Die Inzidenz des Diabetes mellitus (DM) nimmt weltweit jährlich zu und erlangt somit große Bedeutung für die Entwicklung der globalen Gesundheit. Die WHO rechnet bis zum Jahr 2030 mit ungefähr 366 Millionen erwachsenen Diabetikern. Es ist bekannt, dass Männer mit Diabetes mellitus Typ 1 (DMI) und 2 (DMII) häufiger an Subfertilität leiden, wobei dies möglicherweise auf erhöhte Apoptoseraten und vermehrte DNA-Fragmentierungen auf zellulärer Ebene zurückzuführen ist. Die Bedeutung der ROS als Regulatoren von physiologischen und pathologischen Signaltransduktionswegen ist bekannt. Demzufolge könnte die Aktivität der Stickstoffmonoxid-Synthetase (NOS) in diesem Zusammenhang eine Bedeutung haben. Das Ziel der vorliegenden Untersuchungen war es, die Auswirkungen von Apoptose und DNA-Fragmentierungen sowie die Bedeutung der NOS im Zusammenhang mit der Fertilitätsfähigkeit von Spermienzellen von DMI und DMII Patienten zu erfassen und damit erste Erklärungsansätze zur Pathophysiologie der diabetesassoziierten Subfertilität zu liefern. Samenproben von Normalspendern und Diabetikern wurden durch Dichtegradientenzentrifugation in Subpopulationen separiert und mittels fluoreszenzbasierten Assays zur Analyse von apoptoseassoziierten Parametern wie dem Zusammenbruch des mitochondrialen Membranpotentials (MMP), Aktivierung von Caspase-3 (CP3), DNA-Fragmentierungen und reaktiven Sauerstoffspezies (ROS) im Flowzytometer (FACS) untersucht. Die Ergebnisse zeigen eine signifikante Erhöhung von Apoptosemarkern (gestörtes MMP, aktivierte CP3), ROS und DNA-Fragmentationsraten in Spermien von DMI und DMII Patienten im Vergleich zu gesunden Normalspendern. Der Effekt ist bei DMII Patienten verstärkt ausgeprägt. Alle gemessenen Parameter korrelieren umgekehrt mit dem Fertilitätspotential der Spermien, gemessen anhand etablierter Spermiogramm-Analysen, womit ein möglicher Erklärungsmechanismus für die Subfertilität bei Diabetikern geliefert werden kann.

Silicon-based electric chip arrays were developed for characterization of Bacillus cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all chemical reactions until and including the enzymatic reaction took place in an eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control cells of B. subtilis. The 16 positions of the silicon chip array were activated by immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation. / QC 20101111

<p>Eurycoma longifolia (Tongkat Ali / TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report&nbsp / regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods This study&nbsp / encompasses two parts (part 1: on spermatozoa / part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups,&nbsp / washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 &mu / g/ml) for 1 hour at 37&deg / C. A sample without addition of TA served as control. After incubation with TA,&nbsp / the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen&nbsp / species (ROS / dihydroethidium test / DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (&Delta / &psi / m) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells&nbsp / incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 &mu / g/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were&nbsp / evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant&nbsp / dose-dependent trends were found&nbsp / for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 &mu / g/ml, yet, no significance could be&nbsp / observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor &Delta / &psi / m.</p>

No description available.

