EM - IUEM - Técnicas Laboratoriais Forenses
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- Toxicological profile of JWH-018 and JWH-018 N-(3-Hydroxypentyl) metabolite in the HEK 293 T cell linePublication . Sultan, Haider; Quintas, Alexandre; Dias, Mário JoãoThe rise in use of synthetic cannabinoids as herbal smoking mixtures has been attributed to a number of reasons; primary among them being their “legality” and their promotion as being safer than the natural alternatives. As their emergence has been quite recent, their pharmacological and toxicological profiles are not known completely. One of the earliest synthetic cannabinoid to be detected in these herbal mixtures was JWH-018. In the present study, the cell toxicity of JWH-018 and its N-(3-hydroxypentyl) metabolite was investigated using human cell line HEK-293 T. The cell viability was assessed using the MTT assay after treatment with standard JWH-018 and its N-hydroxymetabolite in a concentration range between 1ng/ml to 5000ng/ml of both respectively. The results show that the highest concentration of 5000ng/ml shows statistically significant decrease in cell viability for both the parent compound and the metabolite when compared to cell controls. In addition, the metabolite also showed a statistically significant decrease in cell viability at a concentration of 2500ng/ml. There were also significant differences observed between the smaller concentrations (1ng/ml and 10ng/ml) when compared to the highest concentration (5000ng/ml) for both the parent compound and metabolite. The cytotoxic effect was seen at levels which are above the expected tissue levels after use of JWH-018 either by ingestion or smoking. The cytotoxicity of both the parent compound and the metabolite highlights the adverse effects these drugs may have with consequent need for further research and regulation.
- Application of SERS with raman reporter-labelled metallic nanoparticles for latent fingermark enhancementPublication . Echeonwu, Bobmanuel Chimaroke; Croxton, Ruth S.The importance of human fingermark identification and individualization for forensic purposes cannot be overemphasized. Consequently, the need for robust and effective techniques explains the continued and intensified research in this field of forensics despite the fact that there are already numerous operational techniques available to forensic identification experts. Novel approaches have recently been adopted to understand the chemistry of latent fingermarks and produce reagents targeting some of these constituents of latent fingermarks. Nanotechnology and surface-enhanced Raman spectroscopy (SERS) are gaining more attention in this area of endeavor, where nanoparticle-based reagents are increasingly being developed and optimized for application in latent fingermark enhancement and chemical imaging of such latent fingermarks. In this study, surface-enhanced Raman spectroscopy has been used to investigate the applicability of Raman reporter-labeled metallic nanoparticles to the enhancement of latent fingermarks. Rhodamine 6G and 4-mercaptobenzoic acid have been investigated as Raman reporters adsorbed on either gold or silver nanoparticles. The functionalized nanoparticles were deposited onto latent fingermark samples and characteristic Raman vibrational bands of the reporter molecules when adsorbed on nanoparticles (1076 cm-1 and 1583 cm-1 for 4-mercptobenzoic acid; 610 cm-1, 1360 cm-1 1505 cm-1 and 1648 cm-1 for rhodamine 6G) were monitored and showed very low variability of peak position between samples (coefficient of variation generally < 0.5%). Adsorption of reporter molecules on nanoparticles was characterized using UV-vis absorption spectroscopy as well as the behaviour of characteristic Raman shifts of the molecules which gave an indication of successful adsorption. Rhodamine 6G in this study however had a weak adsorption onto silver nanoparticles as a result of suboptimal amounts of the dye molecule used. Preferential deposition of the Raman reporter-labeled nanoparticles on fingermark ridges was also observed with fingermarks deposited on non-porous surface (glass slide). This study demonstrates the potential for SERS chemical imaging of latent fingermarks based on characteristic SERS vibrational bands from reporter molecules adsorbed on metallic nanoparticles and deposited onto latent fingermarks.
