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Identifier 000457342
Title Διερεύνηση φασματικών δεικτών κατάλληλων για την εκτίμηση της παρουσίας γενετικού υλικού σε αρχαιολογικά οδοντικά δείγματα βάσει δεδομένων φασματοσκοπίας σκέδασης Raman και εκπομπής φθορισμού
Alternative Title In search of spectral indicators, indicative of the presence of genetic material in archaeological teeth, based on data obtained via Raman or fluorescence emission spectroscopy, applied in situ
Author Μάμαλη, Αγγελική Ν.
Thesis advisor Άγγλος, Δημήτριος
Reviewer Παυλίδης, Ιωάννης
Πουλακάκης, Νικόλαος
Abstract The study of ancient genetic material (DNA) and proteins in archaeological human or animal remains (bones, teeth) is of particular interest, as it provides information about the age and gender of individuals, the living conditions of individuals or groups of individuals, but also more broadly, migration and interaction among communities and populations. The analysis of archaeological samples presents several challenges, which stem from the fact that biological and bio-organic remains are often found in small or even trace amounts, as well as the presence of exogenous contamination and the large number of samples available for analysis. The analysis of ancient genetic material is based on the application of PCR (Polymerase Chain Reaction, PCR) methods, while paleoproteomics studies rely on the use of liquid chromatography coupled to high-resolution molecular mass spectrometry (Liquid Chromatography Mass Spectrometry, LC-MS) techniques. The aforementioned palaeogenetic or paaleoproteomic methods are of high sensitivity and specificity, but they are characterized by rather high costs and analysis time, while their application causes partial or total destruction of samples, which may have significant archaeological value. These factors make it expensive, time-consuming and often prohibitive to study a sufficient number of samples and thus draw statistically safe conclusions through the analysis. Therefore, it becomes apparent the need to develop alternative analytical approaches that will allow rapid examination of archaeological samples at a preliminary level and then selection of only those with a higher probability of being rich in information for further analysis with the specialized methods of palaeogenetics and/or palaeoproteomics. Based on such an approach it is possible to significantly limit the analysis of samples with negligible content in ancient organic remains. Biological dental remains constitute a source of endogenous organic material (protein and genetic) found mainly in the cementum tissue, which surrounds the part of the tooth that is inside the jaw. It is understood that the endogenous organic material of a tooth is directly affected by the burial conditions (grave, soil, etc.) and the environmental ones in the burial area. In the context of this Master's thesis, a study was carried out on the applicability of Raman scattering spectroscopy and fluorescence emission spectroscopy in terms of estimating the amount of endogenous genetic material in samples of ancient teeth, indirectly, through the determination of their content in protein material. It is noted that the presence of collagen residues in the teeth reflects the preservation of the tooth and in turn the endogenous genetic material in it, as has been shown in recently published studies. Raman spectroscopy and fluorescence emission spectroscopy have certain features that make them suitable for this application. More specifically, the two techniques provide the possibility and carry out measurements in a short time (typical analysis time < 5 min), while they can, potentially, be applied in field measurements, given the development of portable spectrometers. The techniques are non-destructive and are applied directly onto the sample surface without the need for any pre-treatment (extraction, fragmentation), only direct optical contact with it is required. During the Raman study, a total of 16 teeth of archaeological origin were examined and spectra were recorded in two different spectrometers providing excitation at 1064 nm and 785 nm. Raman spectra show bands related to the mineral content of the teeth, which consists mainly of hydroxyapatite, and arise from the characteristic vibrations of the phosphate group. Accordingly, the organic content, which is mainly proteinaceous in nature, usually collagen residues, shows bands related to the characteristic vibrations of the peptide bond and the vibrations of the methyl and methylene side groups of the amino acids. To evaluate the samples as regards their protein content, Raman spectra were studied, which were recorded with excitation at 