<p>Eurycoma longifolia (Tongkat Ali / TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report&nbsp / regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods This study&nbsp / encompasses two parts (part 1: on spermatozoa / part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups,&nbsp / washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 &mu / g/ml) for 1 hour at 37&deg / C. A sample without addition of TA served as control. After incubation with TA,&nbsp / the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen&nbsp / species (ROS / dihydroethidium test / DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (&Delta / &psi / m) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells&nbsp / incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 &mu / g/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were&nbsp / evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant&nbsp / dose-dependent trends were found&nbsp / for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 &mu / g/ml, yet, no significance could be&nbsp / observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor &Delta / &psi / m.</p>

Vitamin A and its metabolites retinal and retinoic acid are important molecules for the regulation of normal cellular growth, differentiation and other important functions. Retinoids are known to exert mutagenic as well as antimutagenic activity, although conflicting reports are known. All-trans retinoic acid (ATRA) is used in the treatment of many diseases such as acne, psoriasis and ichthyosis. It is also used in differentiated therapy of acute promyelocytic leukemia; however, it is frequently observed that relapses occur when ATRA is prescribed as maintenance therapy. Therefore, understanding the mechanism of action of ATRA in cells would be helpful in the development of high potent and low toxic chemotherapeutic agents. EA-4 is a newly synthesized steroidal analogue of ATRA and is considered as a promising agent for the inhibition of human leukemic cell growth. The study of genotoxicity is an important parameter for the design and development of new chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to their possibility of inducing secondary tumours in cancer patients. Therefore, it is important to determine the genotoxic potential of a drug that will be used in chemotherapy, particularly in native human cells. Taking into consideration the above referred, it would be of interest to evaluate the genotoxic potential of EA-4 in comparison to ATRA, as to their ability to provoke micronucleus (MN) generation, due to both chromosome breakage and chromosome delay. Micronuclei originate from chromosome fragments or whole chromosomes, which lag behind at anaphase during nuclear division. According to our knowledge, there is no information on the ability of all-trans retinoic acid (ATRA) to induce micronucleus formation. To investigate the ability of ATRA and its steroidal analogue EA-4 to enhance micronucleation on human lymphocytes cultured in vitro, the Cytokinesis Block MicroNucleus (CBMN) assay was conducted. By this assay, the cytotoxic effect of the two retinoids was also estimated. To clarify the mechanism by which micronuclei are generated due to ATRA and EA-4 treatment, CBMN was combined with Fluorescence In Situ Hybridization (FISH) using an α-satellite pancentromeric probe to detect centromere inclusion and thus intact chromosome(s) in micronuclei or acentric chromosome fragments. ATRA and EA-4 were shown to be cytotoxic by decreasing CBPI (Cytokinesis Block Proliferation Index) to statistically significant levels in relation to untreated cells. A statistically significant increase in micronucleus frequency was also observed for both investigated compounds. ATRA