Dissertationen zum Thema „Cytogenetic study“

Ulcerative colitis and Crohn's colitis are chronic inflammatory bowel diseases that occur with a high frequency in the North-East of Scotland. They are diseases of adolescents and young adults, although they may have their onset at any age. The aetiology is largely unknown. Oral sulphasalazine is used as the treatment of choice for the prevention of relapses. At present there is no satisfactory alternative and so, on present practice, patients may take the drug indefinitely after diagnosis of the disease. Inflammatory bowel disease itself does not increase the levels of sister chromatid exchange and micronuclei observed in the lymphocytes of the patients. Patients receiving sulphasalazine therapy, however, have significantly elevated levels of sister chromatid exchange and micronuclei in their lymphocytes compared to control individuals. In addition, patients show a significant elevation in sister chromatid exchange and micronuclei frequencies after commencing sulphasalazine therapy. The length of time on sulphasalazine is influential in determining the sister chromatid exchange frequency as is the acetylator phenotype of the patient. Sister chromatid exchange frequencies appear to remain elevated for many months after cessation of therapy, suggesting that the lesions produced by the sulphasalzine therapy are long-lived. In vitro studies have shown that sulphasalazine induces both sister chromatid exchange and micronuclei in human lymphocytes, whereas sulphapyridine and its acetylated metabolites induce only sister chromatid exchanges. 5-aminosalicylic acid, the active moiety of sulphasalazine, and its acetylated metabolite do not induce sister chromatid exchange or micronuclei at the concentrations tested. The present and future use of sulphasalazine therapy should be evaluated in light of these results, and the use of new drugs, based on the 5-aminosalicylic moiety should be encouraged to reduce the potential genetic risk to the patients.

Sister chromatid exchange (SCE) and micronuclei (MN) were studied in peripheral human lymphocytes, treated in vivo with atenolol, azathioprine (AZA) and Chlorpropamide (CPA), and from patients on long term therapy with these drugs. The clinical study consisted of samples from 18 atenolol, 8 AZA and 9 CPA treated patients, and 35 age and sex matched controls. The CPA patients' controls were matched for disease (diabetes). Additional controls monitored variation between culture time. 20 metaphases were scored for SCEs and 2000 cells for MN per person. Statistical comparison of treated and untreated groups were with two-sample t-tests (SCE data) and 2x2 contingency χ² tests (MN data). There were two significant results. The atenolol treated had more MN (p< 0.01) and the AZA treated more SCEs (p< 0.02) than their control groups. The CPA treated and control groups did not differ. In the in vitro study concentrations used were, Atenolol 1.3, 2.7, 6.7 and 13.3ug/ml, AZA 5, 25, 50 and 100ug/ml and CPA and Diabinese (DIA) (pharmaceutical preparation of CPA) 250, 500 and 1000ug/ml. All culture lengths were 72 hours after mitotic stimulation (0 hours). Several exposure regions were used, they were: With metabolising enzymes (S9), all drugs for 6 hours at 48 hours. Without S9, Atenolol for 72 hours, AZA for 24 hours (at times - 24 hours and 48 hours), CPA and DIA for 24 hours at 48 hours. 20 metaphases were scored for SCEs and 4000 cells for MN. Dosed cultures were compared statistically with the control cultures using analyses of variance (SCE data) and linear regression analyses (MN). The significant results were; AZA, increases in SCEs in all experiments, increases in MN with S9 and without S9 (experiment b); CPA increase in MN with S9, decrease in MN without S9. No effects were found with atenolol or DIA treatment.

This study involved the combination of molecular-cytogenetic data and phylogenetic approaches to infer pathways by which chromosome numbers and sizes may have changed during the course of evolution. The two systems for which I generated new data are the monocot plant family Araceae and Coccinia, a genus of Cucurbitaceae. Araceae have about 3800 species in 118 genera, and chromosome numbers range from 2n = 168 to 2n = 8, the latter the lowest number so far and newly reported in my study. The small genus Coccinia includes C. grandis, with the largest known Y chromosome in plants, as documented in my work. The thesis comprises four published or submitted papers. The first paper reports the result of phylogenetic modeling of chromosome number change along a phylogeny for the Araceae with 113 genera represented. I used a maximum likelihood approach to find the most likely combination of events explaining today’s chromosome numbers in the 113 genera. The permitted events were chromosome gains (i.e. breaks), losses (i.e. fusions), doubling (polyploidization), or fusion of gametes with different ploidy. The best-fitting model inferred an ancestral haploid number of 16 or 18, higher than previously suggested numbers, a large role for chromosome fusion, and a limited role of polyploidization. The sparse taxon sampling and deep age (at least 120 Ma) of the events near the root of Araceae caution against placing too much weight on “ancestral” numbers, but inferred events in more closely related species can be tested with cytogenetic methods, which I did in two further studies (papers 2 and 3). I selected Typhonium, with 50-60 species, a range of 2n = 8 to 2n = 65 chromosomes. The family-wide study had suggested a reduction from a = 14 to 13 by fusion in Typhonium, but had included relatively few of its species. I built a phylogeny that included 96 species and subspecies sequenced for a nuclear and two chloroplast markers, and then selected 10 species with 2n = 8 to 24 on which to perform fluorescence in situ hybridization (FISH) with three chromosomal probes (5S rDNA, 45S rDNA, and Arabidopsis-like telomeres; paper 2). The results supported chromosome fusion in two species where I found interstitially located telomere repeats (ITRs), which can be a signal of end-to-end fusions, and polyploidization in one species where I found multiple rDNA sites. I then extended my cytological work to other lineages of Araceae, selecting 14 species from 11 genera in key positions in the family phylogeny, which I enlarged to 174 species, adding new chromosome counts and FISH data for 14 species with 2n = 14 to 2n = 60 (paper 3). With the new data, I confirmed descending dysploidy as common in the Araceae, and I found no correlation between the number of rDNA sites and ploidy level (which would have pointed to recent polyploidy). I detected ITRs in three further species, all with 2n = 30. I also discovered gymnosperms-like massive repeat amplification in Anthurium. Similar ITRs are only known from Pinus species. Paper 4 presents molecular-cytogenetic data for Coccinia grandis, one of a handful of angiosperms with heteromorphic sex chromosomes. The male/female C-value difference in this species is 0.09 pg or 10% of the total genome. My FISH and GISH results revealed that the Y chromosome is heterochromatic, similar to the Y chromosomes of Rumex acetosa, but different from the euchromatic Y chromosome of Silene latifolia; it is more than 2x larger than the largest other chromosome in the genome, making C. grandis an ideal system for sequencing and studying the molecular steps of sex chromosome differentiation in plants.

