Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

No description available.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Thesis (Ph.D.) - University of Glasgow, 1996. / Ph.D. thesis submitted to the Faculty of Medicine, Department of Division of Developmental Medicine, University of Glasgow, 1996. Includes bibliographical references: p. 118-132. Print version also available.

Οι μικροσωληνίσκοι της μιτωτικής ατράκτου είναι υπεύθυνοι για τον αποχωρισμό των αδελφών χρωματιδίων κατά την Ανάφαση. Τροποποίηση ή καταστολή της δυναμικής του δικτύου των μικροσωληνίσκων συνεπάγεται αναστολή της κυτταρικής διαίρεσης μέσω ενεργοποίησης του σημείου ελέγχου της Μίτωσης. Έτσι, ενώσεις που προσδένονται στο μόριο της β-τουμπουλίνης και σταθεροποιούν τους μικροσωληνίσκους, όπως το paclitaxel και η griseofulvin, ή τους αποσταθεροποιούν, όπως η nocodazole, αποτελούν αποτελεσματικά φάρμακα έναντι του καρκίνου, αλλά και άλλων ασθενειών. Η πρόσδεση των φαρμάκων στους μικροσωληνίσκους έχει ως αποτέλεσμα, την αναστολή της αλληλεπίδρασης μικροσωληνίσκων-κινητοχώρου, βλάβες στη λειτουργία της μιτωτικής ατράκτου και ανώμαλο χρωμοσωματικό αποχωρισμό, με συνέπεια την εμφάνιση ανευπλοειδικών κυττάρων. Στόχος της παρούσας εργασίας είναι η συγκριτική διερεύνηση της ανευπλοειδογόνου δράσης των φαρμακευτικών ενώσεων nocodazole, paclitaxel και griseofulvin σε τρία κυτταρικά συστήματα in vitro: στους μυοβλάστες ποντικού C2C12, στους ινοβλάστες ανθρώπου HFFF2 και στα καρκινικά επιθηλιακά κύτταρα ανθρώπου MCF-7. Η συγκριτική ανάλυση πραγματοποιήθηκε με τη μελέτη επαγωγής μικροπυρήνων και του μηχανισμού προέλευσης τους, μέσω της διπλής ανοσοσήμανσης πρωτεϊνών του κινητοχώρου (CREST) και του δικτύου του κυτταροσκελετού (α- τουμπουλίνη). Ακολούθησε μελέτη της ακεραιότητας της μιτωτικής συσκευής, μέσω συνδυασμένης ανοσοσήμανσης πρωτεϊνών του κεντροσώματος (γ- τουμπουλίνη & Aurora A) και του δικτύου των μικροσωληνίσκων (β- τουμπουλίνη). Τέλος, πραγματοποιήθηκε μελέτη της έκφρασης πρωτεϊνών που συμμετέχουν στο χρωμοσωματικό αποχωρισμό, όπως οι Aurora A, β- και γ-τουμπουλίνη, μέσω της μεθόδου της ανοσοαποτύπωσης (Western Blot Analysis). Με την ολοκλήρωση της μελέτης παρατηρείται ότι και οι τρεις χημικές ενώσεις: επάγουν το φαινόμενο της χρωμοσωματικής καθυστέρησης, κατάσταση που υποδηλώνει την ύπαρξη ολόκληρου χρωμοσώματος, με αύξηση της συχνότητας των μικροπυρήνων με κινητοχώρο στο εσωτερικό τους και στις τρεις κυτταρικές σειρές. Επίσης, προκαλούν αύξηση του ποσοστού πολυπύρηνων μεσοφασικών κυττάρων και στα τρία κυτταρικά συστήματα. Προκαλούν αύξηση του μιτωτικού δείκτη και συσσώρευση των κυττάρων στο στάδιο της Μετάφασης στα κύτταρα C2C12, HFFF2 και MCF-7. Η συσσώρευση αυτή συνοδεύεται με ταυτόχρονη μείωση των Ανατελοφάσεων. Αποδιοργανώνουν, ακόμα, το δίκτυο των μικροσωληνίσκων, τόσο στα μεσοφασικά όσο και στα μιτωτικά κύτταρα. Ειδικότερα, η nocodazole επάγει την αύξηση του ποσοστού μεσοφασικών κυττάρων με πολύ συμπυκνωμένο δίκτυο και κατεστραμμένο (στικτό) γενετικό υλικό στα κύτταρα ποντικού C2C12. Επίσης, και οι τρεις ενώσεις επηρεάζουν τον κεντροσωματικό πολλαπλασιασμό, με την εμφάνιση ανώμαλων Μεταφάσεων και στις τρεις κυτταρικές σειρές. Η nocodazole επάγει το σχηματισμό μονοπολικών Μεταφάσεων, αντίθετα το paclitaxel σχηματίζει πολυπολικές Μεταφάσεις. Η griseofulvin παρουσιάζει κυτταρική εξειδίκευση με την πρόκληση μονοπολικών Μεταφάσεων στα κύτταρα C2C12, πολυπολικών Μεταφάσεων στα καρκινικά κύτταρα MCF-7 και κατά πλειονότητα μη-ομαδοποιημένων Μεταφάσεων στα κύτταρα HFFF2. Οι εν λόγω μελετηθείσες χημικές ενώσεις τροποποιούν επίσης την έκφραση των πρωτεϊνών Aurora A, β- & γ-τουμπουλίνης και στα τρία κυτταρικά συστήματα. Κύριο χαρακτηριστικό της δράσης της nocodazole είναι η μείωση της έκφρασης των πρωτεϊνών Aurora A και β-τουμπουλίνης. Αντίθετα το paclitaxel και