generated micronuclei mainly via chromosome breakage while a mild effect on chromosome delay was also apparent. On the other hand, EA-4 generated micronuclei exclusively via chromosome breakage. To verify ATRA and EA-4 genotoxicity, micronucleation was investigated in a second biological system coming from a different organism, C2C12 mouse cells. Micronucleus analysis was achieved by α-tubulin/CREST immunostaining for the visualization of microtubules and the detection of kinetochore inside micronuclei and hence the inclusion of whole chromosome(s) or acentric chromosome fragments. Additionally the effect of ATRA and EA-4 on cell proliferation was investigated by the estimation of Mitotic Index (M.I.). We found that ATRA and EA-4 exerted cytotoxic activity in C2C12 mouse cells by reducing the cell proliferation rate at significant levels, as evaluated by the decrease of M.I. A statistically significant elevation in the frequency of interphase cells with micronuclei was shown. CREST analysis confirmed the clastogenic activity of the studied retinoids that was indicated in human lymphocytes. Micronucleation due to ATRA was mediated mainly by chromosome breakage and in a lesser extent by chromosome delay. EA-4 was shown to induce chromosome breakage as well as chromosome delay, as opposed to human lymphocytes at which only clastogenic effect was shown. These observations suggest that, ATRA and EA-4 are able to provoke chromosome fragmentation, but additionally and in a lesser extent to disturb chromosome segregation at anaphase due to chromosome lagging. Cell cycle analysis showed that ATRA and EA-4 accumulated cells at ana-telophase. The analysis of ana-telophases revealed micronucleation, nucleoplasmic bridges and multinucleation, phenomena that may explain the dual genetic activity of ATRA and EA-4. Multinucleated and multimicronucleated interphase cells were also apparent, the second ones generated due to both chromosome delay and breakage. To further investigate the mechanism of genotoxic activity of ATRA and EA-4 we proceeded our research on two axes based on their aneugenicity and clastogenicity. Thus we studied the effect of ATRA and EA-4: i) on the integrity of mitotic spindle, as a target of aneugens by using double immunofluorescence staining of β- and γ-tubulin in C2C12 mouse cell line, which is a convenient system to apply this experimental procedure, and ii) to investigate the ability of the studied retinoids to induce double-strand breaks on DNA by using neutral Single Cell Gel Electrophoresis (SCGE assay-Comet assay) in two different cell lines, C2C12 mouse cells and HL-60 human leukemic cells. Analysis of mitotic spindle has shown that the studied retinoids affect chromosome orientation during metaphase by inducing bipolar metaphases with non-congressed genetic material due to abnormal microtubule network. In addition defects on centrosome duplication and/or separation were observed due to the presence of monopolar metaphases. Ana-telophases as well as interphases with supernumerary centrosomes were also apparent. Additionally, interphase cells with abnormal microtubule network were observed. The above findings may explain aneugenic as well as clastogenic activity of the studied retinoids. Comet assay revealed that ATRA and its steroidal analogue EA-4 provoke DNA migration due to double strand DNA fragmentation in both C2C12 mouse cells and HL-60 human leukemic cells. EA-4 was shown to be the stronger inducer of DNA fragmentation. These results confirm the findings from FISH and CREST analysis indicating that the studied retinoids show high clastogenic activity. . Taking into account the above, we may say that our findings clarify the cytotoxic and genotoxic activity of retinoic acid and the