Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Bone loss from the cranio-maxillofacial region is a major clinical problem affecting patients worldwide. Conventional treatment strategy includes the use of autogenous or allogeneic bone, biomaterials, and osteogenic growth factors. However, there has been no effective therapy for most cases so far. Stem cell-based gene therapy is the latest research method with possible applications in humans. The present study aims to (1) characterize rabbit mesenchymal stem cells (MSCs) relating to growth pattern, surface antigens, and the potential for multi-differentiation; (2) determine the transduction efficiency and duration of recombinant adeno-associated virus2 carrying enhanced green fluorescent protein (rAAV2EGFP) reporter gene in rabbit MSCs and study the effects of rAAV2EGFP transduction on stem cells’ phenotype and capacity of multi-differentiation; (3) evaluate the differentiation characteristics of rabbit MSCs following recombinant adeno-associated virus 2 carrying bone morphogenetic protein 2 gene (rAAV2BMP2) transduction; (4) investigate whether MSCs transduced by rAAV2BMP2 could successfully induce bone regeneration in rabbit critical-size cranial defects. MSCs were isolated from bone marrows of rabbit tibias and cultured. Cell counting and colony-forming assays demonstrated that growth rates of MSCs dropped substantially with increasing passages. Flow cytometry on MSCs at passage 1 showed that cells expressed high level of CD49a and low level of CD44 as well as stage-specific embryonic antigen 4 (SSEA4). Multi-differentiation and reverse transcriptase-polymerase chain reaction (RTPCR) tests demonstrated that rabbit MSCs were capable to differentiate into osteocytes, chondrocytes and adipocytes. Immunofluorescence microscopy showed that rabbit MSCs produced a series of hematopoietic growth factors, including stem cell factor (SCF), vascular endothelial growth factor-A (VEGFA) and granulocyte macrophage colony-stimulating factor (GMCSF). Subsequently, rabbit MSCs were transduced with rAAV2EGFP in vitro. By comparing the transduction efficiency with different doses of rAAV2EGFP particles, multiplicity of infection (MOI) of 1 x 10 4 was identified as an optimal parameter for the transduction of rAAV2 in rabbit MSCs. Fluorescent microscopy demonstrated long-term expression of EGFP in rabbit MSCs after transduction both in vitro and in vivo. In addition, cell proliferation assay, adipogenic induction test and flow cytometry showed that rAAV2EGFP transduced MSCs exhibited a similar pattern with non-transduced cells on the cell growth, capacity of adipogenic differentiation and expression of surface antigens, indicating that rabbit MSCs maintain their stem cell properties after rAAV2EGFP transduction. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1994.

Orientador: Helena Coutinho Franco de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T19:02:01Z (GMT). No. of bitstreams: 1 Casquero_AndreaCamargo_D.pdf: 1109200 bytes, checksum: 0d66891096f39e7b47251643d7d9d885 (MD5) Previous issue date: 2005 / Resumo: As diferenças entre gêneros quanto ao perfil de lipoproteínas (LP) plasmáticas e risco de doenças ateroscleróticas são geralmente atribuídas aos efeitos protetores dos estrógenos e/ou pró-aterogênicos dos andrógenos. Neste trabalho realizamos três estudos dos efeitos de variações da concentração de testosterona sobre o metabolismo das LP e aterosclerose em camundongos que expressam a proteína de transferência de colesteril éster (CETP) com ou sem deficiência parcial do receptor de LDL. No primeiro estudo, mostramos que a deficiência de testosterona aumentou os níveis plasmáticos de LDL e de anticorpos anti-LDL oxidada e duplicou o tamanho médio da lesão aterosclerótica da raiz da aorta. A expressão da CETP neste modelo levou a um menor aumento de LDL e redução significativa do tamanho da lesão aterosclerótica. No segundo trabalho, comparamos os efeitos da suplementação com testosterona, na sua forma metabolizável ou não. Mostramos que a testosterona, seja metabolizável ou não, induziu elevações significativas de LDL e HDL. Somente a testosterona metabolizável aumentou a expressão da CETP e reduziu a de lipase hepática. No terceiro estudo, investigamos a interação entre os efeitos da testosterona não metabolizável (Mesterolona) e do exercício físico. Verificamos que o exercício físico aumentou HDL, de