η griseofulvin οδηγούν σε υπερέκφραση των Aurora A, β- και γ-τουμπουλίνη. Και οι τρεις φαρμακευτικές ενώσεις παρουσιάζουν κυτταρική εξειδίκευση ως προς τον επηρεασμό της έκφρασης των πρωτεϊνών που μελετήθηκαν. Τα ευρήματα αυτά υποδηλώνουν τη συμμετοχή των τριών πρωτεϊνών στην εκδήλωση της ανευπλοειδογόνου δράσης των τριών χημικών ενώσεων. Τα παραπάνω συμπεράσματα επιβεβαιώνουν και ερμηνεύουν την ανευπλοειδογόνο δράση των φαρμακευτικών ενώσεων, nocodazole, paclitaxel και griseofulvin. / The microtubules of the mitotic apparatus are responsible for the chromosome segregation during Anaphase. Modification or inhibition of the dynamics of the microtubules results to the activation of the mitotic checkpoint. Chemicals that bind at the molecule of β-tubulin and stabilize microtubules, like paclitaxel and griseofulvin or destabilize them, like nocodazole, act as drugs against cancer or other diseases. This binding results to the inhibition of microtubule-kinetochore interactions, damage of the organization of the mitotic spindle, abnormal chromosome segregation and thus the generation of aneuploid cells. In the present study we comparatively investigated the aneugenic potential of three chemical compounds, nocodazole, paclitaxel and griseofulvin in three cell lines in vitro: C2C12 mouse fibroblasts, HFFF2 human myoblasts and MCF-7 human epithelial cancer cells. The comparative analysis was established by three different experimental procedures. The study of micronucleus induction and their generation mechanism was accomplished by CREST analysis in combination with immunostaining of α- tubulin. The ability of the chemicals to affect the organization of mitotic apparatus was investigated by double immunofluorescence of microtubule network (β-tubulin) and centrosomes (γ-tubulin & Aurora A) in all cell lines. Finally, to understand further the mechanisms by which nocodazole, paclitaxel and griseofulvin exert their aneugenic potential, we examined the ability of these compounds to modulate the expression of proteins that participate in chromosome segregation, such as Aurora A, β- and γ-tubulin, by Western blot analysis in C2C12, HFFF2 and MCF-7 cells. Our results revealed that the three chemical compounds:  Induce chromosome delay, showing a high frequency of micronuclei with kinetochore, indicating the presence of intact chromosome in every studied cell line. In addition, all the drugs evoke induction of multinucleated cells in all cell lines.  Increase the mitotic index with simultaneous Metaphase arrest. The cell accumulation at the Metaphase is accompanied with reduction of Anatelophases in all cell lines.  Promote disorganization of the microtubule network. Especially, nocodazole causes induction of abnormal interphase cells with very condensed network (bundled cells) and punctuated genetic material in C2C12 mouse cell line.  Affect the centrosome proliferation, increasing the frequency of abnormal Metaphases in the three cell lines. Nocodazole induces high frequency of monopolar Metaphases, whereas paclitaxel generates polypolar Metaphases. Griseofulvin produces monopolar Metaphases in C2C12 cells, polypolar Metaphases in cancer MCF-7 cells and mainly non-congressed Metaphases in HFFF2 cells, exhibiting cell specificity.  Modify the expression of the proteins Aurora A, β- and γ-tubulin in all cell lines. Nocodazole reduces Aurora’s A expression in C2C12, HFFF2 and MCF-7 cell lines. The same is observed for β- tubulin’s expression, as for C2C12 and MCF-7 cells. Paclitaxel exerts its aneugenic potential by enhancing the expression of Aurora A and γ-tubulin in all cell lines, whereas enhancing β- tubulin’s expression is only noticed in C2C12 and MCF-7 cells. Griseofulvin increases the expression of Aurora A and β-tubulin in human cancer cells MCF-7. As for γ-tubulin’s expression, is elevated only in the human cell lines HFFF2 and MCF-7. Thus, we suggest that the activity of the pharmaceutical compounds on the expression of the above proteins, exhibit cell specificity. These findings implicate that the aneugenic potential of the studied drugs is mediated through changes in the expression of these proteins. The conclusions above confirm and explain the aneugenic potential of the pharmaceutical compounds, nocodazole, paclitaxel and griseofulvin.