by Wang Wei.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1991.
Includes bibliographical references (leaves 154-168).
ACKNOWLEDGMENTS --- p.v
SUMMARY --- p.vi
PUBLICATIONS --- p.viii
STATEMENT OF ORIGINALITY --- p.ix
LIST OF ABBREVIATIONS --- p.x
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.5
Chapter 2.1 --- Chromosome --- p.6
Chapter 2.2 --- Chromosome and Human Disease --- p.9
Chapter 2.3 --- Chromosome and Tumour --- p.12
Chapter 2.4 --- Chromosome in Gynaecologic Tumours --- p.17
Chapter 2.4.1 --- Cervical tumour --- p.17
Chapter 2.4.2 --- Uterine corpus tumour --- p.20
Chapter 2.4.3 --- Ovarian tumour --- p.23
Chapter 2.5 --- Methodology in cytogenetics --- p.26
Chapter 2.5.1 --- Materials --- p.27
Chapter 2.5.2 --- Methods of chromosome preparation --- p.28
Chapter 2.5.3 --- Karyotype analysis --- p.34
Chapter 2.6 --- Problems of cytogenetic analysis in solid tumour --- p.42
Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.47
Chapter 3.1 --- Chemicals and Solutions --- p.48
Chapter 3.2 --- Chromosome preparation from solid gynaecologic tumours --- p.50
Chapter 3.2.1 --- Solid tumour specimens --- p.50
Chapter 3.2.2 --- Chromosome preparation --- p.54
Chapter 3.3 --- Chromosome preparation from an established ovarian carcinoma cell line --- p.61
Chapter 3.3.1 --- Origin of OCC1 cell line --- p.61
Chapter 3.3.2 --- Characteristics of OCC1 cell line --- p.61
Chapter 3.3.3 --- Maintaining of OCC1 cell line --- p.62
Chapter 3.3.4 --- Chromosome preparation --- p.62
Chapter 3.4 --- Karyotype analysis --- p.65
Chapter CHAPTER 4 --- RESULTS --- p.66
Chapter 4.1 --- Cytogenetic features of gynaecologic solid tumour --- p.67
Chapter 4.1.1 --- Cervical cancer --- p.67
Chapter 4.1.2 --- Uterine corpus cancer --- p.94
Chapter 4.1.3 --- Ovarian cancer --- p.104
Chapter 4.2 --- Cytogenetic features of OCC1 ovarian carcinoma cell line --- p.114
Chapter CHAPTER 5 --- DISCUSSION --- p.123
Chapter 5.1 --- Methodology of chromosome preparation in solid tumour --- p.124
Chapter 5.2 --- Chromosome changes in gynaecologic solid tumour --- p.126
Chapter 5.2.1 --- Cervical cancer --- p.126
Chapter 5.2.2 --- Uterine corpus cancer --- p.132
Chapter 5.2.3 --- Ovarian cancer --- p.138
Chapter 5.2.4 --- In summary --- p.141
Chapter 5.3 --- Chromosome changes in an OCC1 ovarian carcinoma cell line --- p.143
Chapter CHAPTER 6 --- CONCLUSION --- p.148
REFERENCES --- p.154

