Crustacyanin, the blue carotenoprotein of the lobster carapace was obtained in a state homogeneous in cellulose acetate electrophoresis. It was crystallised and shown to be a globulin of large particle size, free from lipid and carbohydrate. The minimum molecular weight of the protein calculated on the carotenoid content, was found to be one-tenth of that expected from its size. Reversible changes in spectrum occurring on removal of salt were shown to be attended by dissociation into subunit of the size expected from the minimum molecular weight. Apocrustacyanin was shown to be of the subunit size and heterogeneous in cellulose acetate electrophoresis. Native cnustacyanin was reconstituted from the apoprotein and astaxanthin. Possible modes of binding of the carotenoid are suggested to account for the spectral changes occurring in urea. Electrophoretic similarities between crustacyanin treated with urea and apocrustacyanin are discussed in relation to the number of different subunits. It is suggested that crustacyanin is composed of a specific geometrical association of several small subunits, possibly different, and that the carotenoid stabilises the subunits in a configuration essential for the association and possibly also assisting in the binding. Ovoverdin, the green carotenoprotain of lobster ovary, was obtained homogeneous in cellulose acetate electrophoresis and shown to be a glycolipoprotein, precipitating at low ionic strength out soluble in distilled water. The possibility of two molecules of carotenoid being associated with each molecule of protein has been investigated. Astaxanthin-proteins were purified from the carapaces of Eriphia spinifrons, Carcinus maenas, Astacus astacus and Palinurus vulgaris. Their absorption spectra and properties arc compared with those of crustacyanin and ovoverdin. Glycolipoproteins containing a number of carotenoids have been purified from the ovaries of Carcinus maenas, Pecten maximus and Palinurus vulgaris, and from the eggs of Palinurns vulgaris. Their properties have been investigated and the ode of association of the carotenoids is discussed.

Since their first clinical application in the 1980’s recombinant proteins have become an increasingly larger section of the drug market, growing into a multi-billion dollar global market. The pursuit of improving the design, production, and application of recombinant proteins for biotherapeutic uses is a key driver in industry and academia. The majority of the recombinant biotherapeutic proteins used in the clinic are produced in mammalian cell expression systems due to their capability to undertake human-like complex post translational modifications. The currently ‘gold standard’ mammalian cell expression system for the production of recombinant proteins is the Chinese hamster ovary (CHO) cell line. However, even with advances in mammalian cell expression technology, there is still a high cost and a long development period required for a recombinant protein therapeutic to go from design to market. As such, bottlenecks include the time taken for mammalian cells to grow and divide with slow doubling times compared to microbial systems and limited capacity to synthesise and secrete recombinant proteins. One of the cellular processes that underpins both cell growth and recombinant protein production is the translation of mRNA. Translation consists of three distinct steps: initiation, elongation and termination. One major cell signalling pathway that is considered a master regulator of both initiation and elongation of translation is mTOR, also involved in regulating ribosome biogenesis and cell proliferation. During the process of polypeptide elongation (mRNA translation), elongation factor 2 (eEF2) is a key control point that regulates protein synthesis via its de/phosphorylation. Phosphorylation of eEF2 results in its inactivation; slowing or halting elongation resulting in the attenuating of protein synthesis. This study set out to establish if manipulation of the mTOR signalling pathway and/or manipulation of phosphorylation of elongation factor 2 and the kinase that inactivates eEF2, eEF2K, in CHO cells impacts upon CHO cell growth and recombinant protein production yields. Transient expression of wild type and a Thr56Ala eEF2 mutant in CHOK1 cells affected the short term (24-48 hour) phosphorylation of eEF2 but did not appear to have an effect upon intracellular recombinant protein production and cellular growth in culture over 96 hours. Stable over expression of the wild type eEF2 construct in CHO cells resulted in a 2-fold increase in expression of eEF2 and a decrease in phosphorylation of eEF2 at the protein level; but the there was no change in the levels of total eEF2 mRNA expression. Stable expression of the Thr56Ala and Thr56Glu eEF2 mutants had a greater effect upon eEF2 expression resulting in a 3-5 fold increase in total eEF2 expression, however the phosphorylation of eEF2 was almost unchanged in an Ala56 eEF2 cell line, whereas it was reduced in the Glu56 eEF2 mutant cell 20 line. Growth of the CHO cells lines expressing the eEF2 mutants show that over expression of any of the eEF2 mutants resulted in a change in growth, but the Ala56 eEF2 mutant showed the largest change in cellular growth. Short term transient expression of recombinant firefly luciferase in the stable eEF2 cell lines revealed that the Thr56Ala mutant greatly increases the CHO cells total recombinant protein production. Further, mutation of eEF2 to Ala56 or Glu56 had little effect upon the mis-incorporation of amino acids during translation. The transient knockdown of eEF2K was achieved and this was shown to prevent eEF2 phosphorylation. However, CHO cells do not appear to tolerate the knockdown of eEF2K stably or at only very low levels. This suggests that a sustained, high level of eEF2K knockdown is lethal to the cell; which would result in the loss of eEF2 regulation for an extended period of time. Transient expression of the eEF2K shRNA into CHOK1D6 cells stably expressing firefly luciferase, a nonsecreted protein, resulted in a 5-fold increase in luciferase expression showing that knockdown of eEF2K increased the short term productivity of these cells. Interestingly, the study of CHO cell lines with varying recombinant monoclonal antibody protein production capacities revealed that levels of total and phosphorylated eEF2 did not appear to change in correlation with the mAb titre. These data suggest that eEF2 activity is tightly regulated across cell lines and is not directly related to recombinant protein secretion in these industrially used CHO cell lines The addition of 2 µM of PMA, an activator of mTOR signalling, increased antibody production in a low producer cell line but had no effect upon the antibody production from a high producing cell line. Together the data presented here shows that manipulation of eEF2 and eEF2K activity can enhance cellular growth and recombinant protein production from CHO cells. As such, new engineering approaches that allow the manipulation of both elongation and polypeptide synthesis combined with the secretory capacity of the cell are likely to yie ld new CHO host cells with more predictable recombinant protein capacity and further advance our understanding of the role of mRNA translation in controlling cell proliferation, and both global and recombinant protein synthesis.

