by Peter, Wei Gong Chang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 103-124). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Human genome project --- p.5 / Chapter 1.3 --- Human adult heart cDNA library --- p.7 / Chapter 1.4 --- Human fetal heart cDNA library --- p.8 / Chapter 1.5 --- Sequencing of a human heart cDNA clone --- p.9 / Chapter 1.6 --- Knowledge of the role of selenium --- p.13 / Chapter 1.7 --- Mouse 56kDa selenium binding protein and acetaminophen-binding protein --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Plating out the cDNA library --- p.20 / Chapter 2.1.1 --- "Mediums, buffers and solutions" --- p.20 / Chapter 2.1.2 --- Bacteriophage clones preparation --- p.21 / Chapter 2.2 --- cDNA clone amplification by PCR --- p.23 / Chapter 2.3 --- Cycle sequencing of PCR products --- p.25 / Chapter 2.3.1 --- "Media, buffers and solutions" --- p.25 / Chapter 2.3.2 --- Preparation of sequencing reaction --- p.25 / Chapter 2.4 --- Gel electrophoresis using an automated A.L.F sequencer --- p.27 / Chapter 2.5 --- DNA sequence analysis --- p.29 / Chapter 2.6 --- Preparation of competent E. coli for transformation --- p.30 / Chapter 2.7 --- Transformation of plasmid into competent E. coll --- p.31 / Chapter 2.8 --- Mini-preparation of plasmid DNA --- p.32 / Chapter 2.9 --- Large scale plasmid DNA preparation by QIAGEN --- p.34 / Chapter 2.10 --- Cloning the human 56 kDa selenium binding protein (hSP56) into the pAED4 vector --- p.36 / Chapter 2.10.1 --- Bacterial strains and vector --- p.36 / Chapter 2.10.2 --- "Media, buffers and solutions" --- p.38 / Chapter 2.10.3 --- Primers design and PCR --- p.42 / Chapter 2.10.4 --- Purification of PCR products by Geneclean --- p.43 / Chapter 2.10.5 --- Restriction digestion of purified PCR product and pAED4 --- p.44 / Chapter 2.10.6 --- Ligation and transformation of hSP56 --- p.45 / Chapter 2.10.7 --- Screening and purification ofpAED4hSP56. --- p.47 / Chapter 2.11 --- Expression of hsp56 --- p.50 / Chapter 2.11.1 --- Induction of hSP56 expression --- p.50 / Chapter 2.11.2 --- SDS-PAGE and protein detection --- p.51 / Chapter 2.12 --- Northern hybriddization of hSP56 --- p.53 / Chapter 2.12.1 --- Animals & human tissue --- p.53 / Chapter 2.12.2 --- "Mediums, buffers and solutions" --- p.53 / Chapter 2.12.3 --- Preparation of total RNA --- p.56 / Chapter 2.12.4 --- Formaldehyde agarose gel electrophoresis --- p.57 / Chapter 2.12.5 --- Preparation of radioactive probe --- p.58 / Chapter 2.12.6 --- RNA transfer and Northern hybridization --- p.59 / Chapter 2.13 --- Chromosomal mapping of the hSP56 gene --- p.62 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- The sequencing results of 553 cDNA clones --- p.63 / Chapter 3.2 --- Catalogues of genes expressed --- p.65 / Chapter 3.3 --- Sequence analysis of hSP56 --- p.71 / Chapter 3.4 --- Northern hybridization of hSP56 --- p.84 / Chapter 3.5 --- Cloning of hSP56 into pAED4 --- p.87 / Chapter 3.6 --- Expression of the hSP56 in E. coli --- p.89 / Chapter 3.7 --- Chromosomal mapping of the hSP56 gene --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- General discussion --- p.94 / Chapter 4.2 --- The possible roles of hSP56 and mSP56 --- p.101 / Chapter 4.3 --- Future prospects --- p.102 / REFERENCES --- p.103 / APPENDIX 1 --- p.125 / APPENDIX.2 --- p.127

No description available.

