Graduation date: 1993

The effects of several hormones and inducers of intracellular messengers on plasminogen activator (PA) production and changes in embryonic size by cultured bovine embryos were evaluated. Day 8 embryos were cultured in Ham's F-12 with 1.5 mg/ml bovine serum albumin (BSA) containing different levels of progesterone (P), estradiol -17fl (E₂), dexamethasone (Dex), retinoic acid (RA), dibutyryl cyclic AMP (dbcAMP), or phorbol myristate acetate (PMA) for 5 days under paraffin oil in a humidified atmosphere of 5% CO₂ in air at 37°C. The concentrations of PA in the conditioned media were determined by a caseinolytic assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography were used to determine the molecular weight of PA in the medium and in the embryo homogenate. Changes in embryonic size were determined by measuring overall embryo diameter (OD) at 24-h intervals. None of the hormones and agents tested herein had a significant effect on PA production. Dimethyl sulfoxide (DMSO) which was used to dissolve PMA significantly inhibited PA production during the first 72 h of culture. Time of culture, however, exerted a significant effect on PA production by cultured embryos. The production of this protease was low during the first 48 h, increased during 72 and 96 h, and either remained high or slightly decreased toward the end of the culture period. Furthermore, the peak production of PA was attained 48 h after hatching. The molecular weight of PA in the conditioned medium and embryo tissues suggested that the bovine embryo at this developmental stage produced an urokinase-type PA. With the exception of dbcAMP and PMA, the hormones tested in this study did not affect embryonic size. While dbcAMP decreased OD later in culture, PMA enhanced OD throughout culture. The mechanism by which dbcAMP and PMA modulated embryonic size is not clear. These results suggest that cultured bovine embryos produce urokinasetype PA in a time dependent manner and the production of this enzyme is independent of exogenous hormonal regulation. / Graduation date: 1990

Plant transformation provides a promising methodology of introducing new genes that encode desirable traits to a wide range of crop plants. Success in genetic transformation has been achieved in many of the important crop species, such as soybean, cotton, rice, corn. However, wheat, one of the major crops of the world, has been considered to be difficult to transform via either Agrobacterium or biolistic bombardment (Rakszegi et al., 2001). There have been limited studies on A. tumefaciens-mediated transformation of cereals, including wheat, because of the overall refractory character of host-pathogen interactions between Agrobacterium and the cereal plants (Gould et al., 1991; Hiei et al., 1994; Cheng et al., 1997). While the genetic transformation of rice using Agrobacterium has become routine, only a few successful studies of Agrobacterium- mediated transformation of wheat have been reported, and these involved a model spring wheat, Triticum aestivum cultivar Bobwhite (Cheng et al., 1997). Model genotypes are developed for ease of plant regeneration in tissue culture and both Agrobacterium and biolistic mediated transformation methods require regeneration of plants in tissue culture. More success has been achieved in obtaining fertile transgenic wheat plants by particle bombardment, or biolistics method (Vasil et al., 1992; Weeks et al., 1993; Becker et al., 1994; Zhou et al., 1995; Altpeter et al., 1996). Wheat plants of the model system cultivar Bobwhite were used in most of these studies as well. The primary objective of this study was to use the callus-based transformation procedures mentioned above with a non-model cultivar of hexaploid spring wheat Saratovskaya-29, widely grown in Kazakhstan, to test the genotype dependence of the previously developed transformation protocols with respect to stable transfer of DNA and regeneration of transgenic plants. The spring wheat cultivar Saratovskaya-29 (Albidum-24/ Lutescens-55-11) was chosen for the study as being one of the most widely grown wheat cultivars both in Russia and Kazakhstan. It was bred in early 50??s in the Research Institute of the South-East, Saratov. Because of its drought resistance and good baking quality traits, Saratovskaya-29 reached a peak of nearly 21.2 mln ha in the former USSR in 1996 (Martynov and Dobrotvorskaya, 1996). Economical importance of this cultivar makes it an appropriate candidate for further improvement of economically significant traits. Another objective of the study described was to compare the transformation efficiencies and inheritance in the transgenic plants produced.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Despite recognition of aldehyde dehydrogenase (Aldh) as a surrogate marker in isolating primitive hematopoietic stem and progenitor cells (HSPC) [1], its role in HSPC regulation, particularly during embryonic development, remains unclear. In this study, we examined the role of Aldh during embryonic hematopoiesis in zebrafish, which has emerged as a model for hematopoietic studies. [2] Wild--?type and transgenic [Tg(gata1:gfp),Tg(fli1:gfp)] zebrafish embryos were microinjected with anti--?sense morpholinos (MO) at 1--?cell to 4--?cell stage and evaluated by morphology, flow cytometry, in situ hybridization (ISH) and Q-RT-PCR. In addition, human CD34+ cells, which were enriched with hematopoietic stem cells (HSC), were isolated from umbilical cord blood samples for analysis of ALDH16A1 expression. It was subsequently compared with CD34- cells which were devoid of HSC activity. When aldh16a1 was knocked down by anti-sense morpholino (the embryos were referred herewith aldh16a1MO embryos), gene expression associated with erythropoiesis was significantly reduced at 18hpf .(gata1:0.70±0.03fold; p=0.002) (α-embryonic hemoglobin: 0.48±0.04fold; p=0.003) (β-embryonic hemoglobin: 0.56±0.03fold; p=0.001). Angiogenesis was also perturbed at 48 and 72hpf. Furthermore, human ALDH16A1 was significantly upregulated (4.79±1.00fold; p=0.00006) in CD34+ (enriched with HSC) as compared to CD34- (devoid of HSC) populations in umbilical cord blood. Aldh16a1 is important for the maintenance of primitive hematopoiesis at early (18hpf) and angiogenesis at later (48,72 hpf) embryonic stages. As angiogenesis plays an important role in pathophysiology of malignancies, novel therapy against ALDH16A1 might be exploited in therapeutic intervention in cancer treatment. Moreover, a specific role of zebrafish aldh16a1 in primitive erythropoiesis and a higher level of ALDH16A1 expression in human HSC-enriched cells suggested a conserved mechanism whereby ALDH regulates hematopoiesis. / published_or_final_version / Medicine / Master / Master of Research in Medicine