Chromosome copy number errors (or aneuploidy) of gametes and embryos occurs in humans more frequently than in any other studied species, with a spectrum of manifestations from implantation failure to affected live births. It is predominantly problem arising in maternal meiosis with at least 20% of oocytes being aneuploid, a proportion that increases dramatically with advancing maternal age. Currently the only intervention to reduce the chances of transmitting aneuploidy is by invasive embryo biopsy procedures in high-risk groups (mainly patients with advanced maternal age) undergoing in-vitro fertilisation. Despite the severity of this problem, aneuploidy of the human preimplantation embryo is relatively poorly understood. With this in mind the purpose of this thesis is to explore the premise underpinning the use of preimplantation genetic screening (PGS) in human embryos and investigate its clinical applications and current methodologies. A series of published works demonstrate what I believe to be a significant contribution to the development of applications for studying human preimplantation aneuploidy, also providing insight into its origins and mechanisms at the earliest stages of human development. Specifically, I present a novel standard set of protocols as a general reference work from practitioners in the fields of embryo biopsy and array comparative genomic hybridisation (CGH - the current ‘gold standard’ for preimplantation aneuploidy screening). I present a summary of work encapsulated in three published clinical papers using a linkage based analysis of Single Nucleotide Polymorphism (SNP) karyotypes (Karyomapping). Karyomapping was designed as a near-universal approach for the simultaneous detection of chromosomal and monogenic disorders in a PGS setting and these results demonstrate the utility of the technique in three separate scenarios. In order to study the underlying mechanisms of female meiosis I present my findings on the use of a calcium ionophore to activate human oocytes artificially. An algorithm based on Karyomapping (termed MeioMapping) is demonstrated for the first time specifically to investigate human female meiosis. By recovering all three products of human female meiosis (oocyte, and both polar biopsies – herein termed “Trios”) using calcium ionophore, I present a novel protocol (commissioned by Nature Protocols) to allow exploration of the full extent of meiotic chromosome recombination and segregation that occurs in the female germline. Finally I present a published set of experiments using this protocol to provide new insight into meiotic segregation patterns and recombination in human oocytes. This work uncovers a previously undescribed pattern of meiotic segregation (termed Reverse Segregation), providing an association between recombination rates and chromosome mis-segregation (aneuploidy). This work demonstrates that there is selection for higher recombination rates in the female germline and that there is a role for meiotic drive for recombinant chromatids at meiosis II in human female meiosis. The work presented in this thesis provides deeper understanding of meiotically derived maternal aneuploidy and recombination. More importantly it provides a vehicle within an ethical framework to continue to expand our knowledge and uncover new insights into the basis of meiotic errors that may aid future reproductive therapies.