1064 nm, and through them, two spectral indices were calculated, referred to as Am/P, C/P, representing ratios of values of the integrated intensity of spectral peaks corresponding to organic and inorganic material in the enamel and cementum tissues. In particular, the Am factor is associated with the characteristic vibration of the amide bond of the protein, so it reflects the collagen content of the tooth, while the P factor is related to the vibration of the phosphate group, so it represents the inorganic matrix of the tooth. Therefore, the spectral index Am/P expresses the ratio of the organic protein residue in the tooth to the main inorganic component. The C/P index is used to describe the alteration that hydroxyapatite crystals undergo due to the addition of carbonate ions (CO32-). The C factor is calculated from the characteristic peak intensity due to carbonate ions. It was observed that the values of the Am/P index are significantly different in the cases of enamel tissue versus cementum tissue, having distinctly higher values in the case of cementum, which is indeed richer in protein material. Accordingly, the values of the C/P ratio appear higher in the case of cementum due to the increased porosity of the specific tissue and its low crystallinity. By taking together the values of the Am/P and C/P ratios, calculated for each tooth, specific criteria were adopted, the application of which allows the assessment of samples in terms of their content of endogenous genetic material and, by extension, the distinction of the well preserved teeth against the rest. In particular, using the C/P index, a 90% rejection of poorly preserved teeth is obtained, while using the Am/P index, a 40% rejection is achieved. Additionally, the CH/P spectral ratio obtained from Raman spectra recorded with excitation at 785 nm was also examined. The CH term corresponds to the integrated intensity of the methyl and methylene vibrational bands, so the CH/P ratio reflects, correspondingly to the Am/P ratio, the tooth content in organic material. A satisfactory correlation of the CH/P ratio with the endogenous DNA content of the teeth was found, but due to the reduced sensitivity of the spectrometer, it was only possible to analyze 6 of the samples. In the context of the study of ancient teeth by use of fluorescence emission spectroscopy, a portable spectrometer was employed that provides excitation at 375 nm through the use of an LED source. UV excitation results in fluorescence emission from the proteinaceous material which can be indirectly correlated with the endogenous DNA content of the cementum. Spectral data from the study of enamel (A) and cementum (O) showed that there is a small shift in the wavelength corresponding to the maximum of the fluorescence emission banmax (O) / λmax (A). The values of the ratio λmax (O) / λmax (A) showed a rather weak correlation (R2 = 0.42) with the amount of endogenous genetic material. By setting appropriate criteria, it became possible to reach a rejection rate of about 85% as regards the poorly preserved samples. It is noteworthy that applying the criteria for the Am/P and C/P indices in combination with the λmax (O) / λmax (A) index leads to the rejection of all ancient teeth with endogenous genetic material < 10%, while the acceptance is achieved of all well-preserved tooth specimens. The results, mainly those obtained based on Raman spectroscopy, are in agreement with recently published work using infrared absorption spectroscopy (FT-IR) to estimate the protein content of archaeological bones after sampling a small amount of bone tissue and grinding it before analysis. Despite the small number of samples, examined in this preliminary study, results are very encouraging and suggest that the methodology proposed can offer a reliable sample screening tool in the context of ancient DNA analysis. As a continuation of the present study, measurements of a larger number of samples are planned to confirm the preliminary results that satisfactorily correlate Raman and fluorescence spectral indices with the percentage of endogenous genetic material in archaeological teeth and bones.
Language Greek
Subject Ancient teeth
Fluorescence spectroscopy
Genetic material
Raman spectroscopy
Spectral indicators
Αρχαιολογικά δόντια
Γενετικό υλικό
Φασματικοί δείκτες
Φασματοσκοπία Raman
Φασματοσκοπία φθορισμού
Issue date 2023-07-27
Collection   School/Department--School of Sciences and Engineering--Department of Chemistry--Post-graduate theses
  Type of Work--Post-graduate theses
Permanent Link https://elocus.lib.uoc.gr//dlib/a/0/c/metadata-dlib-1689590554-844153-29781.tkl Bookmark and Share
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