碩士
長庚醫學暨工程學院
基礎醫學研究所
85
Median Cerebrofacial Malformations (MCFM) is a common genetic defect during the fetal development. The patients demostrate the loss of median anatomical structures in the forebrain and midface regions. Some genetics named all the MCFMs as holoprosencephaly, but differentiated them into 4 types accordings to defective chromosomes. The minimun critical regions of the candidate genes are located at chromosome 21q22.3 for HPE1, 2p21 for HPE2,7q36 for HPE3, and 18p11.3 for HPE4. Six MCFM patients were collected and grouped as HPE4 according to the facial appearance with cleft lip and palate, deficicncy of nasal septum, and hypoplasia of prolab and premaxilla. Two of them have been proved with defects on the short arm of chromosome 18 by karyotyping. Furthermore, two patients with microcephaly and mental retardation other than the defects described above were categorized as HPE2. To prove the grouping,eight patientswe examined the blood cells cultured from the venous blood in this study with a molecular cytogenetic technique,fluorescence in situ hybridization (FISH). The probes for HPE4 were a serial of lambda phages clones on the 18p adapted from literature. Those for HPE2 were cloned by ourselves. The PCR fragments were cloned into plasmid vector, and multiplied as the probes for screening human lambda genomic library for the correct clones, which were applied as the probes for FISH. These probes were used for detecting the possible deletions on the patient chromosomes. We localized the deleted region of two patients. One lost the segment from 18p11.2 to pter; the other with ring chromosome 18 lost 18p11.32 to pter, which is a smaller deletion than those of any published cases. With this approach of the gene HPE4. Moreover, with FISH it is not necessary to examine metaphase chromosomes for detecting the deletion of the DNA segment, thus the method can be applied clinically on the prenatal screening. We think examining the interphase nuclei of amniocytes with FISH would be more accurate and much prompter than traditional karyotyping.

碩士
國立臺灣大學
病理學研究所
85
Solid tumor cytogenetic study had been quite underdeveloped compared with l eukemia and lymphoma. There were many factors,including difficulties in tumor culture and getting metaphase, and complex chromosome patterns. The rapid adva nce in molecular cytogenetics, including fluorescence in situ hybridization an d comparative genomic hybridization, has make great breakthoughin the study of the solid tumor cytogenetic. My thesis included studies ina series of solid t umors by using FISH technique. The first and second study allused chromosome p ainting probes to identify the chromosome component of the marker chromosomes in pulmonary hamartoma and dermatofibrosarcoma protuberans respectively.The th ird study was chromosome aberrations in prostate cancer. We use Mega-YACprobe for specific chromosome sites to study the deletion patterns. Our results reve aled that chromosome 8p deletion is associated with the development of prostat e cancer. The fourth study was chromosome aberrations in hepatocellular carcin oma.We used 8 mega-YAC probes and 6 centromere probes on 17 hepatoma and one h epatic adenoma. The results revealed chromosome 4q as the most frequent deleti on site (76.5%),Chromosome 8p as the second most frequent site (59%). These t wo chromosomesites should be the most important site harboring tumor suppresso r genes related to the tumorigenesis of hepatoma.

Ng Heung-ling, Margaret.
"April 2003."
Thesis (M.D.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (p. 216-237).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.

碩士
國立臺北科技大學
生化與生醫工程研究所
101
According to the research, renal cell carcinoma and bladder cancer incidence in Taiwan are rising year by year. APTX plays a major role in single/double strand DNA repair. In recent years, APTX gene becomes a biomarker of the cancer for irinotecan chemotherapy. One of the mutation hotspot of LRRK2 is G2019S, which plays a major role in both neurodegenerative disease and cancer. In current study, we collected 8 RCC and 5 TCC cell lines. The abnormality of genes APTX, LRRK2 and MET were observed by fluorescence in situ hybridization (FISH). Q-PCR and western blot were used to observe APTX mRNA and protein expression. FISH analysis showed amplification of APTX gene, only presented in one RCC cell lines (RCC52) with four gene copies, however the rest of the cell lines showed normal gene copy number. We then further examined the mRNA expression level of APTX in all cell lines. Interestingly, the result of RCC52 cell line remained consistence with high expression of RNA when compared to the normal renal cells, on the other hand, other cell lines presented lower expression of APTX gene while compared to the normal renal cell. The result of protein level shows different expression of Aprataxin in clear cell subtype. We suggest that APTX gene most likely play as the function of tumor suppressor gene in RCCs. Once a mutation occurred on this gene sequence, it might indirectly trigger tumoral formation. Although LRRK2 gene copy number was normal, we suggest that mutation of gene LRRK2 can be identified by sequencing.

Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2019
Os gliomas são os tumores primários mais frequentes do Sistema Nervoso Central, representando 50% de todos os casos de tumores cerebrais. Incluem, entre outros, os astrocitomas (AT) e os oligodendrogliomas (OLG). Uma designação comum para estes tumores, quando se localizam num ou mais lobos cerebrais, na região supratentorial é a de gliomas difusos e atingem, caracteristicamente, jovens adultos. Esses dois tipos de gliomas são considerados de baixo grau e são infiltrativos, de crescimento lento. Os gliomas de baixo grau – grau II, pela Organização Mundial de Saúde – tem uma forte tendência para a progressão maligna, para gliomas anaplásicos (grau III, pela OMS) e até glioblastomas secundários (grau IV, pela OMS), o que, muitas vezes, ocorre após alguns anos, geralmente, entre cerca de 4 a 5. Muitas vezes, após uma resseção cirúrgica de gliomas de baixo grau, as células neoplásicas deixadas no cérebro podem originar um tumor recidivante, que muitas vezes se transforma em glioma de alto grau, com prognóstico variável a longo prazo e uma taxa de sobrevida de entre 5 a 8 anos. A nova classificação da OMS, no que diz respeito a Tumores do Sistema Nervoso Central, revista em 2016, introduziu parâmetros moleculares como mutações IDH, co-deleções de 1p/19q e perdas de ATRX, agrupando os tumores em categorias de acordo com seus perfis genéticos, além dos padrões histológicos usados até então. Sendo assim, é possível classificar os gliomas difusos em astrocitomas, IDH mutantes; oligodendrogliomas, IDH mutantes e 1p/19q co-deletados; astrocitomas IDH wild-type; glioblastomas IDH mutantes; glioblastomas IDH wild-type; oligodendrogliomas sem outras especificações; astrocitomas sem outras especificações; oligoastrocitomas sem outras especificações e glioblastomas sem outras especificações. Os critérios usados para definir o grau de anaplasia dos gliomas, definidos pela OMS, são polimorfismo nuclear e hipercromasia, índice mitótico, proliferação endotelial da microvascularização tumoral e necrose do parênquima tumoral. Estes critérios permitem aos patologistas classificar os gliomas difusos em diferentes graus de malignidade, desde grau II, o menos maligno, até aos graus III e IV, os mais malignos, dos quais o glioblastoma é o mais comum. As alterações moleculares no processo de tumorigénese levam à ativação de oncogenes ou à inativação de genes supressores de tumor. Alguns dos marcadores genéticos de grande relevância no processo de tumorigénese e na determinação do tipo e grau de anaplasia dos gliomas difusos são o 1p/19q (co-deleção), o EGFR, o PTEN e o CDKN2A. A presença de mutação do ATRX ajuda ao diagnóstico dos AT com IDH mutante, distinguindo-os dos OLG. Os AT anaplásicos com mutações combinadas no ATRX e no IDH têm melhor prognóstico do que os que só têm a mutação do IDH. As mutações no codão 132 do gene IDH ocorrem cedo e a uma frequência elevada em AT, OLG de graus II e III, e em glioblastomas secundários desenvolvidos a partir de AT. Os objetivos deste estudo são comparar o perfil imunohistoquímico e as alterações citogenéticas encontradas em 16 casos de doentes com cirurgia a gliomas primários de baixo grau, e a recidiva de grau mais elevado, resultando num total de 32 amostras – 18 oligodendrogliomas, 10 astrocitomas, 3 oligoastrocitomas e 1 gliossarcoma; avaliar e quantificar as alterações e identificar subpopulações baseadas em marcadores, nas amostras de tumores de ambas as cirurgias. Para isso, obtiveram-se lâminas de imunohistoquímica e fizeram-se blocos de Tissue Micro Arrays, dos quais se obtiveram lâminas para Hibridação In Situ por Fluorescência. As proteínas estudadas foram GFAP, IDH1, KI-67, ATRX e Olig-2, e os genes foram CDKN2A, p53, EGFR, PTEN, 1p e 19q. Realizou-se a técnica de imunohistoquímica para os marcadores IDH, ATRX e GFAP, posteriormente fotografados no microscópio ótico, e analisados com programa Image-J (plugin Colour Deconvolution). Às áreas de interesse foram-lhes atribuídas cores secundárias (cada uma associada a um marcador) e as imagens resultantes foram sobrepostas originando áreas de cores primárias. Foi calculado o rácio de pixéis de cada cor de interesse. Realizou-se igualmente a técnica de imunohistoquímica para os marcadores Olig-2 e KI-67, também fotografados no microscópio ótico e analisados no programa Image-J (plugin ImmunoRatio). Realizou-se ainda Hibridação in situ de fluorescência, para analisar os genes CDKN2A, p53, EGFR, PTEN, 1p e 19q, em lâminas de Tissue Micro Array, que foram fotografadas no microscópio de fluorescência e analisadas no programa Image-J (plugin Cell Counter para contagem dos núcleos). Este estudo incluiu 18 oligodendrogliomas, 10 astrocitomas, 3 oligoastrocitomas e 1 gliossarcoma. A análise foi feita inicialmente para os marcadores individuais e, em seguida, para as subpopulações definidas, baseadas na classificação actual de gliomas difusos. Nenhuma das amostras estudadas apresentou deleção do gene supressor tumoral PTEN. O estudo de EGFR mostrou amplificação em apenas 6 das 32 amostras, sendo 4 exclusivamente nas recidivas e 1 exclusivamente num tumor primário de