<p> The molecular gatekeepers of nearly all gene expression in living cells are the proteins that function in the process of transcription. Transcription occurs when a cell must respond to a signal. These signals can be in the form of metabolic responses, signals for growth or differentiation, signals to defend against stress or pathogenic invasion, to name a few. The fundamentals of transcription have been extensively studied in bacterial systems and model organisms, but technical limitations have hindered their studies in mammalian and human systems. Recent developments in mass spectrometric methodologies, next-generation sequencing and techniques to study difficult-to-detect post-translational protein modifications are extensively reviewed here to highlight an important regulatory network through which gene expression is regulated. In addition, I present two vignettes: the first, a study of the regulatory mechanisms of monomethylation of the HIV-1 Tat protein in regulating HIV-1 gene expression and latency; the second, a study investigating the role of acetylation in regulating RNA Polymerase II protein modifications and gene expression in mammalian systems. Together, these studies combine new mass spectrometric techniques, modification-specific antibodies, protein purification methods, and next generation sequencing to better understand the role of these modifications in regulating the transcriptional response in mammalian systems. These findings can be applied to better understand mechanisms that regulate HIV-1 viral latency, along with fundamentally shifting the field of mammalian transcription by pinpointing unique modes of regulation only found in higher eukaryotes relevant to HIV-1 infection and cancer.</p><p>

No description available.

Sepsis is the overwhelming inflammatory response to infection, especially bacterial infection and associated bacterial products. It has major healthcare impacts, affecting an estimated 19-30 million persons/year worldwide with a mortality of 30-70%. Despite intense research, no specific therapy has been established for sepsis and in addition to the high mortality, the associated economic costs are very high. For example, recent data shows that the annual cost of patients with sepsis is more than $20 billion in the USA, and £2.5 billion in the UK. Therefore, novel targets and new therapies for sepsis are required which will have an important impact on both mortality and economic benefits. Recent work has demonstrated that the phopsholipid modifying enzyme, LPCAT, has a role in the regulation of inflammatory responses to bacterial infections. However, the mechanism of action in this regard is not well understood. This project aimed to identify the role of LPCAT-2 in inflammatory response to infections. This project has utilized the RAW264.7 murine macrophage cell line as an experimental model and LPS or Pam3CSK4 as infectious stimuli to investigate the role of overexpressing LPCAT-2 as well as silencing the over-expressed LPCAT-2 using siRNA technique. RAW264.7 cells transiently or stably transfected with the LPCAT-2 gene were used to study the role of LPCAT-2 in the inflammatory responses of macrophages. LPCAT-2 was successfully over-expressed in RAW264.7 cells and the overexpression was successfully confirmed with real time polymerase chain reaction (RT-PCR) and western blotting. The overexpression of LPCAT-2 significantly upregulated the pro-inflammatory cytokines TNF-α and IL-6, at both gene expression, and protein level, while the anti-inflammatory cytokine, IL-10, was down regulated in these cells. Moreover, overexpression of LPCAT-2 significantly decreased the expression of TLR4, peroxisome proliferator-activated receptors –gamma (PPARγ) and CD206 (a marker of M2 macrophages) while it significantly increased CD14, TLR2, COX-2 and iNOS (M1 markers). LPCAT-2 gene expression was also increased when PPARγ was blocked with the selective (PPAR-γ) antagonist T0070907. Importantly, silencing the transiently over-expressed murine LPCAT-2 resulted in a significant reduction in TNF-alpha and a significant increase in IL-10 gene expression. Both the transient and stably transfected RAW264.7 cells have been used to study the role of LPCAT-2 in regulating inflammatory responses in macrophages. The results have significantly added to knowledge of the role of LPCAT-2 in the inflammatory response and will aid in the development of novel therapies for inflammatory conditions such as sepsis.