Thesis (M.Sc.), Memorial University of Newfoundland, 1998. / Restricted until June 1999. Bibliography: leaves 85-97.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves : 117-131). Also available on the Internet.

Made available in DSpace on 2016-12-08T16:24:06Z (GMT). No. of bitstreams: 1 PGCV08MA028.pdf: 640511 bytes, checksum: 7739954e4535ca25f1e1be5d8d3266a8 (MD5) Previous issue date: 2008-05-26 / Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure per se being one of the potential determining factors for the appearance of the problems. Embryo aggregation may be an alternative to minimize such effects during cloning. The aim of this study was to determine the effect of the heteroplasmy caused by fusion of hemi-cytoplasts or by aggregation of hemi-embryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by handmade cloning (HMC), parthenogenesis or in vitro fertilization (IVF). In vitro-matured bovine oocytes, selected by the presence of a polar body, were manually bisected, followed by fluorescence screening of hemi-cytoplasts. One or two enucleated hemi-cytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemi-embryos were in vitro-cultured in microwells (WOW system), being allocated to one of six experimental groups, based on the volume of cytoplasm used for embryo reconstruction either by fusion of hemi-cytoplasts or by aggregation of hemi-embryos or embryo, on a per WOW basis, as follows: single clone or parthenote hemi-embryos (G1: 1 x 50%); aggregation of two (G2: 2 x 50%), three (G3: 3 x 50%) or four (G4: 4 x 50%) clone or parthenote hemi-embryos; single clone or parthenote embryos (G5: 1 x 100%); or aggregation of two clone or parthenote embryos (G6: 2 x 100%). Zona-intact parthenote or IVF embryos (G7: Zona-intact, 100%), used as controls, were in vitro-cultured in 4-well dishes. Data were analyzed by &#967;2, ANOVA, Pearson s correlation or linear regression, for P<0.05. In general, results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate and cell density. Even though those observations were more pronounced in parthenotes, cell density and embryo quality were superior in clone embryos. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies after transfer to female recipients. Nevertheless, the use of only half of the normal volume did not affect the developmental potential to the blastocyst stage, but reduced cell number. In conclusion, despite the increase in embryo development and cell density per WOW, embryo aggregation did not improve blastocyst yield or cell number, on a hemi-cytoplast basis / A clonagem animal tem sido associada a anormalidades de desenvolvimento, com o nível de heteroplasmia causados pela técnica per se sendo um dos potenciais fatores determinantes do aparecimento dos problemas. A agregação embrionária pode ser uma alternativa para minimizar tais efeitos causados pela clonagem. O objetivo deste estudo foi determinar o efeito da heteroplasmia pela fusão de hemi-citoplastos ou da agregação de hemi-embriões, variando-se o volume citoplasmático final, no desenvolvimento e na densidade celular de embriões produzidos por handmade cloning (HMC), partenogênese ou fecundação in vitro (FIV). Oócitos bovinos maturados in vitro, selecionados pela presença do corpúsculo polar, foram bisseccionados manualmente, seguidos da seleção de hemi-citoplastos por fluorescência. Um ou dois hemi-citoplastos enucleados foram pareados e fusionados a uma célula somática cutânea. Embriões e hemi-embriões de clonagem e de partenogênese (sem zona pelúcida) foram ativados e cultivados in vitro em micropoços (sistema WOW), sendo alocados para um de seis grupos experimentais, baseado no volume de citoplasma utilizado para a reconstrução embrionária pela fusão de hemi-citoplastos ou agregação de hemi-embriões e embriões, por micropoço (WOW), conforme a seguir: um único hemi-embrião de clonagem ou de partenogênese (G1: 1 x 50%); agregação de dois (G2: 2 x 50%), três (G3: 3 x 50%) ou quatro (G4: 4 x 50%) hemi-embriões de clonagem ou de partenogênese; um único embrião de clonagem ou de partenogênese (G5: 1 x 100%); ou agregação de dois embriões de clonagem ou de partenogênese (G6: 2 x 100%). Embriões intactos oriundos de FIV ou de partenogênese (G7: Zona intacta, 100%), utilizados como controle, foram cultivados in vitro em placas de 4 poços. Os resultados foram analisados pelos testes do &#967;2, ANOVA, correlação de Pearson ou regressão linear, para P<0.05. Em termos gerais, os resultados indicaram que o aumento no número de estruturas agregadas em cada micropoço foi seguido por um aumento linear nas taxas de clivagem, de blastocisto e na densidade celular. Mesmo que tais observações tenham sido mais pronunciadas nos partenotos, a densidade celular e a qualidade embrionária foram superiores em embriões clonados. O aumento no volume citoplasmático por fusão ou agregação apresentou um efeito positivo no desenvolvimento embrionário, dando suporte ao estabelecimento de prenhezes após a transferência para fêmeas receptoras. No entanto, a utilização de apenas metade do volume normal não afetou o potencial de desenvolvimento até o estádio de blastocisto, mas reduziu o número de células. Em conclusão, apesar de haver um aumento no desenvolvimento e densidade celular em embriões por micropoço, a agregação embrionária não melhorou a taxa de blastocistos ou o número de células, com base no número de hemi-citoplastos utilizados por embrião