- Contribution to portuguese urban fire investigations: forensic fire debris analysisPublication . Oliverio, Stefania; Noronha, João Paulo; Carvalho, AntónioFire scene investigation and fire debris analysis (FDA) are two tightly linked and mutually exclusive key elements of a forensic arson investigation. While the fire scene investigation is performed by a trained fire investigator, FDA is a task undertaken by a fire debris analyst, which is normally a forensic chemist with expert knowledge in analytical chemistry. To solve a case efficiently and successfully, cooperation and information- and knowledge-exchange are necessary, especially since this branch of forensic science is defined as one of the most complex and arduous ones due to the highly destructive nature of fires that severely compromises the integrity of the evidence collected at the crime scene. The task of analysing the fire debris for the detection of ignitable liquid residues (ILRs) is further complicated by the large influence and existence of interfering products. Most ignitable liquids (ILs) are made of hydrocarbons, which are also compounds that have been found in the substrate of common household or construction materials as well as being created during the processes of combustion and pyrolysis. The purpose of this study was to identify and characterize background, combustion and pyrolysis products of commonly encountered substrate materials in fire scenes in Portugal and observe the effects on ILRs identification by using headspace solid-phase micro-extraction linked to gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results show that both the background of the substrates and the combustion and pyrolysis products formed during a fire include many compounds that constitute common ILs, such as alkanes and aromatics, that especially in the presence of low amount of ILRs constitute a problem in the conclusive identification of ILs. Even though detailed analysis of the chromatographic and mass-spectral patterns and the combinations of compounds detected in the materials can avoid misinterpretation, in some cases, awareness and knowledge of interfering products is essential.
- Analysis of tobacco and cannabis smoke and its toxicological impact in neuroblastoma tumour cellsPublication . Grafinger, Katharina; Quintas, Alexandre; Costa, Joana Couceiro daThe dried products of Cannabis sativa have been consumed for their psychoactive effects for thousands of years. Almost five hundred different natural cannabinoids could be identified in Cannabis sativa with Δ9-THC being the most psychoactive one. The most effective way of consumption is by smoking, because it avoids digestion and the first pass in the liver. Usually so called "joints" are rolled in a 1:1 ratio of dried cannabis and tobacco filler. The reason for this is that cannabis has a lower burning efficiency and that tobacco is cheaper. Processed Nicotina tabaccum contains about 0.6- 9 % nicotine; an oily, volatile liquid alkaloid. Pyrolysis of tobacco and cannabis leads to the formation of new products whose cytotoxicity is not yet established. This study investigates the effect of smoke of tobacco, cannabis and their mixture in similar conditions as humans uptake it. For production of smoke an in-house developed smoking device was used which trapped the water-soluble and organic-soluble molecules separately. Qualitative analysis of the smoke was performed using GC-MS and measurement of cytotoxicity on neuroblastoma tumour cells SH-SY5Y was performed using the MTT assay. SH-SY5Y cell line selectively expresses Cannabinoid 1 (CB1) receptor and Nicotinic acetylcholine receptor (nAChR). The results allow us to identify several pyrolysated products which are hydroxylated forms of nicotine and Δ9-THC. Moreover, MTT analysis strongly suggests different toxicological impact between smoke of tobacco, cannabis and their mixture. In fact our results points smoke from the mixture of tobacco and cannabis having the higher impact on cell viability followed by the smoke of tobacco which in turn is more toxic than smoke of cannabis, the least toxic compound out of these three. This study shows that at 100 μM cannabis has a cell viability of 80 % while tobacco and the mixture have around 40 %.
- Towards the understanding of the impact of pH on age estimationPublication . Oliveira, Irina Filipa Patrício; Noronha, João Paulo; Pereira, Cristiana PalmelaThe first steps in human remains identification are the determination of age, sex, ancestry and stature. Of these, determination of age is crucial as it facilitates the development of a biological profile be it in archaeological remains or recent forensic matters. Teeth with their hard exterior can protect useful information and store this information for years after death, though environmental conditions may affect the information within. In addition to developing and optimizing a method for quantifying and determining the proportional ratio of amino acids present in teeth by Gas-Chromatography-Mass spectrometry. An introduction of new techniques for the diastereoisomeric separation of D- and Lenantiomers of aspartic acid were tested for use with GC-MS with and achiral column and GCFID with a chiral column. On top of this a novel technique was used to determine the racemic mixture percentage of D- and L- aspartic acid. Circular Dichroism was used to measure the racemic content in samples which were submitted to various temperatures in pH solutions of 3, 5 and 9 for a time interval of 72hours. Results obtained demonstrate occurrence of racemization in temperatures of 80 ℃ and above as well as the differential effect produced by acidic and alkaline pH solutions. It would be interesting to discover if with a more sensitive technique, such as GC-MS, and an experiment with a longer time interval a significant change between pH could be demonstrated as well as comparing the difference in racemization rate between free and bound aspartic acid.
