<p>Cancer cells often have elevated frequencies of chromosomal aberrations, and it is likely that loss of genome stability is one driving force behind tumorigenesis. Deficiencies in DNA replication, DNA repair, or cell cycle checkpoints can all contribute to increased rates of chromosomal duplications, deletions and translocations. The human ATM and ATR proteins are known to participate in the DNA damage response and DNA replication checkpoint pathways and are critical to maintaining genome stability. The <italic>Saccharomyces cerevisiae</italic> homologues of ATM and ATR are Tel1p and Mec1p, respectively. Because Tel1p and Mec1p are partially functionally redundant, loss of both Tel1p and Mec1p in haploid yeast cells (<italic>tel1 mec1</italic> strains) results in synergistically elevated rates of chromosomal aberrations, including terminal duplications, chromosomal duplications, and telomere-telomere fusions. To determine the effect of Tel1p and Mec1p on chromosome aberrations that cannot be recovered in haploid strains, such as chromosome loss, I investigated the phenotypes associated with the <italic>tel1 mec1</italic> mutations in diploid cells. In the absence of induced DNA damage, <italic>tel1 mec1</italic> diploid yeast strains exhibit extremely high rates of aneuploidy and chromosome rearrangements. There is a significant bias towards trisomy of chromosomes II, VIII, X, and XII, whereas the smallest chromosomes I and VI are commonly monosomic. </p> <p> The telomere defects associated with <italic>tel1 mec1</italic> strains do not cause the high rates of aneuploidy, as restoring wild-type telomere length in these strains by expression of the Cdc13p-Est2p fusion protein does not prevent cells from becoming aneuploid. The <italic>tel1 mec1</italic> diploids are not sensitive to the microtubule-destabilizing drug benomyl, nor do they arrest the cell cycle in response to the drug, indicating that the spindle assembly checkpoint is functional. The chromosome missegregation phenotypes of <italic>tel1 mec1</italic> diploids mimic those observed in mutant strains that do not achieve biorientation of sister chromatids during mitosis. </p> <p> The chromosome rearrangements in <italic>tel1 mec1</italic> cells reflect both homologous recombination between non-allelic Ty elements, as well as non-homologous end joining (NHEJ) events. Restoring wild-type telomere length with the Cdc13p-Est2p fusion protein substantially reduces the levels of chromosome rearrangements (terminal additions and deletions of chromosome arms, interstitial duplications, and translocations). This result suggests that most of the rearrangements in <italic>tel1 mec1</italic> diploids are initiated by telomere-telomere fusions. One common chromosome rearrangement in <italic>tel1 mec1</italic> strains is an amplification of sequences on chromosome XII between the left telomere and rDNA sequences on the right arm. I have termed this aberration a "schromosome." Preliminary evidence indicates that the schromosome exists in the <italic>tel1 mec1</italic> cells as an uncapped chromosome fragment that gets resected over time.</p> / Dissertation

Mutations in the presenilin 1 gene account for most early-onset familial Alzheimer's disease (FAD). The presenilins and AD may also be related through a common involvement in the cell cycle. Here we report that one important aspect of the cell cycle---proper chromosome segregation---is dependent on presenilin function and therefore may be involved in AD pathogenesis. Specifically we find that FAD mutations in presenilin 1 (M146L and M146V) lead to chromosome missegregation and aneuploidy in vivo and in vitro: 1) Both metaphase chromosome analysis and in situ hybridization reveal significant aneuploidy in the lymphocytes and neurons of PS-1 transgenic mice. 2) Transiently transfected human cells expressing normal and, especially, mutant PS-1 develop aneuploidy within 48 hours, including trisomy 21, while cells transfected with dominant negative PS-1 genes lacking ?-secretase activity have no effect on chromosome segregation. 3) Analysis of mitotic spindles in the transfected cells reveals abnormal microtubule arrays and lagging chromosomes. The possible mechanisms by which cell cycle defects and chromosome missegregation induced by y-secretase may contribute to Alzheimer's disease will be discussed.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Additional advisors: Vithal K. Ghanta, Denise R. Shaw, Stephen A. Watts, Bradley K. Yoder. Description based on contents viewed Feb. 20, 2009; title from PDF t.p. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references (leaves 164-190). Also available online.