<p> <i>Candida albicans</i> is a yeast-like fungal pathogen that can cause infections ranging from superficial to life-threatening systemic candidiasis. Current treatments for systemic candidiasis are available but often ineffective and toxic. Consequently, it is necessary to develop new therapeutic approaches. The purpose of this study was to construct antibody-based fusion proteins that can bind to <i>C. albicans</i> cells and eliminate them. Two such fusion proteins were constructed. Each one is composed of M1 Fab as the antibody component that binds to <i> C. albicans</i> mannan and the antifungal peptide HPRP-A1. HPRP-A1 was attached via a 15-amino acid linker to either the C-terminus of the constant light chain of M1 Fab (M1 Fab-HPRP-CL) or the N-terminus of the variable light chain of M1 Fab (M1 Fab-HPRP-VL). Binding of the fusion proteins to purified <i> C. albicans</i> mannan was assessed with enzyme-linked immunosorbent assay and the half maximal effective concentration (EC₅₀) for each fusion protein was estimated. EC₅₀ for M1 Fab-HPRP-CL was 273.6 compared to 74.1 for the original M1 Fab (<i>p</i> &lt; 0.05), whereas M1 Fab-HPRP-VL did not show any binding activity, indicating a negative impact on the antibody binding by the linked peptide. Similarly, M1 Fab-HPRP-CL also showed reduced binding for <i>C. albicans</i> cells when compared to M1 Fab as determined with immunofluorescence microscopy and flow cytometry. The effect of M1 Fab-HPRP-CL on the growth of <i>C. albicans</i> cells was analysed using microdilution and absorbance. At 16 &micro;M, the growth of yeast cells treated with M1 Fab-HPRP-CL was 47.1 % of the growth control, compared to 43.5 % for M1 Fab (<i>p</i> > 0.05) and to 1.9 % for HPRP-A1 by itself (<i>p</i> &lt; 0.001). Moreover, HPRP-A1 killed <i>C. albicans</i> at 32 &micro;M and 64 &micro;M, while M1 Fab and M1 Fab-HPRP-CL did not, indicating a loss of the antifungal activity of HPRP-A1 when attached to the antibody. These data together provide valuable insights into the development of novel antibody-based therapeutics as an alternative treatment for candidiasis.</p><p>

<p> Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels, control abnormal vasculatures and prevent recurrence. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties, lay down vascular patterned networks reminiscent of host endothelial blood vessels and served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands in the event of the ensuing hypoxia resulting from AAT. In our studies, we have demonstrated that AAT accelerates VM. We observed that Vatalanib (a VEGFR2 tyrosine kinase inhibitor) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer side. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at the periphery of the tumors. During our extensive studies to understand the tumor-inherent mechanisms of AAT-induced resistance, we identified a crucial chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. IL-8 has been well established as a highly prevalent cytokine in GBM with potent pro-migratory and pro-angiogenic functions. AAT-treated groups had significantly higher populations of CXCR2+ glioma stem cells and endothelial-like subpopulations and these populations were decreased following treatment with HET0016 and SB225002 (a CXCR2 antagonist). CXCR2+ GBM tumor cells were shown to form VM-like vascular channels carrying functional RBCs. Knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that HET0016 and SB225002 have potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.</p><p>

Pronounced opsonic activity has been demonstrated in the coelomic fluid of the Oligochaete Eisenia foetida. This humoral factor enhances the uptake of particles by Eisenia phagocytes by 94% in the case of S. cerevisiae. 31.5% inE.coli and by 34.6% in B.megaterium. In order to determine whether agglutinin molecules are responsible for this activity, the 20,000D haemagglutinin was purified. This agglutinin was found to be heat sensitive, (15 min at 60&deg;C destroying activity completely), partially dependent on calcium ions and with an optimal pH range of 5-8. The agglutinin was found to be active towards a range of vertebrate erythrocytes and various fungal and bacterial cells but is not responsible for opsonic activity. Polyclonal antibodies to this purified agglutinin were raised in rabbits and used in immunocytochemical studies to determine the source of this molecule. It was found that the cell membranes of Eisenia phagocytes bear agglutinin or agglutinin like molecules and their possible roles in internal defence mechanisms is discussed. Eisenia coelomocytes were found to release agglutinins in vitro though the site of synthesis of these molecules is yet to be established.

Two molecular forms I and II (separable by gel filtration) from mature Vicia faba seeds have been studied. The extractability of the enzymes and the in vitro conversion of the low MW form, II, to the larger oligomer, I has been examined over a range of salt concentrations. Specific and total activities of the preparations were high when strong salt solutions were used for extraction. It would appear that α-galactosidase I, in comparison with II, is best extracted from the seeds if solutions of high ionic strength are used. The effects of these salt solutions on the relative levels of the two forms have been investigated by gel filtration. Interpretation of the gel elution profiles obtained is, however, complicated by the in vitro conversion of form II to form I, which is favoured by high salt concentrations and some routine procedures in the purification of α-galactosidases, such as ammonium sulphate fractionation. The relationships between the multiple forms have been studied. α-galactosidase II can be resolved into enzymes II1 and II2 by CM-cellulose chromatography, a-Gal actosidases I, II1 and II2 have been highly purified. Form I (MW 160,000) is a tetramer of enzyme II2 as shown by SDS-PAGE. Immunological studies on the three forms have been carried out and the evidence suggests that they are structurally related, although forms I and II2 are more closely related than forms I and II. On hydrolysis,monosaccharides are released from the three purified enzymes, suggesting they are glycoproteins. The three α-galactosidases also agglutinate red blood cells indicating that they possess lectin activity.