mechanism of its action by indicating its ability to induce chromosome breakage via double-strand DNA breaks and secondary its ability to provoke chromosome delay due to defects in microtubule network and mitotic spindle integrity. / Η βιταμίνη Α και οι μεταβολίτες της, ρετινόλη και ρετινοϊκό οξύ είναι ισχυροί παράγοντες για τη ρύθμιση σημαντικών λειτουργιών, όπως της κυτταρικής ανάπτυξης, διαφοροποίησης και άλλων. Τα ρετινοειδή είναι γνωστά για την μεταλλαξιγόνο αλλά και αντιμεταλλαξιγόνο δράση τους, αν και έχουν αναφερθεί αντικρουόμενα ευρήματα. Το all-trans ρετινοϊκό οξύ (ATRA) χρησιμοποιείται στη θεραπεία πολλών ασθενειών, όπως η ακμή, ψωρίαση, ιχθύωση, αλλά και στη θεραπεία κακοηθειών όπως η μυελογενής λευχαιμία. Συχνά σε περιπτώσεις όπου το ATRA αποτελεί τη βασική θεραπεία παρατηρούνται υποτροπιάσεις Έτσι, η κατανόηση του μηχανισμού δράσης του ATRA στα κύτταρα θα αποτελέσει χρήσιμο εργαλείο για την ανάπτυξη νέων, ισχυρών και μη-τοξικών θεραπευτικών παραγόντων προερχόμενων από αυτό. Το EA-4 είναι ένα πρόσφατα συντεθέν στεροειδικό ανάλογο του ATRA, που θεωρείται υποσχόμενος παράγοντας για την αναστολή της ανάπτυξης ανθρώπινων λευχαιμικών κυττάρων. Η μελέτη της γονιδιοτοξικότητας αποτελεί σημαντική παράμετρο για το σχεδιασμό και την ανάπτυξη νέων θεραπευτικών παραγόντων. Οι γονιδιοτοξικές επιπτώσεις αντικαρκινικών φαρμάκων σε μη-καρκινικά κύτταρα είναι ιδιαίτερης σημασίας, και αποτελούν πιθανή αιτία εμφάνισης δευτερογενών όγκων σε ασθενείς. Έτσι, είναι σημαντικό να μελετηθεί η γονιδιοτοξική δράση ενός φαρμάκου που θα χρησιμοποιηθεί στη χημειοθεραπεία. Λαμβάνοντας υπόψη όλα τα παραπάνω, θεωρήθηκε ενδιαφέρον να εκτιμηθεί η γονιδιοτοξικότητα του EA-4 σε σύγκριση με το ATRA ως προς την ικανότητά τους να προκαλούν την εμφάνιση μικροπυρήνων (MN) είτε μέσω της χρωμοσωματικής θραύσης είτε μέσω της χρωμοσωματικής καθυστέρησης. Οι μικροπυρήνες προέρχονται από χρωμοσωματικά θραύσματα ή ολόκληρα χρωμοσώματα, τα οποία καθυστερούν κατά την ανάφαση της μείωσης ή της μίτωσης. Σύμφωνα με όσα μέχρι σήμερα γνωρίζουμε, δεν φαίνεται να υπάρχουν στοιχεία που αφορούν την ικανότητα του all-trans ρετινοϊκού οξέος (ATRA) να επάγει το σχηματισμό μικροπυρήνων. Για τη διερεύνηση της ικανότητας του ATRA και του στεροειδικού αναλόγου του EA-4 να επάγει την εμφάνιση μικροπυρήνων, πραγματοποιήθηκε η μέθοδος αναστολής της κυτταροκίνησης (CBMN assay) σε ανθρώπινα λεμφοκύτταρα in vitro. Με την ίδια μέθοδο εκτιμήθηκε και η κυτταροτοξικότητα των δύο ρετινοειδών. Για την διευκρίνιση του μηχανισμού δημιουργίας των μικροπυρήνων από τη δράση των ATRA και EA-4, η μέθοδος CBMN συνδυάστηκε με την in situ υβριδιποίηση με φθοροχρώματα (FISH) και χρήση α-δορυφορικού (α-satellite) πανκεντρομερικού ανιχνευτή για την επισήμανση του κεντρομέρους και την ανίχνευσή του σε μικροπυρήνες. Η παρουσία σήματος υβριδοποίησης στους μικροπυρήνες υποδηλώνει την ύπαρξη άθικτου χρωμοσώματος στο εσωτερικό τους. Το αντίθετο υποδεικνύει την παρουσία άκεντρου χρωμοσωματικού θραύσματος. Τα αποτελέσματα έδειξαν ότι και οι δύο χημικές ενώσεις προκαλούν στατιστικά σημαντική αύξηση της συχνότητας των μικροπυρήνων Το ATRA οδηγεί στην δημιουργία μικροπυρήνων κυρίως μέσω χρωμοσωματικής θραύσης, και σε ηπιότερο βαθμό μέσω χρωμοσωματικής καθυστέρησης. Αντίθετα, το EA-4 επάγει το σχηματισμό μικροπυρήνων αποκλειστικά μέσω χρωμοσωματικής θραύσης. Επίσης το ATRA και το EA-4 παρουσάζουν ισχυρή κυτταροτοξικότητα, όπως φάνηκε από τη στατιστικά σημαντική μείωση του κυτταρικού δείκτη πολλαπλασιασμού (CBPI), σε σύγκριση με τις καλλιέργειες του μάρτυρα. Προκειμένου να επιβεβαιωθεί η γονιδιοτοξικότητα του ATRA και του EA-4, διερευνήθηκε η ικανότητά τους να προκαλούν αυξημένες συχνότητες μικροπυρήνων σε ένα δεύτερο βιολογικό σύστημα, την κυτταρική σειρά ποντικού C2C12. Η ανάλυση των MN