- Evaluation of current and novel methods for the storage of DNA-extractsPublication . Sjoukema, Pieterjan; Barroso, Maria Helena; Carvalho, RaquelSecure preservation of DNA-extracts has been an issue at forensic laboratories for a long time. DNA-extracts are commonly stored at either -20°C or -80°C, but storage at these temperatures is not without risk of failure, environment unfriendly and costly. The Laboratório de Polícia Científica (LPC; Forensic Science Laboratory) of the Polícia Judiciária (PJ; Judicial Police) in Lisbon, Portugal is currently looking into new ways of storing their DNA-extracts at room temperature. Both DNAstable® and GenTegra-DNA®, two commercially available products for the storage of DNA-extracts at ambient temperatures, have been evaluated. DNA samples with extremely low concentrations of DNA (0.1 ng/μL, 0.05 ng/μL and 0.025 ng/μL) and DNA-extracts extracted from buccal swabs with the SwabSolution™ kit (Promega, Madison, USA) have been stored in standard polypropylene tubes, DNAstable® tubes and GenTegra-DNA® tubes for 8 days at four different conditions, i.e., -20°C, 4°C, at room temperature and at 60°C. Besides this, randomly selected DNA-extracts that have been stored at -20°C since 2006, 2009 or 2013 have been reanalyzed with the currently at the LPC used techniques in order to assess the level of degradation after long-term storage at -20°C. Results showed that DNA degrades over time at -20°C, with recovery levels found as low as 20% after three years of storage at -20°C. DNAstable® and GenTegra-DNA® both showed to be able to preserve DNA dried at room temperature and 60°C. Given the short period of storage full DNA-profiles have also been obtained from samples stored in standard polypropylene tubes at room temperature. Significant differences were found between DNA-extracts stored at 60°C with either DNAstable® or GenTegra-DNA® and those stored in unprotected tubes, showing that both DNAstable® and GenTegra-DNA® are capable of storing DNA at room temperature for long periods of time with a similar or even higher level of protection compared to those achieved with conventional storage at -20°C. Nevertheless, more research is needed before a switch can be made from frozen storage at -20°C to the storage of DNA-extracts at room temperature with products such as DNAstable® and GenTegra-DNA®.
- The cytotoxic effect of THJ-018 in human neuroblastoma SH-SY5Y cellsPublication . Khosasih, Vivia; Quintas, Alexandre; Costa, Joana Couceiro daSince its first appearing on 2004, synthetic cannabinoidsm the market of the so-called recreational drug, have regained popularity among adolescents and young adults. Current in vitro studies show that synthetic cannabinoids have stringer binding affinity to cannabinoids 1 (CB1) receptor than the Δ9-tetrahydrocannabinal. Therefore, it suggested that synthetic cannabinoids possess stronger psychotropic effect than cannabis. Albeit it has been increasingly abused as the legal alternative of cannabis, the pharmacological and toxicological profiles of synthetic cannabinoids remais poorly understood. One of the earliest synthetic cannabinoids to be identified was JWH-018. This study was performed to explore the toxicological effect of synthetic cannabinoids THJ-018 as the legal analog to JWH-018 on human cells model. SH-SY5Y cell lineage was used as the human cell model in this study since it has been reported to express cannabinoids 1 receptor (CB1). Seven designated concentrations of synthetic cannabinoids THJ-018, such as 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM and 100 μM were exposed for 24 hours to human neuroblastoma SH-SY5Y cell lineage. The cell viability was evaluated by performing the 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay. The MTT results on SH-SY5Y cells show a statiscally significant increase on cell viability (* p<0.05, ** p<0.01 or *** p<0.001) for the concentration of 10, 25, 50, 75 and 100 μM of THJ-018. This results indicated that the THJ-018 is not toxic to the SH-SY5Y cell line. Furthermore, the cytotoxicity effect of THJ-018 combustion product, which is performed to evaluate the mimicry of the process of smoking as the common route of administration for synthetic cannabinoids, show a decrease of cell viability when SH-SY5Y cell exposed with THJ-018 combustion product.