baixo grau. O p53 estava mutado em 6 das 32 amostras estudadas, sendo que 4 desses 6 tumores com mutação eram recidivas. Analisando as alterações de primários para recidivas, encontraram-se 4 casos com p53 wild-type no primário e mutação nas recidivas e 1 caso que manteve a mutação em ambos os grupos. O CDKN2A estava deletado no primário e na recidiva em simultâneo, em apenas 1 caso. 6 casos tinham deleções nos primários e apenas 3 tinham deleções nas recidivas. A expressão de KI-67 apresentou valores mais elevados nas recidivas do que nos primários. Relativamente à expressão de Olig-2, observou-se o contrário, sendo os valores mais elevados nos primários do que nas recidivas. Não se encontraram diferenças major nas três subpopulações estudadas (IDH1mut/ATRXloss, IDH1mut/ATRX/1p/19q co-deletadas e IDH1wt) entre as amostras de tumores primários e as suas respectivas recidivas. Descobriram-se mais células tumorais IDH1mut/ATRXloss em recidivas (9799±24384) do que em primários (5053±10116), e mais células tumorais IDH1- em primários (671939±180448) do que nas recidivas (609653±284091). Contudo, estas diferenças não foram muito evidentes. Células IDH1mut/ATRX/1p/19q co-deletadas foram encontradas em apenas um dos dezasseis casos estudados. As proteínas e genes estudados cobrem a maioria das principais vias de sinalização molecular que levam ao desenvolvimento de carcinomas. Contudo, a ausência de variações muito evidentes entre os dois grupos comparados (primário e respetiva recidiva) indica que poder-se-á não estar a estudar os marcadores mais relevantes para esta evolução. Outros marcadores que poderão ser relevantes são o PDGF e o Ras. O PDGF é um agente mitogénico de células mesenquimais, incluindo células gliais e já foi associado a glioblastomas (vias PI3k/AKT e Ras-Raf-Mek-Erk). O gene Ras foi o primeiro oncogene humano a ser identificado e sabe-se que está mutado em cerca de um terço dos carcinomas. Estes são apenas dois exemplos de outras moléculas que podem ser estudadas nos gliomas. Este estudo é um passo noutra direcção, no que diz respeito aos marcadores biológicos em gliomas. Os resultados obtidos levantam variadas questões face aos marcadores estudados e oferecem uma série de sugestões de outros a considerar; e, uma vez que não há bibliografia de suporte, seria interessante continuar a estudar esta linha de progressão tumoral do primário para a recidiva. O objetivo não seria apenas caracterizar subgrupos histológicos de gliomas do ponto de vista genético e molecular mas também, e acima de tudo, tentar compreender a evolução do tumor primário e os mecanismos e ferramentas biológicas presentes no próprio, que lhe permite recidivar com um nível de malignidade superior. Se se conseguir prever estas transformações no tempo, poder-se-á tentar controlar a progressão da doença logo desde o momento do diagnóstico.
The most frequent primary tumors of the Central Nervous System are gliomas, representing 50% of all brain tumor cases, which include, among others, astrocytomas and oligodendrogliomas. Those two types of gliomas, considered low-grade gliomas, are infiltrative and slow-growing. Low-grade gliomas (World Health Organization grade II) have a strong tendency for malignant progression to anaplastic gliomas (World Health Organization grade III) and even secondary glioblastomas (World Health Organization grade IV), which often takes place after a few years, usually about 4 to 5. Indeed, after a surgical resection of LGG, cancerous cells left in the brain can give rise to a recurrent tumor, often transformed in a high-grade glioma, with variable long-term prognoses and a survival rate between 5 to 8 years. The new World Health Organization Classification of Tumors of the Central Nervous System, revised in 2016, introduced molecular parameters, such as IDH mutations, 1p/19q co-deletions and ATRX losses, for grouping tumors into categories according to their genetic profiles besides the histologic patterns used until then. The aim of this study is to compare immunohistochemical profile and cytogenetic changes in 16 cases with two different surgeries from the same patient, treated for recurrences, evaluate and quantify those changes and identify marker based subpopulations in tumour samples from both surgeries. The proteins studied were GFAP, IDH1, KI-67, ATRX and Olig-2, and the genes were CDKN2A, p53, EGFR, PTEN, 1p and 19q. We did not found major differences in the populations we studied (IDH1mut/ATRXloss, IDH1mut/ATRX/1p/19q co-del and IDH1wt) between primaries and their relapses. However, differences were found in Ki-67, Olig-2, EGFR and CDKN2A between the two groups studied (primary tumors and relapses). All proteins and genes studied cover most of the main pathways that lead to cancer development, which may lead us to think that we are looking at the wrong set of markers. We found some results that suggest it should be interesting to continue this type of research of comparing primary tumors with their own relapses, and try to understand what makes tumors relapse in a much more aggressive form, in order to control the progression of the disease right in the moment of the diagnosis.