πραγματοποιήθηκε με τη μέθοδο διπλού ανοσοφθορισμού α-τουμπουλίνης/CREST, για την ανίχνευση σήματος κινητοχώρου στο εσωτερικό του μικροπυρήνα κι έτσι την παρουσία ολόκληρου χρωμοσώματος. Επίσης,η κυτταροτοξικότητα τους διερευνήθηκε με την εκτίμηση του μιτωτικού δείκτη. Με τη ίδια μέθοδο αναλύθηκε η πρόοδος του κυτταρικού κύκλου. Παρατηρήθηκε ότι το ATRA και το EA-4 παρουσιάζουν κυτταροτοξική δράση στα κύτταρα C2C12 μειώνοντας το ρυθμό κυτταρικού πολλαπλασιασμού σε στατιστικά σημαντικά επίπεδα. Επιπλέον αποκαλύφθηκε στατιστικά σημαντική αύξηση της συχνότητας κυττάρων με μικροπυρήνες. Η επισήμανση του κινητοχώρου επιβεβαίωσε τη θραυσματογόνο δράση των υπό μελέτη ρετινοειδών που παρατηρήθηκε στα ανθρώπινα λεμφοκύτταρα. Η δημιουργία μικροπυρήνων μέσω του ATRA ήταν αποτέλεσμα κυρίως χρωμοσωματικής θραύσης και σε μικρότερη έκταση χρωμοσωματικής καθυστέρησης, σε συμφωνία με τα ευρήματα από τα πειράματα στις καλλιέργειες ανθρώπινων λεμφοκυττάρων. Αντίθετα, παρατηρήθηκε ότι το EA-4, πλην της ισχυρής θραυσματογόνου δράσης, προκαλεί και χρωμοσωματική καθυστέρηση. Οι παρατηρήσεις αυτές υποδεικνύουν ότι το ATRA και το EA-4 είναι ισχυροί θραυσματογόνοι παράγοντες, αλλά σε μικρότερο βαθμό είναι ικανοί να διαταράξουν και τον χρωμοσωματικό αποχωρισμό κατά την πυρηνική διαίρεση. Η μελέτη του κυτταρικού κύκλου έδειξε ότι τόσο το ATRA και όσο και το EA-4 προκαλούν καθυστέρηση συσσωρεύοντας τα κύτταρα στα στάδια ανάφασης και τελόφασης της πυρηνικής διαίρεσης. Κύτταρα που συσσωρεύονται στα παραπάνω στάδια χαρακτηρίζονται από την εμφάνιση πυρηνοπλασματικών γεφυρών, την παρουσία περισσότερων του ενός πυρήνων, αλλά και την παρουσία μικροπυρήνων, φαινόμενα τα οποία είναι σύμφωνα με τη διττή γενετική δράση των ATRA και EA-4. Επίσης, παρατηρήθηκαν πολυπύρηνα μεσοφασικά κύτταρα και μεσοφασικά κύτταρα με πολλαπλούς μικροπυρήνες, με τον δεύτερο τύπο κυττάρων να προέρχεται τόσο από χρωμοσωματική θραύση όσο και από χρωμοσωματική καθυστέρηση. Έτσι, φαίνεται ότι τα δύο υπό μελέτη ρετινοειδή μπορούν να χαρακτηρισθούν μόρια με θραυσματογόνες αλλά και ανευπλοειδογόνες ιδιότητες. Για τη λεπτομερέστερη ανάλυση του μηχανισμού δράσης του ATRA και του EA-4 σχεδιάσθηκαν πειράματα σε δύο βασικούς άξονες που αφορούσαν την περαιτέρω μελέτη τόσο της ανευπλοειδογόνου όσο και της θραυσματογόνου δράσης τους. Έτσι, μελετήθηκε η επίδραση του ATRA και του EA-4 αντίστοιχα ως προς: α) την ακεραιότητα της μιτωτικής συσκευής, η οποία αποτελεί κυτταρικό στόχο ανευπλοειδογόνων ενώσεων. Η μελέτη πραγματοποιήθηκε στην κυτταρική σειρά C2C12, μέσω της μεθόδου διπλού ανοσοφθορισμού για τη β- και γ-τουμπουλίνη, δομικά στοιχεία των μικροσωληνίσκων και του κεντροσώματος, και β) την δημιουργία δίκλωνων ρηγμάτων στο DNA μέσω της μεθόδου ηλεκτροφόρησης μοναδιαίων κυττάρων (SCGE assay-Comet assay) σε δύο διαφορετικές κυτταρικές σειρές, στα κύτταρα ποντικού C2C12 και στα λευχαιμικά κύτταρα ανθρώπου HL-60. Τα αποτελέσματα μας έδειξαν ότι τα υπό εξέταση ρετινοειδή επηρεάζουν τον χρωμοσωματικό προσανατολισμό κατά τη μετάφαση με την εμφάνιση διπολικών μεταφάσεων με τα χρωμοσώματα μη-διατεταγμένα στο ισημερινό πεδίο, λόγω ανωμαλιών του δικτύου των μικροσωληνίσκων. Επίσης, φάνηκε ότι προκαλούν ανωμαλία στον πολλαπλασιασμό και πιθανόι στον αποχωρισμό των κεντροσωμάτων, παρατήρηση που δικαιολογείται από την παρουσία μονοπολικών μεταφάσεων, καθώς και ανάτελοφάσεων αλλά και μεσοφασικών κύττάρων με υπεράριθμο κεντροσωματικό αριθμό. Επιβεβαιώθηκε επίσης η επίδρασή τους στην πορεία του κυτταρικού κύκλου με συσσώρευση των κυττάρων στα στάδια ανάφασης-τελόφασης. Επιπρόσθετα, φάνηκε ότι το ΕΑ-4, στη μεγαλύτερη συγκέντρωση, διακόπτει τον κυτταρικό κύκλο στο στάδιο της μετάφασης. Παράλληλα, παρατηρήθηκε διαταραχή στη δομή του δικτύου των μικροσωληνίσκων. Όλα τα παραπάνω ευρήματα ερμηνεύουν τόσο την