- “Charlie” (buphedrone/ethcathinone) behavioural patterns after stimulated binge administrationPublication . Sola Sancho, Sofía; Lopes, Álvaro TeixeiraSince the entry of XXI century, the consume, availability and variety of new psychoactive substances also called “legal highs” “legal drugs” or “design drugs,” have been rising enormously. This tendency has increased the concern of both, health care providers and legal authorities about this family of drugs. This unique study is a first toxicological approach to a drug named “Charlie”. Effects for a repeated administration of a singular dose of 45mg/kg has been assessed. “Charlie” is a combination of two synthetic cathinones (Buphedrone and Ethcathinone) with no toxicological or behavioural studies done, until the date. For developing the study a binge administration was performed in a ten days study with the combination of the drug with ethanol in a dose of 2g/kg. For the toxicological assessment, adolescent male CD1 mice were used and four behavioural tests were chosen, Functional Observational Battery (FOB), Open Field (OF) test, Marble burying test and T-maze test. Results showed the significant differences between controls and mice under the influence of “Charlie” or under the mixture of “Charlie” and ethanol at all performed tests. The combination cathinone-ethanol showed mayor effects in the variables for FOB and OF than when the drug was administered alone ad different behavioural patterns in Marble and T-Maze tests. The study of the weights and food and water intake before and during the experiments was also assessed and results pointed the variations in weight and food intake during the days of the experiment suffering a marked decrease in those days. With this study it is demonstrated the influence of “Charlie” in the behaviour and physiological conditions of the individuals under its effects by using behavioural tasks for the assess and opening a new “file” in the cathinones behavioural assessment.
- A contribution for medico-legal estimation of age in portuguese population using the London atlas of tooth developmentPublication . Pavlovic, Strahinja; Pereira, Cristiana Palmela; Santos, Rui Filipe Vargas de SousaChronological age estimation from the dental parameters is becoming one of the fundaments of Forensic Science. Numerous body traits can be used for this purpose, but dental parameters are considered the most reliable ones. The London atlas of tooth development is the most recent developed method for dental age estimation and represents a modification of the previous older methods. The aim of this study was to evaluate the accuracy of the London atlas in a sample of a Portuguese population. The sample included 736 radiographic images (498 females and 238 males) of Portuguese origin, patients of Dental Clinic of Superior Institute of Health Sciences Egas Moniz and Dental Medicine Faculty, Univeristy of Lisbon. The age range of the individuals was between 3 and 24 years. Several tests were applied in order to evaluate the London atlas. All the statistical analysis were based on the paired t-test. The results showed that there was no statistically significant difference between left and right side of the jaw (p > 0.05). Both sides showed an average overestimation of age by one month approximately. Moreover, the significant difference between chronological and estimated age was not observed in the females. However, the significant difference was observed in a sample coming from males (right: p = 0.008; left: p = 0.003). Additionally, there was a significant difference in a population coming from Institute Egas Moniz (p <0.05). Our results indicated that the London atlas can be potentially used as a tool for age estimation, especially in cases when all teeth finished their development. However, the difference between sexes clearly suggests that separate charts should be made for each sex. Further studies, which will have as a final goal development of a new method for age estimation using dental parameters, are needed.
- Production of HIV-1 polypeptidesPublication . Bojadzija, Gorenka; Barroso, Maria HelenaHIV causes acquired immunodeficiency syndrome known as AIDS. HIV-1 and HIV-2 are the etiologic agents of AIDS, and despite very similar structural and genomic organization, HIV-1 and HIV-2 infections are very different regarding immunological and clinical outcomes. Neutralizing antibody responses, both autologous and heterologous is more frequent in HIV-2 than in HIV-1 infection. HIV-1 can escape naturally occurring antibodies (Nabs) due to sequence variability and epitope masking by glycosylation, which can cause difficulties and challenges in vaccine development. Even though is still debatable, region in HIV-2 that may contain broadly neutralizing epitotes is the V3 region. Furthermore, in HIV-2 envelope complex C2 and C3 regions are unprotected and therefore under strong diversifying selection, which can indicate that, like in HIV-1, they may harbor neutralizing epitopes. The hypothesis beneath this project is that an chimeric envelope surface glycoprotein containing the C2, V3 and C3 regions of HIV-2 and the remaining regions of env of recombinant AE subtype HIV-1 92TH019 might elicit a broadly neutralizing response against both HIV-1 and HIV-2. Aim of this project is the production of HIV-1 polypeptides (V1V2, C5, C3V4C4, V4) derived from recombinant AE subtype HIV-1 92TH019 which will be further used in immunization boosting in mice with vaccine, as well as detection of antibodies in mice. In this study these regions were successfully amplified and purified. Annealing temperatures that gave best amplification results were 49°C (C3V4C4 region), 50°C (V1V2 region), 50°C (C5 region) and 49°C (V4 region). To my best knowledge this is first time that these regions were amplified from this subtype. Due to the lack of time, cloning and expression of these regions was not possible, however research will be continued.