Introduction: In recent years, the average age of first reproduction has risen significantly, the mean now standing at around 30 years in many countries. The adverse effects of maternal age on fertility and reproduction have been well documented. However, the influence of paternal age on fertility, reproduction and postnatal health is relatively poorly understood, and 50% of all male infertility cases are classed as idiopathic or unexplained infertility. Methods: The aim of this study was to investigate factors that contribute to male infertility, split into two main parts. The first part focused on analysing data collected from patients who had undergone fertility treatment to assess the influence of different factors on infertility, especially at the genome level. The second part attempted to deal with some of the technical challenges of screening and diagnostic methods to study the genome, with the aim of providing tools that would assist future studies in pinpointing genetic factors responsible for infertility, especially in cases of idiopathic infertility. Results: Based on data from the first part of the study, it was determined that advanced paternal age can affect sperm progressive motility, sperm DNA integrity and the fertilisation rate of in vitro fertilisation (IVF) cycles, as well as the development of embryos. Direct analysis of sperm DNA fragmentation (SDF) and degradation levels revealed an association between elevated SDF and impaired embryo development. Furthermore, a correlation was shown between chromosome aneuploidy and variance in SDF and sperm DNA degradation. Moreover, aneuploidy can influence abnormal sperm morphology and consequently also progressive motility. Also, embryo development rate of IVF cycles on day three, demonstrated a significant decline in cycles where the sperm used for fertilisation had a high aneuploidy rate, which can highlight the reduced developmental capacity of aneuploid embryos. From the lifestyle factors assessed, only alcohol consumption significantly correlated with the sperm DNA damage. Therefore, poor semen quality may highlight damage that has been incurred by the sperm DNA. When the semen quality is suboptimal, the intracytoplasmic sperm injection (ICSI) technique is suggested as a standard strategy to improve the prognosis of ART. However, when the progressive motility is poor, the ICSI approach is not as effective. Based on our findings and in line with other studies, the only sperm parameter that can be affected by paternal age is sperm motility, which could be an indicator of SDF. Therefore, the decline in ICSI fertilisation rate in patients with impaired sperm progressive motility could be due to sperm DNA damage, and even ICSI cannot improve the fertilisation rate considerably. Discussion: The aim of the second part of this project was to establish a robust workflow for whole- genome amplification (WGA) and whole-genome sequencing of single cells to improve the coverage rate and fidelity, with the aim of providing means of detecting any mutation in the genome that might be responsible for reduced embryonic developmental competence. Towards this end, the efficiencies of two different WGA protocols (REPLI-g and TruePrime) were compared. Multiple technical factors required optimisation in order to create a suitable protocol. Our results demonstrated the overall superiority of REPLI-g compared to TruePrime in almost all the assessed parameters. The amplification rate of REPLI-g was much faster than that of TruePrime, and prolonged incubation led to overamplification and an increased duplication rate. However, the TruePrime method has a slower amplification rate and therefore, by increasing the incubation time, it was possible to improve the quality of the data. The modified protocol with reduced volume also had the most promising outcome in terms of the data produced, and could fulfil our expectations by being fast, cost-effective and efficient. Conclusion: In conclusion, the results from the first part of this study confirmed the negative impact of male age on assisted reproductive treatments, which can result in decreased success rates of fertilisation. Other factors such as sperm DNA damage may also contribute to this age effect, suggesting that assessing this parameter prior to fertility treatment, and attempting to mitigate elevated levels of sperm DNA damage, may be of value to older patients. Additionally, overcoming the technical challenges in studying genetic contributory factors in infertility is a promising step toward better understanding of the mutations and variations that are involved in this phenomenon.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-03-30T13:52:07Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Adriana_Bispo_final_ficha catalografica.pdf: 4400523 bytes, checksum: b3e9eb79c58483144a4659be5ab2d45c (MD5) / Made available in DSpace on 2016-03-30T13:52:07Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Adriana_Bispo_final_ficha catalografica.pdf: 4400523 bytes, checksum: b3e9eb79c58483144a4659be5ab2d45c (MD5) Previous issue date: 2015-09-15 / A síndrome de Turner (ST) é caracterizada primariamente pelo cariótipo 45,X, mas podem ocorrer linhagens celulares incluindo o cromossomo Y. A precisa identificação do cromossomo Y nessas pacientes é de grande importância clínica devido a um aumento no risco de tumores gonadais. A alta frequência de mosaicismo na ST faz dessa síndrome um importante modelo para investigação do efeito dos polimorfismos dos genes da rota do folato como fatores de risco à não disjunção cromossômica somática. Alterações no metabolismo do folato podem promover aneuploidias por um efeito indireto sobre os padrões de metilação do DNA. Neste trabalho reportamos a frequência de mosaicismo críptico do cromossomo Y e sua associação clínica, como também a descrição de uma alteração cromossômica rara. Adicionalmente, foi investigada uma possível associação entre os polimorfismos de genes da rota do folato e o risco de não disjunção cromossômica somática na ST. A presença de mosaicismo oculto do cromossomo Y foi detectada em 2,7% dos casos, os quais mostraram genitália feminina normal sem sinais de virilização ou desenvolvimento tumoral. Assim, a busca de sequências do Y deve ser realizada na ST independente do cariótipo e/ou sinais clínicos. Não foi possível estabelecer uma associação entre os polimorfismos dos genes MTHFR, MTR, RFC1 e TYMS, independentes ou combinados, modulando