Zootechnics has the mission to improve animal production, according to the human needs, that will influence market demand and animal and human health. Animal reproduction gives an important contribution giving rise to an "extreme Tango" in which genetics is the crazy score with a solid melody but which can change depending on the context in which this music is played. This thesis reports the research studies carried out during the doctorate. The Disorders of Sexual Development are the main topic of the first chapter. These are genetic diseases involving the reproductive system that cause sterility to the carriers and economic losses to the breeders. The animals involved in this study are dogs and horses, they were examined clinically, cytogenetically and genetically in order to identify DSD etiopatogenesis of each subject. The main topic of the second chapter is the improvement of stallions’ sperm quality using phytotherapic drugs. This study has a dual purpose, to improve the natural breeding and the techniques of artificial insemination. Morphometric analyzes and chromatin fragmentation tests were performed on the ejaculates in addition to the qualitative and quantitative evaluations routinely carried out. The Third Chapter describes a work born by a collaboration among different research groups. Exploiting the training acquired on the morphometric evaluation of the spermatozoa of different mammals’ species we have applied the same techniques on the spermatozoa of Apis mellifera Ligustica, in order to characterize its semen.

碩士
國立陽明大學
遺傳學研究所
94
Amniocentesis is the most common, although invasive, test for prenatal diagnosis of fetal chromosome abnormalities. Indications for fetal chromosome analysis include advanced maternal age, abnormal maternal serum screening results, abnormal ultrasound findings, a previous child with congenital anomaly, family history of chromosome aberrations, and other minor purposes. We collected 101,528 amniocentesis cases for fetal chromosome analysis performed in a five-year interval, from year 2000 to 2004, in Taiwan. Among these, 59.84% were referred for advanced maternal age, 24.92% for abnormal maternal serum screening results, 7.45% for abnormal ultrasound findings, 1.89% for a previous child with congenital anomaly, 0.90% for a family history of chromosome aberrations, and 5.01% for other purposes. Chromosome aberrations were detected in 2,670 cases (2.63%), among these 44.76% cases were autosomal aneuploidy (25.24% were Down syndrome), 19.89% were sex chromosome aneuploidy, and 35.36% were structural abnormalities. Abnormal maternal serum screening results have a higher positive rate to detect cases with Down syndrome and structural abnormalities. Abnormal ultrasound findings were the most informative for Trisomy 18 and Turner syndrome. Sex chromosome abnormalities (SCA) are less damaging to the appearance and intelligence. Couples faced with a very difficult decision-making process when informed about their fetus’ diagnosis of SCA. We reported parental decisions following the prenatal diagnosis of SCA during these five years and compared with the outcome of prenatal SCA in other countries. Parents’ notion and perception were retrospectively interviewed to investigate what kind of information they had obtained during genetic counseling and what additional resources would have been helpful during their decision making. In this retrospective study, we also used semi-structured interview and questionnaires to examine patterns of genetic counseling, parents’ immediate emotion, and their satisfaction with counseling based on 50 parents of children with Turner syndrome. From these results, we are able to know their experience and difficulty, and what additional help and information should have been provided. These information can be beneficial for further improvements in both prenatal as well as postnatal genetic counseling.

Bibliography: leaves 70-85. The aim of this study was to develop the midget chromosome as a model system to isolate structural elements from a cereal chromomsome. The midget is a diminutive chromosome found in wheat plants that contain a substituted rye cytoplasm.

碩士
國立臺灣師範大學
生命科學研究所
95
Five known species of Japalura lizards are found in Taiwan, including J. swinhinis, J. brevipes, J. makii, J. luei, and J. polygonata xanthostoma. Great variation have been found among inter- and intra- species, however, no cryptic specie can be detected based on their morphologies, mitochondria RFLP and 12S rRNA except for the chromosome data. Cytogenetic analysis is proved to be useful in establishing animal phylogeny, especially in the identification of Japalura. The present study focus on building reconstructing the phylogenetic relationships of all Japalura including new cryptic species found in Taiwan via cytogenetical approaches. Seven species including two new species of Japalura lizards from Taiwan were successfully karyotyped. One new specie is Japalura sp. 1 distributed in the low mountains in Taichung and Nantou Counties, another is Japalura sp. 2 distributed in the Hsueshan Mountain Range. Chromosome numbers of Japalura sp. 1 and Japalura sp. 2 are 2N = 46 and 36, respectively. The sex-determining mechanism of J. sp. 1 is ZZ/ZW type, and the others have no sex chromosome. The C-bands of chromosomes in all Taiwanese Japalura lizards studied are not successfully stained thus is not included in the present study. There is only one pair of Ag-NORs in the karyotypes that are located at the distal end of the 18th pair in Japalura swinhonis and J. brevipes, the 13th pair in J. makii and J. luei, the 21th pair in J. p. xanthostoma, the 14th pair in J. sp. 1 and the 17th pair in J. sp. 2. The macrochromosomes of all taxa showed remarkable G-bands that are species specific. Karyotypic patterns among four out of seven species, Japalura brevipes, J. makii, J. luei and J. sp. 2, are relatively similar, especially in the 1st and 2nd pairs of chromosomes, that infers the close relationship among these four species. Japalura brevipes and J. sp. 2 have more similar banding patterns and are more closely related. Two hypothesis about evolutions of the genus Japalura from Taiwan are proposed according to cytogenetic analysis. The fusion hypothesis proposed the present karyotypes with low diploid chromosome numbers and high biarmchromosome numbers were derived from a primitive karyotype with 2N = 46 and all telocentric chromosomes by Robertsonian fusions, while the fission hypothesis derived from a primitive karyotype with 2N = 36 that are suspected to Robertsonian fissions to form the present karyotypes. All the morphological data, the ribosomal 12S rRNA, and the present NORs-carrying marker chromosomes support the fusion hypothesis and against the fission hypothesis. In summary, the Japalura species in Taiwan can be separated into two sister clusters: the Japalura swinhonis group (J. swinhonis and J. sp. 1) and the Japalura polygonata group (J. p. xanthostoma, J. makii, J. luei, J. brevipes, and J. sp. 2).