Asbesto é um nome geral dado a seis tipos de fibras minerais encontradas naturalmente na crosta terrestre. Estas fibras vêm sendo exploradas industrialmente desde 1970, porém diversos trabalhadores expostos às fibras apresentaram patologias no trato respiratório, como fibroses e carcinomas. Alguns tipos de fibra foram banidos do mercado, porém o tipo de asbesto crisotila ainda pode ser comercializado na maioria dos países. Estudos in vivo e in vitro tentam elucidar as alterações causadas pela exposição à asbesto nos tecidos e nas células que possam estar relacionadas ao aparecimento de doenças, e foi verificado que a exposição às fibras leva a quebras na dupla fita de DNA, estresse oxidativo, formação de células micronucleadas e células aneuploides. O presente estudo teve como objetivo verificar a presença de alterações causadas em células em cultura expostas à crisotila por 48 h e recuperadas em meio livre de fibras por 48 h, 4 dias e 8 dias, além de observar por microscopia em tempo real divisões aberrantes após a exposição as fibras por 24 e 48h. Foram verificadas alterações que permaneceram na cultura mesmo após 8 dias de recuperação, quando não foram mais observadas fibras na cultura, como formação de células aneuploides, diminuição de frequência de células em G0/G1, aumento de células em G2/M e aumento relativo de células em metáfase quanto à porcentagem de células em fases mais tardias da fase M do ciclo. Já aumento da frequência de células micronucleadas ocorreu apenas nos períodos quando foram observadas fibras na cultura. Para análise da formação de mitoses multipolares e destinos destas células foram construídos vetores para expressão de tubulinas fusionadas a proteínas fluorescentes RFP e GFP, padronizadas as condições de transfecção e de aquisição de imagens para que as células tratadas com crisotila fossem observadas por time-lapse. Alguns destinos de mitoses multipolares causadas pelo tratamento com crisotila foram observados, como morte em metáfase, divisão gerando duas ou três células filhas, fusão de células durante a telófase e retenção em metáfase. Os dados sugerem também a indução da amplificação centrossômica, que parece ocorrer inicialmente em células interfásicas, e também devido à fusão de células. / Asbestos is a general name given to six different fibrous silicate minerals found naturally in the earth\'s crust. These fibers are being exploited industrially since 1970, but several workers exposed to the fibers developed diseases in the respiratory tract, such as fibrosis and carcinomas. Some types of fiber were banished from the market, but the type of asbestos chrysotile can still be marketed in most countries. Studies in vivo and in vitro are trying to elucidate the asbestos effects in tissues and cells that could be related to the development of diseases, and these studies verified that asbestos exposure lead to DNA double strand breaks, oxidative stress, multinucleated and aneuploid cell formation. The present work aimed to verify the alterations in culture cells exposed to chrysotile for 48 h and recovered in fiber-free medium for 48 h, 4 days and 8 days, and also observe aberrant mitosis using time-lapse microscopy after 24 h and 48 h of chrysotile exposure. Some alterations were observed and remained in