ανευπλοειδογόνο όσο και τη θραυσματογόνο δράση των δύο ρετινοειδών. Με τη μέθοδο ηλεκτροφόρησης μοναδιαίων κυττάρων δείχθηκε ότι το ATRA και το στεροειδικό του ανάλογο EA-4 προκάλεσαν τη δημιουργία «κομητών», δηλαδή πυρήνων με ανώμαλη μορφολογία μέσω του σχηματισμού δίκλωνων θραυσμάτων DNA. Το φαινόμενο αυτό παρατηρήθηκε τόσο στα κύτταρα ποντικού C2C12 όσο και στα λευχαιμικά κύτταρα ανθρώπου HL-60, με το EA-4 να παρουσιάζει ισχυρότερη επαγωγή θραύσης του DNA. Τα αποτελέσματα αυτά επιβεβαιώνουν τα ευρήματα των μεθόδων FISH και CREST, υποδεικνύοντας ότι τα υπό εξέταση ρετινοειδή παρουσιάζουν ισχυρή θραυσματογόνο δράση. Λαμβάνοντας υπόψη όλα τα παραπάνω, μπορούμε να ισχυριστούμε ότι τα ευρήματά μας διευκρινίζουν την κυτταροτοξική και γονιδιοτοξική δράση του ρετινοϊκού οξέος. Υποδεικνύουν ιδιότητες ισχυρώς θραυσματογόνων παραγόντων μέσω δημιουργίας δίκλωνων ρηγμάτων στο DNA των κυττάρων. Δευτερογενώς μπορούν να χαρακτηρισθούν ως ήπιες ανευπλοειδογόνες ενώσεις που προκαλούν ανώμαλο χρωμοσωματικό αποχωρισμό μέσω ανωμαλιών τόσο του δικτύου των μικροσωληνίσκων όσο και της ακεραιότητα της μιτωτικής συσκευής.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-01T13:22:09Z No. of bitstreams: 1 thiagopereiradasilva.pdf: 8248708 bytes, checksum: 846bfbc45d8748f458221b2646af213c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T16:00:55Z (GMT) No. of bitstreams: 1 thiagopereiradasilva.pdf: 8248708 bytes, checksum: 846bfbc45d8748f458221b2646af213c (MD5) / Made available in DSpace on 2016-07-13T16:00:55Z (GMT). No. of bitstreams: 1 thiagopereiradasilva.pdf: 8248708 bytes, checksum: 846bfbc45d8748f458221b2646af213c (MD5) Previous issue date: 2012-03-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O bacterioplâncton é um dos componentes da cadeia trófica em ecossistemas aquáticos, sendo regulado por fatores como disponibilidade de nutrientes, temperatura, predação e infecção viral. A morte de bactérias é caracterizada como o processo de perda funcional e morfológica da célula e tem função de controlar a abundância e produção bacteriana, com significado importante na ciclagem de carbono e nutrientes nos ecossistemas aquáticos. No entanto, o processo de morte celular em bactérias aquáticas é desconhecido. Ainda não se sabe se a morte celular programada (MPC), processo regulado e bem conhecido em organismos eucariotos, ocorre em bactérias aquáticas. Este trabalho teve por objetivo investigar a ocorrência de MCP em bactérias aquáticas utilizando dois modelos de ecossistemas: lago Batata e lago dos Manacás. O lago Batata é um ecossistema amazônico de inundação e encontra-se divido em duas áreas: impactada e natural. O lago dos Manacás é um ecossistema artificial localizado em Minas Gerais. Três grupos de bactérias, provenientes do lago dos Manacás, lago Batata – área natural e lago Batata – área impactada foram estudados. As amostras de água foram coletadas na sub-superfície dos lagos e processadas para análises da viabilidade celular por microscopia de fluorescência e da fragmentação de DNA por citometria de fluxo. Em paralelo, amostras do lago Batata – área impactada foram processadas para microscopia eletrônica de transmissão (MET) para caracterizar a diversidade ultraestrutural da comunidade bacteriana e investigar a ocorrência de alterações celulares bacterianas. Os resultados da viabilidade celular utilizando marcadores específicos para integridade de membrana (LIVE/DEAD BacLight) mostraram diretamente a presença de bactérias vivas/viáveis e mortas/inviáveis . O lago Batata apresentou maior proporção de morte celular bacteriana (36,20%) em comparação com o lago dos Manacás (19,66%). As análises de fragmentação de DNA (ensaio TUNEL) mostraram que a MCP constitui um fenômeno presente nos ecossistemas aquáticos