博士
高雄醫學大學
醫學研究所
98
Most of intracranial meningiomas are slow growth and benign. Even meningioma is removed totally; high percentages of recurrence were noted during following-up period. Occasionally meningiomas show histologic progression to a higher grade II and III. In our clinical data, between July 1982 and December 2008, 517 cases of intracranial meningioma received surgical intervention. There were 419 (81%) cases with total resection of tumor. Ninety-two cases recurred, with 22.2%of total resection. Among 92 recurrent cases, there were 80 cases （20.7%） of benign type, 9 cases（33.3%）of atypical type, and 5 cases（45.4% ）of malignant type. The ratio of female to male is 2.2 to 1. Most of cases occurred at 4th to 6th decade. Most of tumors located on convexity or parasagittal/falx. The high recurrence rat in pathobiology of meningioma still is not clear. The genesis of meningiomas has been associated with loss of genetic material on chromosome 22. Twenty-one tumors in our series were examined by means of conventional cytogenetic analysis and fifteen tumors were also examined by comparative genomic hybridization (CGH). Results showed 47.6% of tumors were loss of chromosome 22, loss of the other chromosome including chromosome 9,14, 18, 19 and 21. The CGH findings suggested that loss of 1p is genetic changes implicated in the malignant progression of meningioma. Deletion of 1p, mainly in regions of 1p36, 1p35-p32, and 1p22-p13, is the most frequent progression associated chromosome aberration in meningiomas and is highly correlated with increased tumor recurrence (80%). The gains of losses of chromosomes are potent initiators and promoters of tumorigenesis, chromosome instability (CIN) is a result of chromosome missegregation caused by deregulation of centrosome cycle. Therefore, centrosome deregulation causes sustained CIN, hence providing the link between centrosome and tumorigenesis. The of centosomal protein (centrin, hNinein, and γ-tubulin), Aurora-A and Aurora-B were examined in meningiomas using RT-PCR. There are 29 intracranial meningiomas including 25 WHO grade I and 4 WHO grade II and III. The results showed centrin 2 overexpression in WHO grade I is 96％and WHO grade II, III is 100%. But centrin 3 is all overexpression in meningioma. In γ-tubulin, the overexpression rate in WHO Grade I was is 80%, and then in Grade II, III was 100%. In WHO Grade I, the expression rate of hNinein isoform 1, 2, 5 was 48%, 68% and 72% respectively. In WHO grade II and III the rate was 50%, 75% and 75% respectively. But the isoform 6 was not overexpression. As for the Aurora A and B, in the WHO grade I, the overexpression rate of Aurora A and B was 76% and 96% respectively, and in WHO grade II and III was 75% and 100%. Therefore centrosomal proteins including centrin 2, 3, and γ-tubulin, Aurora A and Aurora B may be attributed to inappropriate centrosome; and then tumorigenesis of meningioma. Proliferative markers have been used to predict the clinical course of meningioma. The purpose of the study is to investigate the utility of identify centrosome abnormalities, as a predictor of increased risk of recurrence in meningiomas after gross total resection. Seventy-five patients had gross total resection. To identity centrosome abnormalities, the immunoflurorescerce technique with γ-tubulin antibody was applied to paraffin-embedded sections. Among all tumors, 27(36%) expressed a centrosome abnormality. These displayed a higher recurrence rate (41%) than those not expressing centrosome abnormalities (8%) (P=0.001). In WHO Grade I meningiomas, the presence of a centrosome abnormality was directly correlated with an increased recurrence rate (38%) compared to those lacking this change (7%) (p=0.002). Meningothelial meningiomas with a centrosome abnormality displayed a significant increase in recurrence rate (42%) when compared to those without (8%) (P=0.007). In conclusion, the current results reveal that expression of a centrosome abnormality directly correlates with an increased recurrence rate among benign meningiomas after gross total resection. The presence of a centrosome abnormality may be a potential indicator of a meningiomas tendency to recur. In microarray survey, down regulated of DLC-1 was found in meningioma. Therefore, we want to study the function DLC-1 in primary meningioma cells growth. The relation of DLC-1 between benign and non-benign was also examined using immunohistochemical stain. Primary meningioma cell culture was established. The primary meningioma cells were transfected with AdDLC-1, AdGFP. In order to check for inhibition of meningioma cell growth by direct cell counting and by crystal violet staining, the result is the DLC-1 adenoviral vector significantly suppressed cell numbers compared to equivalent titer of AdGFP. In immunohistochemical stain of DLC-1 in paraffin-embedded sections, 22 cases of specimen were stained including 9 cases of grade I and 13 cases of grade II and III. In grade I, among nine cases, two cases showed negative-weak staining of DLC-1 and seven cases showed moderate-strong staining. Then, in grade II, III, among 13 cases, nine cases showed negative -weak staining, and four cases showed moderate-strong staining. In 11 recurrent cases, one case of grade I showed weak staining, and five showed moderate-strong staining. Among grade II, III all five cases showed negative-weak staining. In conclusion, DLC-1 may play the inhibition of tumor cells proliferation in meningioma. Immunohistochemical study showed that grade I meningiomas and recurrence grade I expressed significantly high DLC-1 than those of grade II and III. Increased expression of endothelin 1 (ET1) has been shown in various human malignancy, marked neovascularization is a hall mark of many neoplasm in the nervous system. ET1 has an angiogenic effect through vascular endothelial growth factor (VEGF). For this reason, we want to study the relationship of ET1 and VEGF in serum and meningioma tissue. The serum of ET1 and VEGF showed no positive correlation, but in meningioma tissue using immunohistochmical staining showed positive correlation of ET1 and VEGF. The grade III meningioma showed high express than the others. In conclusion, ET1 and VEGF may play a functional role in tumorigenesis of meningioma. The tumorigensis of meningioma is involved widely. The result of our study in centrosomal protein, Aurora A and B, ET1 and VEGF could do some contribution to the recurrence of meningioma, further investigation is necessary.