maneira mais potente nos animais que expressavam a CETP e nos animais que não foram tratados com Mesterolona. O exercício também reduziu a atividade da CETP, os níveis de triglicérides e ácidos graxos livres, independentemente do tratamento com Mesterolona. O tratamento com Mesterolona aumentou VLDL, independente do genótipo e do estado de atividade física, e reduziu HDL nos animais exercitados / Abstract: Gender differences in lipoprotein profile and atherosclerotic disease risk are generally attributed to an attenuating effect of estrogen and/or to a proatherogenic action of androgens. In this work we studied the effects of dificiency and excess of testosterone on the lipoprotein metabolism and development of atherosclerosis in transgenic mice expressing CETP with or without partial deficiency of the LDL receptor. In the first study, we showed that testosterone deficiency increased the levels of plasma LDL-cholesterol, and the titers of auto-antibodies anti-oxidized LDL and doubled the size of atherosclerotic lesions in the aortic roots. CETP expression in this animal model led to a less pronounced elevation of LDL-cholesterol and reduced significantly the atherosclerotic lesion size. In the second study, we compared the effects of two types of testosterone supplementation, metabolizable or not. We showed that either type of testosterone induced significant elevations in LDL- and HDL- cholesterol. Howerver, only the metabolizable testosterone promoted an increase in the CETP and a reduction in the hepatic lipase expression. In the third work, we investigated the interaction between the effects of non metabolizable testosterone (mesterolone) and physical exercise. We observed that the physical exercise increased HDL, more potently in animals expressing CETP and without mesterolone treatment. Physical exercise also reduced the CETP activity, triglycerides and free fatty acids levels, independently of the mesterolone treatment. The treatment with mesterolone increased VLDL, independently of the genotype and physical active state, and reduced HDL in the exercised mice / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

The first experiment was conducted to determine the optimum in vitro culture system for one-cell bovine embryos. Subsequent experiments compared bisection and biopsy for acquisition of cellular material from bovine morulae for DNA amplification by the polymerase chain reaction technique (PCR), and evaluated the use of DNA microinjection, in vitro culture, morula bisection, and PCR for production and selection of transgenic bovine preimplantation embryos. In vivo fertilized one-cell bovine embryos were cultured in TCM-199 (n=46), co-cultured with bovine oviductal epithelial cells (OEC; n=38), or in explanted immature mouse oviducts (n=54). Of the embryos that cleaved once, 52.6, and 30.4 and 0.0% developed to morulae/blastocysts after culture in OEC, TCM-199, and explanted mouse oviducts. Mean cell number for embryos cultured in OEC (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<.05). Bovine morulae were subjected to bisection (n=44; 20 to 30 cells) or biopsy (n=50; 8 to 12 cells) to assess embryo development in vitro and compare the efficiency of PCR amplification of an endogenous 18S rRNA. Mean development scores (l1=degenerate, 2=morula, 3=blastocyst) and mean cell number after microsurgery and 48 h of culture did not differ between treatments (P>.05; 2.4 ± .1 and 41.8 ± 2.5 versus 2.3 ± .1 and 48.8 ± 2.9, respectively). Frequency of the 18S rRNA amplifications was similar (P>.05) for demi-morulae (78%; 32/41) and biopsies (81%; 39/48). In the third experiment, in vivo fertilized one- (n=155), two- (n=57) and four-cell (n=62) bovine embryos were collected for pronuclear and nuclear DNA microinjection. Approximately 70% of the embryos were injected with DNA and 30% served as controls. Injection did not affect (P>.05) mean development scores after 144 h of cultured in TCM-199 with OEC. Sixty-five (34%) of the DNA injected embryos developed to morulae and were bisected. Injected DNA was amplified by PCR in 29% (19/65) of the demi-morulae. Frequency of DNA detection was more frequent (P<.01) in morulae injected at the 1-cell stage (50%: 16/32) than at the 2-cell (10%; 1/10) or 4-cell (9%: 2/23) stage. Production and selection of transgenic bovine morulae was most successful when one-cell bovine embryos were microinjected. / Ph. D.

