• The effect of cryopreservation on the genome of fish reproductive cells

      Kopeika, Julia (University of BedfordshireUniversity of Bedfordshire, 2003-09)
      Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programs for endangered species. Many studies have been devoted to the mechanisms of cryodamage. However, in spite of growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells often remains poor. It is known that cryopreservation causes extensive damage to membrane, results in decreased metabolism of cells, and disturbs the bioenergetical processes of cells by damage to mitochondria. Nonetheless, it has not yet been identified clearly if cryopreservation causes some disruption in the genetic integrity of reproductive cells and the safety of this approach still needs to be confirmed. The present study was undertaken on the spermatozoa of weather loach (Misgurnus tassilis) and blastomeres cells of zebrafish (Danio rerio). It was shown that survival was decreased for embryos derived from sperm after cryoprotectant treatment or cryopreservation. Some evidence has emerged that this decrease is more likely to reflect some genetic instability caused by cryopreservation of sperm. The present study showed for the first time that the DNA repair system of oocytes was activated after fertilisation with cryopreserved sperm. The effect of DNA repair system was also studied. It was found that incubation of fertilised eggs in caffeine could reverse the detrimental effects of cryopreservation of loach sperm on subsequent embryo development. On the other hand incubation of fertilised eggs with 3-aminobenzamide - inhibitor of the poly (ADP-ribose) polymerase (PARP)- brought further decrease in the survival of embryos derived from cryopreserved sperm. The effect of individual donors of sperm and eggs on overall embryo survival was also studied and these investigations revealed significant differences between different donors. Effect of cryopreservation on zebrafish blastomeres was studied at the DNA molecular level. Mitochondrial DNA was sequenced after cryopreservation and increased level of frequency of the mutation was observed. This finding showed that cryopreservation might potentially increase the instability of mtDNA genome. The significance of these changes on the subsequent function of the cells is to be elucidated. Meanwhile this study suggests that it is important to be cautious in making judgements on the safety of cryopreservation techniques in reproduction.
    • Expression and comparison of tropomyosin isoform actin-binding properties and their resolution within the thin-filament proteome

      Dudekula, Khadar B. (University of BedfordshireUniversity of Bedfordshire, 2015-01)
      Tropomyosins(Tm) are a group of proteins that regulate the actin filaments in both muscle and non-muscle cells. In mammalian cells four Tm species are found: α-Tm (fast) encoded by α-Tm /TPM1 gene, β-Tm, encoded by β-Tm/ TPM2 gene, α-Tm (slow) encoded by γTm gene/ TPM3 and δ-Tm encoded by δTm / TPM4gene. Mutations in Tm are linked to many cardiac and skeletal diseases like hypertrophic cardiac myopathy (TPM1 and TPM2), familial cardiac myopathy (TPM1) and skeletal diseases like nemaline myopathy (TPM2 and TPM3) along with other sarcomere proteins. The hypothesis on which this study is based is, the isoform composition in both muscle and non-muscle cells adapts in response to disease and physiological changes. A significant part of that adaptation is changes in the thin filament protein isoforms expressed and the post translational modifications of these proteins. In this study Tpm3.12st isoform of γTm and other striated muscle tropomyosin isoforms (Tpm1 and Tpm2) and a non-muscle Tmp4 were characterised using a variety of techniques. The aim was to enhance our understanding of the role of tropomyosin interactions in regards to its efficiency of actin binding capacity as well as its effect on actin polymerisation. Human tropomyosin 3 (Tpm3.12st) was expressed in E. coli to produce recombinant protein with three N-terminal sequence variants (Met, MM and (M)ASM). The proteins were characterised for their binding affinity with actin as this isoform has not been well characterised so far. Its properties are compared with other striated muscle tropomyosin Tpm1.1st and Tpm2.2st and non-muscle Tpm4.1cy. The proteins were purified through ion exchange chromatography and the purity was checked by using SDS-PAGE and UV spectrometry. The molecular weights of the recombinant proteins produced were confirmed by mass spectrometry. Cosedimentation assays were performed for their actin binding affinity using ultracentrifugation. The variant of Tpm3.12st with AS N-terminal extension was found to have similar actin affinity to Tpm1.1st in the range of 0.1-0.8 μM (half saturation). However the variants with Met and MM N-termini bound to actin weakly with high half saturation concentration of ~ 6 μM and ~8 μM tropomyosin respectively. Measurement of actin polymerisation kinetics showed it is affected in presence of tropomyosin. From this study it is shown that tropomyosin accelerates the initiation step in actin polymerisation with varying differences within the isoforms in contrast to several previous studies. There have been very few studies of the effect of tropomyosin on actin polymerisation in the last two decades. This work shows that tropomyosin isoforms have a large and variable role in controlling actin polymerisation and understanding tropomyosin function will need further investigation in this area. This study also developed an ELISA screening method using monoclonal antibodies for identification and quantification of Tpm3.12st which was tested against all the four tropomyosin isoforms. None of the twelve antibodies studied showed reactivity only with Tpm3.12st. From the data analysed it is deduced the amino acid residues in the region of 24-43 shows the prospect of designing a monoclonal antibody specific to Tpm3.12st isoform. Accurate quantification of tropomyosin isoforms is key to understanding their function and the effects of modulation of isoform composition in health and disease. A reverse phase liquid chromatography method was developed which is compatible with the analysis of the thin filament proteome using top-down mass spectrometry. Reverse phase liquid chromatography (RPLC) is one of the most popular methods used in mass spectrometry analysis where proteins are separated based on their hydrophobicity. The RPLC method developed in this study gives an efficient separation of major thin filament proteins along with small soluble proteins that is compatible to use for top down mass spectrometry for identification and quantification of proteins, PTMs and isoform composition. With a minimum amount of 2 mg of tissue using chicken and mouse heart and skeletal muscle samples a buffer system was optimized to extract thin filament proteins. With the optimized RPLC method actin, tropomyosin and troponin complex subunits (TnC, TnI and TnI) were successfully separated and the proteins were identified using SDS-page by comparison with the previous research results. This novel method of extraction and the optimised RPLC method will provide a “bird’s eye view” of thin filament proteome providing information of PTMs of all the proteins together within one single extraction, reducing the time for analysis and the sample size. This has the potential to give insight into tissue, muscle and heart adaptations that could act as a prognostic indicator.