We apply the data cloning method developed by Lele et al. (2007) to estimate the model of Smets and Wouters (2007). The data cloning algorithm is a numerical method that employs replicas of the original sample to approximate the maximum likelihood estimator as the limit of Bayesian simulation-based estimators. We also analyze the identification properties of the model. We measure the individual identification strength of each parameter by observing the posterior volatility of data cloning estimates, and access the identification problem globally through the maximum eigenvalue of the posterior data cloning covariance matrix. Our results indicate that the model is only poorly identified. The system displays bad global identification properties, and most of its parameters seem locally ill-identified. / Neste trabalho aplicamos o método data cloning de Lele et al. (2007) para estimar o modelo de Smets e Wouters (2007). O algoritmo data cloning é um método numérico que utiliza réplicas da amostra original para aproximar o estimador de máxima verossimilhança como limite de estimadores Bayesianos obtidos por simulação. Nós também analisamos a identificação dos parâmetros do modelo. Medimos a identificação de cada parâmetro individualmente ao observar a volatilidade a posteriori dos estimadores de data cloning. O maior autovalor da matriz de covariância a posteriori proporciona uma medida global de identificação do modelo. Nossos resultados indicam que o modelo de Smets e Wouters (2007) não é bem identificado. O modelo não apresenta boas propriedades globais de identificação, e muitos de seus parâmetros são localmente mal identificados.