cell culture even after 8 days of recovery when chrysotile fibers were no longer observed - such as aneuploid cell formation, increased frequencies of G2/M cell, decreased frequencies of G1 cells, and increased frequencies of cells in early M phases as metaphase. The induction of micronuclei occurred only during the periods that fibers were observed in cell culture. For the analysis of multipolar mitosis formation and destinies of these cells after chrysotile treatment, DNA vectors for the expression of tubulins fused to fluorescent proteins (GFP and RFP) were constructed, and the conditions for cells transfection and image acquisition for time-lapse microscopy were established. The fate of some multipolar metaphases was observed: cell retention on metaphase, cell cycle progression generating two or three daughter cells, cell fusion during cytokinesis or during telophase after a multipolar anaphase, and cell death. The centrosome amplification was not observed during the M phase of cell cycle, and may occur in interphase, and also despite cell fusion.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

A aneuploidia é uma característica dos tumores sólidos. Ela pode ser resultado de diferentes erros durante a mitose, como a amplificação centrossômica, mitoses multipolares, e anormalidades durante a citocinese. Hoje se sabe que a aneuploidia pode estar relacionada à supressão ou progressão tumoral dependendo do grau da aneuploidia e do contexto genético das células, e assim esforços vem sendo feitos a fim de elucidar quais erros durante a mitose estão relacionados à formação de células aneuploides viáveis e inviáveis. Estudos prévios do nosso grupo mostraram que tratamentos de células em cultura com fibras de crisotila e com vincristina levam a formação de células aneuploides. Agora direcionados nossos esforços para elucidar os mecanismos envolvidos na formação dessas células, investigando alterações nos centrossomos, número de cromossomos, e origens e destinos de mitoses multipolares após o tratamento com crisotila e com vincristina. As fibras de crisotila, em linhagens de células tumorais e normais, levaram a padrões de localização alterados de proteínas relacionadas à abscisão durante a citocinese, e ocorreu a regressão deste processo e consequente formação de apenas uma célula-filha com o dobro do conteúdo de cromossomos e de centrossomos. Nas duas linhagens estudadas essas células tetraploides progrediram no ciclo celular, gerando mitoses multipolares e consequente formação de células aneuploides. O tratamento com vincristina levou a respostas similares e também distintas em células normais e tumorais. Durante a retenção em metáfase ocorreu a fragmentação da matriz pericentriolar, e as células foram encaminhadas à morte celular ou à saída da mitose sem a ocorrência de divisão celular. Entretanto, células de origem normal tetraploides não progrediram no ciclo celular e não formaram mitoses multipolares, enquanto na linhagem tumoral as células apresentaram aumento da expressão de Aurora A e células com conteúdo cromossômico aumentado e aneuploide em mitoses multipolares. As mitoses multipolares formaram uma, duas ou três células e apresentaram diversas anormalidades no processo de divisão. As alterações observadas no número e composição dos centrossomos após o tratamento com as duas linhagens indicaram que processos de amplificação centrossômica ocorreram após o tratamento. Os dados foram compatíveis com a reduplicação dos centrossomos e com a formação de centríolos a partir do aumento da matriz pericentriolar. Os dados reunidos mostram que apenas células tumorais foram capazes de proliferar mesmo após diferentes erros mitóticos, enquanto células normais puderam apenas superar os erros ocasionados pelas fibras de crisotila / Aneuploidy