o risco de não disjunção somática na ST, demostrando que polimorfismos nesses genes, envolvidos na rota do folato, podem não representar uma importante contribuição para os mecanismos de geração das aneuploidias. / Turner syndrome (TS) is primarily characterized by the 45,X karyotype, but can occur cell lines including the Y-chromosome. The precise identification of Y-chromosome in TS patients is of great clinical importance due to an increased risk of gonadal tumors. The high frequency of mosaicism in TS makes this syndrome an important model to investigate the effect of genetic polymorphisms in folate pathway as risk factors to somatic non-disjunction. Changes in folate metabolism can promote aneuploidies by an indirect effect on the DNA methylation patterns. In this work was reported the frequency of Y-chromosome hidden mosaicism and its clinical association, and also described a rare chromosomal alteration. Additionally, a possible association between gene polymorphisms in folate pathway and the risk of somatic chromosome non-disjunction in TS was investigated. The presence of hidden Y chromosome mosaicism was detected in 2.7% of cases, which showed normal female genitalia without signs virilization or tumor development. Thus, the search for Y sequences should be held at TS regardless of the karyotypes and/or clinical signs. We could not establish an association between polymorphisms of MTHFR, MTR, RFC1 and TYMS genes, independent or combined, modulating the risk of somatic non-disjunction in TS, showing that polymorphisms in these genes, involved in folate metabolism, may not represent an important contribution to the generation mechanisms of aneuploidies.

Abstract Studies on families and individuals with sex chromosome abnormalities and 46,XY females, together with molecular research, have provided proof that both X and Y chromosome genes are expressed in human tooth crown growth. The Y chromosome promotes the formation of both permanent tooth crown enamel and dentin, whereas the effect of the X chromosome is seen mainly in enamel formation. In particular, the effect of the Y chromosome on dentin formation explains the expression of sexual dimorphism in crown size. When crown growth is complete, root dentin is formed and requires proliferation of epithelial cells in Hertwig's epithelial root sheath to initiate the differentiation of root odontoblasts. These epithelial cells determine the size, shape and number of the roots. There is a clear sex difference in tooth crown sizes, men have larger teeth than women. The aim of this research was to study completed permanent tooth root lengths in individuals with sex chromosome abnormalities and 46,XY females, an approach which might also provide some clues for a further insight into the development of sexual dimorphism in human growth. The underlying hypothesis was that the effect of the X and Y chromosomes on crown growth is also expressed in root growth. The subjects were participants of L. Alvesalo's research project, Kvantti, and comprised 45,X/46,XX females, 47,XYY and 47,XXY males and female sex reversals with insensitivity to androgens (46,XY females). The root lengths were measured from dental panoramic radiographs with a sliding digital calliper. All available teeth (except third molars) with complete root formation on both sides of the jaws were measured. The results showed longer final permanent tooth root lengths in 47,XYY and 47,XXY males, while the roots in 45,X/46,XX females were shorter compared with the values of normal men and women, respectively. The root lengths of 46,XY females were longer compared to normal women and placed on a level with normal men. The root morphology did not reveal any major deviations from normal variation. In terms of population dental developmental standards it is conceivable that changes in these study groups in final size of their permanent tooth roots become evident during a period beginning eight years after birth and continuing up to the age of 14 years, at least. It became clear that the effect of the Y chromosome on tooth root growth is greater than that of the X chromosome, and this may cause the observed sexual dimorphism, males having longer roots than females. It is suggested that root growth may be affected by the same genes on the X and Y chromosomes which promote crown growth.

One in six couples wishing to start a family are infertile. The many causes of infertility include genetic defects that can be single gene, multifactorial or chromosomal (including Y deletions, karyotype abnormalities and gamete aneuploidy). This thesis is concerned with the association between infertility and increased sperm aneuploidy. Specific questions are: should males be screened for sperm aneuploidy before intracytoplasmic sperm injection (ICSI)? Is there a relationship between individual semen parameters and sperm aneuploidy for specific chromosome pairs? What is the role of genome organisation in male gametes and its association with infertility? Whether use of alternative therapy (in this case, traditional Chinese Medicine (TCM)) can be used to improve sperm disomy levels. Statistical analysis of questionnaire data revealed that infertility specialists believed there to be merit in screening sperm aneuploidy levels before ICSI. Evidence is presented for possible chromosome-specific and semen parameter specific mechanisms for sperm aneuploidy as is evidence of genome organisation that may be perturbed in infertile males. Finally, in six males studied, sperm aneuploidy levels improved significantly coincident with TCM. Closer investigation of the biological activity of individual therapeutic herbs and treatment cocktails revealed strong anti-oestrogenic and anti-oxidant properties. This suggests a possible mechanism of action of the herbs and provides the basis from which future placebo controlled clinical trials might continue. Possible criticisms of the work presented here include the unavailability of blood samples from many of the patients (thus preventing karyotype analysis) and the absence of a second control group in our studies on semen parameters. Nevertheless significant steps have been made towards establishing the need for, and the implementation of, a pre-ICSI screening test. Moreover progress has been made towards further understanding the aetiology of sperm aneuploidy and towards the implementation of a new treatment that may, ultimately, augment, or even replace ICSI.