estudados, com maior ocorrência na área impactada do lago Batata. A MET revelou a presença de bactérias aquáticas com grande diversidade ultraestrutural, representada por diferenças morfológicas quanto ao envoltório celular, cápsula, mesossomos, vesículas membranosas, partículas aderidas e tilacóides. Além disso, 34,28% das bactérias apresentavam vírus no citoplasma. A diversidade ultraestrutural pode representar a ampla diversidade metabólica e adaptativa bacteriana, enquanto a presença de vírus parece relacionada com a morte bacteriana. As análises de integridade ultraestrutural mostraram que (i) a maior proporção de bactérias encontrava-se com alterações ultraestruturais indicativas de processo de morte, (ii) a frequência de ocorrência de cápsula foi menor nas bactérias vazias, (iii) bactérias com alterações apresentaram maior frequência de partículas aderidas, e (v) o número médio de fagos por secção foi significativamente maior nas bactérias alteradas em comparação com as bactérias intactas. A MET mostrou a ocorrência de alterações ultraestruturais típicas de MCP, como retração e condensação citoplasmáticas. Em conjunto, nossos dados ressaltam morte bacteriana como um evento importante atuando na regulação da comunidade bacteriana e demonstra, pela primeira vez, a ocorrência de MCP em bactérias aquáticas. A MCP pode ter significado funcional como um dos mecanismos desenvolvidos para sobrevivência da comunidade bacteriana. / The bacterioplankton is an important component of the food web structure in aquatic ecosystems and it is regulated by many factors such as nutrient supply, temperature, predation and virus infection. Bacterial death is characterized by functional and morphological loss of the bacterial cell with roles in the control process of abundance and bacterial production of aquatic ecosystems and functional meaning in the carbon and nutrient cycles. However, the cell death process in aquatic bacteria remains to be defined. Programmed cell death (PCD) is a regulated process largely known in eukaryotic organisms. There are not studies dealing with PCD occurrence in aquatic bacteria. This study aimed to investigate the occurrence of PCD in free-living aquatic bacteria in two models of aquatic ecosystems: Batata lake and Manacás lake. Batata Lake is located on floodplain Trombetas river in northern of Brazilian Amazon. This ecosystem has been impacted for bauxite tailings over teen years and it is presently divided in impacted and natural stations. Manacás Lake is an artificial system located in Minas Gerais. Three groups of bacteria from Manacás Lake, Batata lake – impacted area - and Batata lake – natural area -were studied. Water samples were collected from the subsurface of these lakes and processed for analyses of bacterial viability by fluorescence microscopy and DNA fragmentation by flow citometry. In parallel, samples from Batata lake – impacted area - were processed for transmission electron microscopy (TEM) for characterization of the bacterial community ultrastructural diversity and investigation of bacteria alterations. Our cell viability results using specific markers for membrane integrity (LIVE/DEAD BacLight) showed directly the presence of live/viable and dead/ not viable bacteria. Batata lake presented a higher proportion of bacterial death (36,20%) compared to Manacás lake (19,66%). DNA fragmentation analyses (TUNEL assay) showed that PCD is a phenomenon occurring in all aquatic ecosystems investigated, with higher frequency in the Batata lakeimpacted area compared to the other ecosystems. Our TEM analyses revealed a great bacterial ultrastructural diversity represented by morphological differences in the cellular envelope, capsule, mesosomes, membrane vesicles, attached particles