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Improving Developmental Competence of Murine Preimplantation Embryos by Supplementation of Anti-apoptotic PeptidesFernandes, Roxanne 30 November 2011 (has links)
Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.
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Improving Developmental Competence of Murine Preimplantation Embryos by Supplementation of Anti-apoptotic PeptidesFernandes, Roxanne 30 November 2011 (has links)
Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.
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Molecular Targets in Autoimmune Polyendocrine Syndrome Type1 and Their Clinical ImplicationsAlimohammadi, Mohammad January 2009 (has links)
Autoimmune diseases occur when the immune system attacks and destroys healthy body tissue. Autoimmunity is known to cause a wide range of disorders, and is suspected to be responsible for many more. Most autoimmune disorders are chronic and cause severe morbidity for the patients, and are also costly for society. A majority of these disorders are today considered as complex diseases with incompletely known etiology. Hence, model systems for studying the pathogenesis of autoimmunity are important to unravel its causes. Autoimmune Polyendocrine Syndrome Type 1 (APS-1), (OMIM 240300), is a rare autoimmune disorder. Patients with APS-1 progressively develop multiple organ-specific autoimmune lesions involving both endocrine and non endocrine tissues. Typical autoimmune disease components in APS-1 are hypoparathyroidism, Addison’s disease, vitiligo, alopecia and type 1 diabetes. The gene preventing APS-1 has been identified and designated Autoimmune Regulator (AIRE). It has been shown that mutations of AIRE cause loss of tolerance to self-structures, resulting in organ-specific autoimmunity. Although APS-1 is a rare syndrome occurring mainly in genetically isolated populations, the disease components of APS-1 are, in isolated forms, not unusual in the general population and affect many patients. Hence, APS-1 is an attractive model disease for studies of molecular mechanisms underlying organ-specific autoimmunity. This thesis concerns investigations in which two novel autoantigens are identified in APS-1 and used in serological diagnosis of the disease. NALP5, is identified as a parathyroid autoantigen - an important finding since autoimmune hypoparathyroidism is one of the cardinal symptoms of APS-1. Additionally, KCNRG is identified as a bronchial autoantigen in APS-1 patients with respiratory symptoms. Finally, studies that compare the immune response in APS-1 patients and the mouse model for APS-1 are presented.
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Involvement of Nlrp5 in the Maintenance of Genome Integrity in Murine OocytesVelummailum, Russanthy 25 August 2011 (has links)
Nlrp5, a maternal-effect gene, is required for embryonic progression and female fertility in mice. Previous work indicated an age-related decline in Nlrp5 transcripts in murine oocytes. As maternal age is associated with increased spindle organization defects, studies in this thesis focused on the analysis of meiotic spindle defects in oocytes of Nlrp5-deficient mice. NALP5 protein showed a novel kinetochore-localization pattern, which was disturbed by spindle poisons. Nlrp5-deficient oocytes displayed a higher frequency of spindle abnormalities and chromosomal misalignment. Upon fertilization, these defects translated into increased incidences of multinucleation. As these phenotypes are associated with deficiencies in genome stability, we examined spindle assembly checkpoint (SAC) components. We found that numerous SAC proteins were dysregulated, implying that NALP5 may be critical in sensing oocyte-related SAC defects. We found that Nlrp5-deficient oocytes may have increased DNA damage. Thus, Nlrp5 may be an integral component responsible for preservation of genome integrity in female gametes.
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Involvement of Nlrp5 in the Maintenance of Genome Integrity in Murine OocytesVelummailum, Russanthy 25 August 2011 (has links)
Nlrp5, a maternal-effect gene, is required for embryonic progression and female fertility in mice. Previous work indicated an age-related decline in Nlrp5 transcripts in murine oocytes. As maternal age is associated with increased spindle organization defects, studies in this thesis focused on the analysis of meiotic spindle defects in oocytes of Nlrp5-deficient mice. NALP5 protein showed a novel kinetochore-localization pattern, which was disturbed by spindle poisons. Nlrp5-deficient oocytes displayed a higher frequency of spindle abnormalities and chromosomal misalignment. Upon fertilization, these defects translated into increased incidences of multinucleation. As these phenotypes are associated with deficiencies in genome stability, we examined spindle assembly checkpoint (SAC) components. We found that numerous SAC proteins were dysregulated, implying that NALP5 may be critical in sensing oocyte-related SAC defects. We found that Nlrp5-deficient oocytes may have increased DNA damage. Thus, Nlrp5 may be an integral component responsible for preservation of genome integrity in female gametes.
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