Abstract The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR’s) for 16 days. At CIDR removal, does were injected with 300 IU eCG. The recipient does were allocated to 3 groups (n = 9 per group), based on the technique of cryopreservation used for the embryos transferred. The in vivo produced embryos used were at blastocyst stage and surgically collected on day 6 following AI from Boer goat donors superovulated with pFSH. The first group received fresh embryos and served as the control, the second group of does received conventional slow frozen/thawed embryos and the third group received vitrified/thawed embryos. Two blastocysts were transferred per doe. A pregnancy rate of 85.7% (n = 6) was obtained following the transfer of fresh embryos and tended to be better than in does receiving slow frozen and vitrified embryos, (n = 4; 50.0% and n = 3; 37.5% does pregnant, respectively). The overall gestation period recorded for all does was 146.3 ± 3.0 d, with an overall litter size of 1.7 ± 0.5 being recorded. The kidding rate of the recipient does declined to 57.0% (4) and 25.0% (2) for fresh and conventional slow frozen groups, respectively. An embryo survival rate of 35.7% (n = 5) for fresh, 25.0% (n = 4) for conventional slow freezing and 31.3% (n = 5) for vitrification was recorded and was not affected by the number of CL’s present on the respective ovaries at the time of transfer. There was a tendency for more females to be born than males (ratio 1 : 2, male : female) but this could not be related to the cryopreservation technique. Although the pregnancy rate following the transfer of fresh embryos was satisfactory, the embryo survival rate following the transfer of either fresh or cryopreserved embryos tended to be less acceptable. More research is warranted with larger numbers of animals, directed at improving the survivability of embryos following fresh and cryopreserved goat embryo transfer.

Abstract The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR’s) for 16 days. At CIDR removal, does were injected with 300 IU eCG. The recipient does were allocated to 3 groups (n = 9 per group), based on the technique of cryopreservation used for the embryos transferred. The in vivo produced embryos used were at blastocyst stage and surgically collected on day 6 following AI from Boer goat donors superovulated with pFSH. The first group received fresh embryos and served as the control, the second group of does received conventional slow frozen/thawed embryos and the third group received vitrified/thawed embryos. Two blastocysts were transferred per doe. A pregnancy rate of 85.7% (n = 6) was obtained following the transfer of fresh embryos and tended to be better than in does receiving slow frozen and vitrified embryos, (n = 4; 50.0% and n = 3; 37.5% does pregnant, respectively). The overall gestation period recorded for all does was 146.3 ± 3.0 d, with an overall litter size of 1.7 ± 0.5 being recorded. The kidding rate of the recipient does declined to 57.0% (4) and 25.0% (2) for fresh and conventional slow frozen groups, respectively. An embryo survival rate of 35.7% (n = 5) for fresh, 25.0% (n = 4) for conventional slow freezing and 31.3% (n = 5) for vitrification was recorded and was not affected by the number of CL’s present on the respective ovaries at the time of transfer. There was a tendency for more females to be born than males (ratio 1 : 2, male : female) but this could not be related to the cryopreservation technique. Although the pregnancy rate following the transfer of fresh embryos was satisfactory, the embryo survival rate following the transfer of either fresh or cryopreserved embryos tended to be less acceptable. More research is warranted with larger numbers of animals, directed at improving the survivability of embryos following fresh and cryopreserved goat embryo transfer.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.