and thylacoids. Moreover, around 34,28% of the bacteria showed virus in their cytoplasm. The ultrastructural diversity may represent the large metabolic and adaptative diversity of aquatic bacteria while the presence of virus may be related to bacterial death. Our ultrastructural integrity analyses showed that (i) the higher proportion of bacteria was in death process or dead (damaged and empty), (ii) the capsule frequency is lower in empty bacteria, (iii) the higher bacteria-particle association was found in altered bacteria, (iv) the mean number of viruses per cell section was higher in altered compared to intact bacteria. TEM also showed the presence occurrence of typical ultrastructural changes indicative of MPC, such as cell retraction and condensation. Altogether, our data demonstrate that PCD occur in aquatic bacteria, and that this event may be a survival mechanism for bacterial communities in these ecosystems.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Introduction: Eurycoma longifolia (Tongkat Ali; TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods: This study encompasses two parts (part 1: on spermatozoa; part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups, washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 μg/ml) for 1 hour at 37°C. A sample without addition of TA served as control. After incubation with TA, the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen species (ROS; dihydroethidium test; DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (Δψm) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 μg/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant dose-dependent trends were found for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 μg/ml, yet, no significance could be observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor Δψm. Part 2: The viability rates and protein production of TM3-Leydig and TM4-Sertoli cells at 48-hour exposure to TA showed increases whereas at 96-hour incubation periods viability and protein production declined especially as from concentration 25 μg/ml TA. Similar results could be seen for TM4-Sertoli cells pyruvate production. The testosterone production at 48-hour exposure marginally increased (P=0.0580) at the highest (50 μg/ml) concentration of TA. However, at 96-hour exposure to TA the testosterone production significantly (P=0.0065) increased. It is also apparent that after 96 hours the concentration of testosterone has increased [12 x 10-4 ng/ml] when compared to 48-hour exposure [6 x 10-7ng/ml] of Tongkat Ali. Conclusion: Part 1: Results indicate that the Tongkat Ali extract has no deleterious effects on sperm functions at therapeutically used concentrations (<2.5 μg/ml). Part 2: The cytotoxic effect of TA are only presented at higher concentration from 25 μg/ml. TM3-Leydig cells appears to be more resilient than TM4-Sertoli cells in viability and protein production yet at prolonged periods of exposure it is detrimental. Testosterone production only increases after 96 hours exposure to TA.

This masters thesis has a few objectives. One of them is to acquaint with the problems of genome engineering, especially with fragmentation of DNA, the macromolecule DNA, the methods for purification and separation of the nucleic acids, the enzymes used for modification of these acids, amplification and get to know with cluster and gradient analysis as well. The next aim is to peruse the existed application and compare it to the layout of the proposed application, that is the third aim. The last one from the objectives is the implementation and the report how was the application tested by the real data. The results will be discussed as well as the possibilities of the further extension.

No description available.