Lee Siu-wah.
Thesis submitted in: December 1999.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves [106]-116).
Abstracts in English and Chinese.
Abstract (in English) --- p.i
Abstract (in Chinese) --- p.iii
Acknowledgements --- p.v
Table of Contents --- p.vi
List of Figures --- p.ix
List of Tables --- p.x
Abbreviations --- p.xi
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.2
Chapter 1.2 --- Etiology of Hepatocellular Carcinoma --- p.5
Chapter 1.2.1 --- Viral Infection --- p.5
Chapter 1.2.1.1 --- Hepatitis B Virus --- p.5
Chapter 1.2.1.2 --- Hepatitis C Virus --- p.7
Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.8
Chapter 1.2.3 --- Aflatoxin --- p.9
Chapter 1.3 --- Genetic Studies of Hepatocellular Carcinoma --- p.9
Chapter 1.3.1 --- Conventional Cytogenetics --- p.9
Chapter 1.3.2 --- Molecular Cytogenetics --- p.12
Chapter 1.3.3 --- Molecular Genetic Studies --- p.12
Chapter 1.3.3.1 --- Proto-oncogenes --- p.12
Chapter 1.3.3.2 --- Tumour Suppressor Genes --- p.13
Chapter 1.3.3.3 --- Cell Cycle Genes --- p.14
Chapter 1.4 --- Background of Study --- p.16
Chapter 1.5 --- Objectives of Study --- p.17
Chapter Chapter 2 --- Material and Methods --- p.18
Chapter 2.1 --- Materials --- p.19
Chapter 2.2 --- Analysis of Chromosomal Imbalances by Comparative Genomic Hybridization --- p.23
Chapter 2.2.1 --- Comparative Genomic Hybridization --- p.23
Chapter 2.2.2 --- Methods
Chapter 2.2.2.1 --- Preparation of Normal Metaphases --- p.30
Chapter 2.2.2.2 --- Extraction of High Molecular Weight DNA --- p.30
Chapter 2.2.2.3 --- Labeling of DNA by Nick Translation --- p.31
Chapter 2.2.2.4 --- Labeling Efficiency --- p.31
Chapter 2.2.2.5 --- Preparation of Probe --- p.33
Chapter 2.2.2.6 --- Hybridization --- p.33
Chapter 2.2.2.7 --- Washing and Detection of Signals --- p.35
Chapter 2.2.2.8 --- Image Acquisition and Analysis --- p.35
Chapter 2.2.2.9 --- Control Experiments --- p.36
Chapter 2.3 --- Positional Mapping of Novel Amplicon by Interphase Cytogenetics --- p.39
Chapter 2.3.1 --- Fluorescence in situ Hybridization --- p.39
Chapter 2.3.2 --- Using Yeast Artificial Chromosomes (YAC) as Probe --- p.41
Chapter 2.3.3 --- Methods --- p.48
Chapter 2.3.3.1 --- Culture of Yeast Artificial Chromosomes --- p.48
Chapter 2.3.3.2 --- Extraction of Total YAC DNA --- p.48
Chapter 2.3.3.3 --- Verification of YAC Clones for Chimerism by FISH --- p.49
Chapter 2.3.3.4 --- Inter-Alu-PCR --- p.49
Chapter Chapter 3 --- Assessment of Genetic Changes in HCC by Comparative Genomic Hybridization (CGH) --- p.57
Chapter 3.1 --- Introduction --- p.58
Chapter 3.2 --- Materials and Methods --- p.58
Chapter 3.2.1 --- Patients and Specimens --- p.58
Chapter 3.2.2 --- Comparative Genomic Hybridization --- p.60
Chapter 3.2.3 --- Statistical Analysis --- p.60
Chapter 3.3 --- Results --- p.61
Chapter 3.3.1 --- Overall Copy Number Aberrations in 67 HCC and Surrounding Cirrhotic Tissues --- p.61
Chapter 3.3.2 --- TNM Staging --- p.61
Chapter 3.3.3 --- Tumour Size --- p.72
Chapter 3.3.4 --- Serum AFP Elevation --- p.72
Chapter 3.3.5 --- Chromosomal Aberrations in HCC arising from Cirrhotic and Non- cirrhotic Livers --- p.72
Chapter 3.4 --- Discussion --- p.73
Chapter 3.4.1 --- Recurrent Gains --- p.73
Chapter 3.4.2 --- Recurrent Losses --- p.75
Chapter 3.4.3 --- Tumour Progression --- p.76
Chapter 3.5 --- Conclusion --- p.78
Chapter Chapter 4 --- Positional Mapping of a Novel Amplicon on Chromosome 1q21-q25 by Interphase Cytogenetics --- p.79
Chapter 4.1 --- Introduction --- p.80
Chapter 4.2 --- Materials --- p.82
Chapter 4.3 --- Methods --- p.82
Chapter 4.3.1 --- Preparation of Paraffin-embedded Tissue Sections --- p.82
Chapter 4.3.2 --- Verification of YAC Probes for Chimerism --- p.83
Chapter 4.3.3 --- Hybridization Efficiency of Test and Reference Probes --- p.83
Chapter 4.3.4 --- Slide Pretreatment and FISH with YAC Probes --- p.88
Chapter 4.3.5 --- Scoring of FISH Signals --- p.88
Chapter 4.4 --- Results --- p.89
Chapter 4.4.1 --- Relative Copy Number Gain --- p.89
Chapter 4.4.2 --- Intratumour Heterogeneity --- p.90
Chapter 4.5 --- Discussion --- p.90
Chapter 4.6 --- Further Studies --- p.104
Chapter 4.6.1 --- Fine Mapping of Chromosomal Region 1 q21 --- p.104
Chapter 4.6.2 --- Isolation of Novel Genes in the Amplicon --- p.105
Chapter 4.6.3 --- Expression Status of the EDC Genes --- p.105
References --- p.106