Une surprolifération cellulaire associée à de l’aneuploïdie est un marqueur couramment retrouvé dans les cancers et une faible instabilité génétique peut-être un élément aggravant (sinon déclencheur) de la tumorigénèse. Récemment, il a été montré sur un modèle de cellules cancéreuses en culture qu’une forte aneuploïdie compromet la prolifération cellulaire en entraînant la mort de ces dernières. Au cours de ma thèse, nous avons souhaité tester si cette hypothèse se vérifiait in vivo en utilisant comme modèle, les tumeurs du système nerveux central de la larve de D. melanogaster. Nous avons fait le choix d’utiliser des mutants pour des gènes impliqués dans la formation du fuseau mitotique et la ségrégation des chromosomes (Sas-4 ou AurA) afin d’induire ces tumeurs. Pour générer l’aneuploïdie, nous avons choisi d’associer les mutations sas-4 ou aurA avec des mutations pour des gènes essentiels du SAC, Mad2 ou BubR1ken. Nous avons ensuite analysé par immunofluorescence et microscopie l’effet de la perte du SAC sur la prolifération des Nb. Pour sas-4, la perte du SAC cause l’apparition d’une forte aneuploïdie et une baisse du nombre de Nb associée à une forte réduction de taille des cerveaux. Cela compromet totalement la capacité des cerveaux mutants à induire des tumeurs lorsqu’on les injecte dans l’abdomen de mouches adultes saines. Dans le cas d’aurA, ni hausse de l’aneuploïdie dans le tissu ni baisse de la prolifération des Nbs n’ont été observés. Par ailleurs, la même forte proportion de mouches injectées avec des cerveaux aurA ou aurA mad2 développant une tumeur a été constaté. Afin de mieux comprendre pourquoi le mutant aurA ne réagit pas comme le mutant sas-4 à la déplétion du SAC, nous avons entrepris une analyse détaillée des mutants aurA et aurA mad2. Nous avons d’abord observé que, malgré la perte du SAC, 1) il existe toujours un délai en mitose dans aurA mad2 et 2) il existe un délai entre la satisfaction du SAC et l’entrée en anaphase dans aurA. Comme l’entrée en anaphase est dépendante de la dégradation de la CycB et de la Sécurine via l’APC/C, nous avons analysé le comportement de la CycB (couplé à une étiquette GFP) par vidéo-microscopie en temps réel et observé un défaut de la régulation de la dégradation de cette dernière dans le mutant aurA ainsi que dans le double mutant aurA mad2. Ces observations nous ont permis de proposer un nouveau rôle pour la kinase AurA dans la régulation de la dégradation de la CycB en fin de mitose. / Cellular overproliferation associated with aneuploidy is a common hallmark of cancers. Low genetic instability may be a contributing factor of tumorigenesis. Recently, it was shown on a cellular cancer model in culture that strong aneuploidy compromises cell proliferation by causing cell death. During my thesis, we have test if this hypothesis was verified in vivo by using as a model, the tumours of the larval central nervous system of D. melanogaster. We decided to use mutants involved in mitotic spindle formation and chromosome segregation (Sas-4 or AurA) to induce these tumours. To generate aneuploidy, we chose to associate these mutations with mutations in genes essential for the SAC, Mad2 or BubR1ken. We then analysed the effect of the SAC depletion on the Nb proliferation. For sas-4, loss of the SAC leads to high aneuploidy and a decrease in Nb number associated with brain size reduction. It completely undermines the ability of mutant brain to induce tumors when injected into the abdomen of healthy adult flies. In the case of aurA, nor increase of aneuploidy in tissue or decrease in nb proliferation have been observed. Moreover, the same proportion of flies injected with aurA or aurA mad2 brains developed tumours. To better understand why the aurA mutant not react as the sas-4 mutant to the SAC depletion, we undertook a detailed analysis of aurA and aurA mad2 mutants. We first observed that despite the SAC depletion, 1) there is always a delay in mitosis in aurA mad2 and 2) there is a delay between SAC satisfaction and anaphase onset in aurA. Since anaphase onset is dependent of the CycB and Securine degradation via the APC / C, we analysed the behaviour of the CycB (coupled with a GFP tag) by real-time videomicroscopy and observed a defect in the regulation of CycB degradation in aurA and in the double aurA mad2 mutant. These observations lead us to propose a new role for AurA kinase in regulating the degradation of CycB at the end of mitosis.