Sistemas de produção in vitro de embriões mamíferos muitas vezes requerem que o cultivo embrionário seja realizado de forma individualizada. Entretanto, os resultados obtidos com o cultivo in vitro (CIV) individual são inconstantes e, por vezes, inferiores ao CIV em grupo. Entre os sistemas que requerem o CIV individual, a técnica de handmade cloning (HMC) se destaca por produzir embriões sem zona pelúcida que não podem ser cultivados agrupados em protocolos convencionais de CIV. O objetivo deste experimento foi determinar as taxas de desenvolvimento in vitro e in vivo de embriões bovinos clonados pela técnica de HMC e submetidos a três diferentes sistemas de CIV em micropoços (Well of the well – WOW), sendo um industrial, confeccionado em polidimetilsiloxano (PDMS), que visou à padronização da configuração do sistema de CIV. Após 11 replicações, de 3.876 complexos cumuli-oócito bovinos maturados in vitro, 3.437 (88,6%) oócitos apresentaram a extrusão do 1º corpúsculo polar. Após a digestão da zona pelúcida, 2.992 estruturas foram bisseccionadas manualmente, com a produção de 2.288 hemi-citoplastos. Reconstruiram-se 1.011 embriões pela adesão de dois hemi-citoplastos a uma célula somática (célula-tronco mesenquimal bovina de uma fêmea adulta da raça Nelore), dos quais 751 (74,2%) embriões fusionaram após eletrofusão, sendo 715 ativados quimicamente. Os embriões clonados (n=705) foram então alocados aleatoriamente em três grupos experimentais para o CIV: Grupo 1 (G1) – micropoço modificado (WOW-modificado, FELTRIN et al., 2006a); Grupo 2 (G2) – micropoço convencional (WOW-convencional , VATJA et al., 2000); e Grupo 3 (G3) – micropoço – PDMS (WOW-PDMS) . Como grupo controle (FIV, Grupo 4, G4), 594 oócitos foram colocados em maturação, fecundação e cultivo in vitro, em paralelo aos grupos clonados. Os resultados das taxas de clivagem no Dia 2, de blastocisto no Dia 7 e de prenhez no Dia 30 de desenvolvimento foram analisados pelo teste do χ2 com nível de significância de 5%. Não houve diferença significativa quanto à taxa de clivagem nos diferentes grupos. Entretanto, a taxa de blastocisto (BL) no G1 (99/239; 41,4%) foi significativamente superior à observada no G2 (72/232; 31,0%) e no G3 (68/234; 29,1%), sendo estas últimas semelhantes entre si. A taxa de BL do grupo controle (315/594; 53,0%) foi superior aos três grupos experimentais. Finalmente, o desenvolvimento in vivo até o Dia 30 de prenhez não diferiu entre os grupos G1 (7/15; 46,7%), G2 (7/13; 53,9%) e G3 (6/16; 50,0%). O sistema em micropoço modificado promoveu melhores condições de CIV a embriões clonados por HMC, traduzido por maiores taxas de BL, não se refletindo, entretanto, em diferenças no desenvolvimento in vivo subseqüente à transferência para fêmeas receptoras. / In vitro production systems for mammalian embryos quite often require embryos to be cultured individually. However, results obtained after individual embryo in vitro culture (IVC) are frequently inconsistent and inferior to IVC in groups. The Handmade Cloning (HMC) procedure is among the systems that require individual IVC, since zona-free embryos cannot be grouped for culture by standard IVC protocols. The aim of this study was to evaluate the in vitro and in vivo development of bovine embryos cloned by HMC, after the IVC in three distinct microwell arrangements, including an industrial chip, manufactured in polydimethylsiloxane (PDMS), which aimed to standardize the pattern of the IVC system. After 11 replications, 3,437 (88.6%) oocytes were selected based on the extrusion of the first polar body, out of 3,876 in vitro-matured bovine cumuli-oocyte complexes. Following zona pellucida digestion, 2,992 structures were manually bisected, generating 2,288 hemi-cytoplasts. A total of 1,011 embryos were reconstructed by the adhesion of two hemi-cytoplasts to a somatic cell (bovine mesenchymal stem cells from an adult Nelore female), with 751 fusing (74.2%) following electrofusion, and 715 being chemically activated. Cloned embryos (n=705) were then randomly allocated to one of three IVC experimental groups: Group 1 (G1) – modified microwells (FELTRIN et al., 2006a); Group 2 (G2) – conventional microwells (VATJA et al., 2000); and Group 3 (G3) – microwells in PDMS. As control group (IVF, Group 4, G4), 594 oocytes were in vitro-matured, fertilized and cultured in parallel to the cloned groups. Cleavage, blastocyst and pregnancy rates evaluated on Days 2, 7 and 30 of development were analyzed by the χ2 test, for a significance level of 5%. No differences in cleavage rates were observed between groups. However, blastocyst rates in G1 (99/239; 41.4%) were significantly higher than in G2 (72/232; 31.0%) and in G3 (68/234; 29.1%), which did not differ between one another. Blastocyst rates in the IVF control group (315/594; 53.0%) were in turn higher than in all cloned experimental groups. Finally, in vivo development to Day 30 of pregnancy was not different between G1 (7/15; 46.7%), G2 (7/13; 53.9%) and G3 (6/16; 50.0%). In summary, the modified microwell system promoted superior development to the blastocyst stage than both the conventional and the DMPS-based microwell systems, with no impact on the subsequent in vivo development after transfer to female recipients.

This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.

University of Macau / Faculty of Law

Thesis (M.A.)--University of Wyoming, 2008. / Title from PDF title page (viewed on Nov. 11, 2009). Includes bibliographical references (p. 89-90).