Four experiments were conducted to investigate methods of producing transgenic bovine embryos entirely in vitro. Experiment 1 examined the effect of DNA microinjection at 11, 15 and 19 h after fertilization (haf) on survival rate and DNA detection frequency by polymerase chain reaction (PCR). There was no difference in transgene detection frequency between treatments (53% at 11; 50% at 15; 48% at 19 haf). Of all injected embryos developing to the morula or blastocyst stage after 7 d in culture, 89% tested positive for the presence of the transgene by PCR. Greater developmental efficiencies can be obtained when injection is performed early in pronuclear formation (7% (11/161) at 11; 4% (61159) at 15; 1 % (1/165) at 19 haf; p<0.05). Experiment 2 examined the effect of microinjection of DNA into the germinal vesicle (gv) of bovine oocytes on subsequent development and detection of the transgene. Injection of the transgene into the gv reduced developmental rates compared to controls (control=23% (89/384); non-injected=9% (23/250); GV injected=5% (12/259); p<O.05). Transgene detection frequency was 64% (37/58). Injection of bovine oocytes before fertilization results in viable embryos containing the transgene, although at low frequencies. Experiment 3 was designed to examine whether the frequency of microinjected DNA detection by peR In whole bovine embryos would decline over a 21 d culture period. At d 0, the transgene was detected in 100% (46/46) of embryos analyzed. At d 7, detection frequency was 84% (51/62) in viable embryos, at d 14 49% (18/37), and at d 21 38% (3/8). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates until after 14 d in culture. Experiment 4 examined microinjected bovine embryos for their potential use as donor embryos in nuclear transfer, or cloning. There was no difference in development between embryos cloned from microinjected donor embryos and those from control donor embryos (injected=11 % (37/377); control=9% (7/81); p>0.05). Of the embryos developing from microinjected donors, 32% (12/37) were PCR positive. Microinjected embryos can be successfully used in a nuclear transfer program to produce more viable embryos, and the resulting embryos may be more reliably screened by PCR. The efficiency of producing viable bovine embryos positive for the injected gene may be increased by performing microinjection early in pronuclear formation, and entering the resulting embryos into a nuclear transfer program. / Ph. D.

No description available.

A series of experiments were used to evaluate three culture media and two incubation temperatures for their ability to support development of DNA microinjected porcine zygotes. Development in vitro was compared between embryos collected from postpubertal and prepubertal donors and between embryos injected with DNA into the pronucleus and the cytoplasm. Additionally, embryos were analyzed by the polymerase chain reaction (PCR) for the presence of the transgene. One-cell embryos (n=458) were recovered from 36 postpubertal gilts in Experiment 1. Injected and control embryos were cultured in modified media NCSU-23 (mNCSU-23), NCSU-37 (mNCSU-37), and CZB at 37°C and 38.8°C for 7 d. In Experiment 2, one-cell embryos (n=245) were collected from postpubertal (n=15) and prepubertal (n=14) gilts, microinjected with DNA, and cultured in medium mNCSU-23. Superovulated prepubertal gilts (n=22) were flushed in Experiment 3 to yield 343 one-cell embryos which had DNA injected into the cytoplasm or pronucleus. Whole embryos were assessed by PCR. Mean percentages of embryos developing to the expanded or hatched blastocyst stage in mNCSU-23 and mNCSU-37 did not differ from each other (p>.05), but both were greater than the development in CZB (p<.05). Development was greater at 38.8°C (p<.05) than at 37° C. Microinjection of DNA decreased the developmental percentage (p<.05) from that of non-injected controls. Embryos collected from postpubertal gilts had a higher percentage (68.0 ± 3.4) of expanded and hatched blastocysts than embryos from prepubertal donors (29.0 ± 4.6, p<.05). No difference was seen in development between embryos injected in the pronucleus or cytoplasm (p>. 05), but development for both was less than for control embryos (p<.05). Results of PCR analysis indicated that 40% of the embryos developing to the expanded blastocyst stage were positive for the transgene compared to a rate of 60% positive for degenerate embryos. These studies show that DNA microinjected porcine zygotes can be cultured to the expanded blastocyst stage in media mNCSU-23 and mNCSU-37 at 38.8°C. Microinjection of DNA decreases survival of embryos collected from both postpubertal and prepubertal sources, but postpubertal embryos exhibit a higher rate of development. Cytoplasmic injection does not improve embryo viability in vitro above that of pronuclear injection. Finally, whole embryo analysis by PCR is possible, but cross specificity of human Protein C and whey acidic protein (WAP) oligonucleotides for endogenous porcine DNA is strong and creates difficulty in applying PCR analysis to embryos microinjected with WAP-PC transgenes. / Master of Science

