvent maltene fractions; 5) there is no definite improvement or decline in the dynamic mix behavior of bio-modified mixes at low temperatures; 6) bio-modified asphalt mixes exhibit better rutting performance than traditional asphalt mixes; 7) bio-modified asphalt mixes have lower susceptibility to moisture damage; 8) more field compaction energy is needed to compact bio-modified mixes.

REMEDIATION OF TETRACYCLINE FROM WATER SOURCES USING VETIVER GRASS (Chrysopogon zizanioides L. Nash) AND TETRACYCLINE-TOLERANT ROOT-ASSOCIATED BACTERIA

Antibiotics are emerging contaminants worldwide. Due to insufficient policy regulations, public awareness, and the constant exposure of the environment to antibiotic sources has created a major environmental concern. Wastewater treatment plants (WWTP) are not equipped to filter-out these compounds before the discharge of the disinfected effluent into water sources (e.g., lakes and streams) and current available technologies are not equipped to remediate these compounds from environmental sources. Hence, the challenge remains to establish a biological system to remove these antibiotics from wastewater. An invitro hydroponic remediation system was developed using vetiver grass (Chrysopogon zizanioides L. Nash) to remediate tetracycline (TC) from water. Comparative metabolomics studies were conducted to investigate the metabolites/pathways associated with tetracycline metabolism in plants and TC-degrading bacteria. The results show that vetiver plants effectively uptake tetracycline from water sources. Vetiver root-associated bacteria recovered during the hydroponic remediation trial were highly tolerant to TC (as high as 600 ppm) and could use TC as a sole carbon and energy source. Growth conditions (pH, temperature, and oxygen requirement) for TC-tolerant bacteria were optimized for higher TC remediation capability from water sources. The plant (roots and shoots) and bacterial species were further characterized for the metabolites produced during the TC degradation process using GC-MS to identify the possible biochemical mechanism involved. Also, the plant root zone was screened for metabolites/enzymes that were secreted during antibiotic degradation and could potentially enhance the degradation process. The root zone was selected for this analysis because this region of the plant has shown a greater capacity for antibiotic degradation compared to the shoot zone. The role of antioxidant enzymes in TC degradation process revealed glutathione-S-transferase (GSTs) as an important group of enzymes in both plant and bacteria potentially involved in TC degradation process. Metabolomics results also suggest potential GST activity in the TC remediation/ transformation process used by plants. This information could be useful in gaining insights for the application of biological remediation systems for the mitigation of antibiotics from waste-water.

Determination of menthol in plasma and urine of rats and humans by headspace solid phase microextraction and gas chromatography--mass spectrometry

A method for the determination of menthol and menthol glucuronide (M-G) after enzymatic hydrolysis in plasma and urine of rats and humans was developed using headspace solid phase microextraction and gas chromatography-mass spectrometry in the selected ion monitoring mode (HS-SPME/GC-MS). The assay linearity for plasma ranged from 5 to 1000 ng/ml. The limit of quantification (LOQ) in plasma was 5 ng/ml. The intra- and inter-day precision for menthol and M-G were < or = 18.1% R.S.D. at the LOQ and < or = 4.0% at higher concentrations. Menthol and M-G were determined in rat and human plasma and urine after administration of menthol.