is a feature of solid tumors. Aneuploid cells result from errors during mitosis, such as centrosome amplification, multipolar mitosis and cytokinesis abnormalities. The capability of aneuploidy to promote and to suppress tumorigenesis has driven the efforts to characterize mitotic errors that form viable and not viable aneuploid cells. We have previously shown that chrysotile, an asbestos fiber, and vincristine, a chemotherapeutic agent, are able to induce aneuploidy. Now we directed our focus to discover possible mechanisms involved in aneuploid cell formation. Herein we evaluated centrosome morphology, chromosome number, and origins and fates of multipolar mitosis after chrysotile and vincristine treatment. Chrysotile fibers, in normal and cancer cells, led to mislocalization of proteins involved in abscission, which resulted in cytokinesis regression and tetraploid cells. These cells were able to enter cell cycle, giving rise to multipolar mitosis and aneuploid cells. Vincristine treatment led to specific and common responses in normal and cancer cells. During metaphase arrest, pericentrosomal matrix was fragmented, and the cells could be conducted to mitotic slippage in both lineages. However, normal tetraploid cells could not progress through cell cycle and neither to form multipolar mitosis, while cancer tetraploid cells showed Aurora A overexpression, structural and numerical centrosome abnormalities, multipolar mitosis and high levels of aneuploidy. The results showed that cancer cells could proliferate even after several mitotic errors, while normal cells could only overcome errors induced by chrysotile treatment

Quantitative fluorescence polymerase chain reaction (QF-PCR) is a molecular genetic method based on the amplification of microsatellites (Short tandem repeats, STR) and measurement of the peak heights of amplicons in the electropherogram. Currently, the QF-PCR deemed reliable, fast, and inexpensive method that is gradually replacing conventional cytogenetic analysis of aneuploidy (examination of long-term cultures of amniotic fluid). However, in certain cases it is impossible to determine the parental origin and meiotic aneuploidy by QF-PCR. The aim of this work was to verify the new dinucleotide STR markers on chromozomes 13, 16, 18, 21, and 22 and further increase the diagnostic efficiency of QF-PCR retaining other STR markers on chromozome 15, 16, 22 and to determine the population and the analytical characteristics of these markers. For all dinucleotide STR markers stutter occurred in high frequency and therefore there were found not to be suitable for routine diagnostics. STR markers for chromozomes 15, 16 and 22 were tested on 100 patients. We selected four informative markers for both chromozome 16 and 22, and three markers for chromozome 15. Thus, I expanded set of diagnostic STR markers in this thesis.

Yes / Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness. / This study was supported by Belgian Science Policy Office (TRIT, P7/41), Flemish Fund for Scientific Research (G.0.B81.12), and Department of Economy, Science and Innovation in Flanders ITM-SOFIB (SINGLE project, to J.C.D.). G.D. and B.C. were supported by the Research Foundation—Flanders (FWO) (grants 12Q8115N and 11O1614N, respectively). V.S., J.M. and P.V. were supported by Czech Science Foundation (project no. 13-07500S) and Charles University (UNCE 204017/2012). J.R.V. was supported by research grants from the KU Leuven (SymBioSys [PFV/10/016]) and the Hercules Foundation (ZW11-14). M.S., M.B., and J.A.C. were supported by the Wellcome Trust through the core support for the Wellcome Trust Sanger Institute (grant no. 098051). G.B., P.P., and G.F.S. were supported by Institut Pasteur strategic fund for the LeiSHield project (to G.F.S.).