Muitas das técnicas utilizadas para gerar animais transgênicos são caras e laboriosas. Neste contexto, a transferência gênica mediada por espermatozóides (TGME) pode ser uma alternativa para a produção em larga escala de animais transgênicos. Estudos de SMGT têm seu foco no número de cópias de DNA incorporada pelo espermatozóide. Por isso, há pouca informação disponível sobre como as moléculas de DNA se comportam durante o processo de fecundação e quais os efeitos dos protocolos de TGME sobre a célula espermática. Neste sentido, com o objetivo de avaliar a existência de sitio de integração preferencial das moléculas exógenas de DNA no genoma hospedeiro, utilizamos a hibridização in situ para acompanhar a veiculação do transgene durante o processo de fecundação. Foram avaliadas as membranas acrossomais, plasmática e potencial de membrana mitocondrial de espermatozóides submetidos a TGME. Para isso, o sêmen de três diferentes touros foram submetidos ao gradiente de Percoll 45-90%. As células viáveis foram incubadas com o vetor recombinante pCX-EGFP (0, 250, 500 ou 1000ng/106 células) seguidas ou não de eletroporação (300v, 35µF e 0,25ms). Os espermatozóides tratados foram utilizados para a produção in vitro de embriões. Os embriões foram cultivados por sete dias até o estágio de blastocisto. Espermatozóides e embriões produzidos in vitro foram submetidos ao ensaio de hibridização in situ, com metodologia descrita Whyte et al. (2000). O potencial de membrana mitocondrial (PMM), integridade de membrana acrossomal (MA) e plasmática (MP) foram avaliados por citometria de fluxo (Guava Technologies, Hayward, CA, USA) utilizando as sondas fluorescentes JC1, FITC-PSA e PI (Molecular Probes), respectivamente. Os dados foram analisados pelo teste paramétrico ANOVA (teste LSD) usando o programa estatístico SAS for Windows, com nível de significância de 5%. A hibridização in situ não foi possível em espermatozóides bovinos, pois não houve hibridação da sonda controle. Blastocistos oriundos de espermatozóides incubados com DNA exógeno apresentaram integração de forma difusa, embriões oriundos de espermatozóides eletroporados apresentaram integração pontual. As diferentes concentrações de DNA não exerceram efeitos deletérios nas MP ou PMM, a adição de 500ng de DNA causou aumento de lesão na MA (p<0,05). A eletroporação não afeta a MP e MA, mais apresenta grande efeito no PMM causando redução da função mitocondrial. Este estudo conclui que maiores esforços são necessários para elucidar o comportamento das moléculas exógenas de DNA durante o processo de fecundação e quais são os efeitos da TGME sobre a célula espermática. / Most techniques used to produce transgenic animals are laborious and expensive. In this manner, sperm mediated gene transfer (SMGT) may be a viable alternative for long-scale production of transgenic animals. Many SMGT studies have focused the DNA internalization and number of DNA copies incorporated by spermatozoa. However, limited data is available about how foreign DNA molecules behave during fertilization and the direct effects of the SMGT technique on sperm cells. Hence, in order to monitor the existence of preferential integration sites by the exogenous DNA at the host genome, in situ hybridization was used to track the transgene conveyance during in vitro fertilization. In addition, acrosome and plasmatic membrane integrity and mitochondrial membrane potential of sperm cells subjected to SMGT were assessed. Briefly, thawed semen from three different bulls was submitted to a 45- 90% Percoll gradient. Viable cells were incubated with recombinant PCX-EGFP vector (0, 250, 500 or 1000ng/106 sperm cells) or incubated and electroporated (300V, 35µF and 0.25ms). Treated sperm cells were then used for in vitro production of embryos. Embryos were in vitro cultured for 7 days until blastocyst stage. Treated spermatozoa and in vitro produced blastocysts were submitted to in situ hybridization assay, as described by Whyte et al. (2000). The mitochondrial membrane potential (MMP), acrosomal membrane (AM) and plasmatic membrane (PM) integrity were assessed by flow cytometry (Guava Technologies, Hayward, CA, USA) using JC1, FITC-PSA and PI probes (Molecular Probes), respectively. Data were analyzed by parametric ANOVA (LSD test) using SAS for Windows software, at a 5% level. The transgene was not observed at the bovine spermatozoa because the control probe could not be hybridized. In situ hybridization revealed that blastocysts produced from incubated sperm cells had a diffuse foreign DNA integration while blastocysts produced from electroporated sperm cells had a punctual DNA integration. No deleterious effects of exogenous DNA concentrations on PM or MMP were observed. However, the addition of 500ng of exogenous DNA caused sperm AM injury (P<0.05). Electroporation did not affect PM or AM integrity, but it had a great effect on MMP, which may cause a reduction of mitochondrial function. This study suggest that more efforts are needed to elucidate the behavior of exogenous DNA during fertilization and the effects of SMGT in bovine sperm cells.