Dissertations / Theses on the topic 'Abortifacient'

Breakdown of a native chlamydial vaccine against ovine enzootic abortion (OEA), coupled with the identification of the protective capacity of the major outer membrane protein (MOMP) of <I>C. psittaci, </I>has encouraged research into the use of recombinant forms of MOMP in a third generation OEA vaccine. The prospect of testing all recombinant forms of MOMP in pregnant sheep trials is a daunting one, both in terms of time and money. To circumvent these problems, it was decided that a model system would be required to assess the efficacies of recombinant MOMP constructs. The initial model developed (active immunisation of mice) did not appear to reflect immunisation in sheep. Difficulties were encountered in eliciting seroconversion in mice vaccinated with recombinant antigens. It also demonstrated a possible H-2 link to the MOMP-specific antibody response. Problems associated with the model were overcome to some extent, by the development of a second model (passive transfer of sheep sera to mice). In this case, it was demonstrated that infection of mice susceptible to <I>C. psittaci,</I> could be reduced by passively transferring sera from sheep which had been vaccinated with recombinant antigens. The mechanism of neutralisation <I>in vivo</I> was also examined for this model. The model was further used to identify protective regions within the MOMP by passive transfer of affinity purified antibodies and monoclonal antibodies. Significant protection was afforded by the VS1 and VS2 regions of the MOMP.

Neospora caninum is an obligate intracellular protozoan parasite, which is the most frequently diagnosed abortifacient in dairy cattle in the UK and is a leading cause of abortion worldwide. Neospora caninum infection in early gestation is associated with foetal death whereas in late gestation, infection can result in the birth of asymptomatic, but persistently infected animals. How the parasite kills the foetus is not fully understood, but it has been suggested that more mature foetuses are better able to mount a stronger immune response to control parasite multiplication and dissemination. The ability of the bovine foetus to respond to various antigens develops in a sequential fashion during the gestation period and foetal immunocompetence starts to develop at approximately 100 days gestation age (dg), but can only fully recognise antigens during mid-gestation at around 150 dg. Chapter 2 assessed the pathological effects of N. caninum on bovine foetuses in early and late gestation (70 and 210 days gestation, respectively) and also in foetuses from naturally infected dams after recrudescence of N. caninum in mid to late gestation. Based on results of an initial histological screen of 35 bovine foetuses and 2 new-born calves, a total of 12 foetuses and calves were selected and subjected to more detailed histological examination. Both haemolymphatic and non-haemolymphatic tissues were used. The distribution of N. caninum antigen, CD3-positive T cells, PAX5-positive B cells, monocytes/macrophages and neutrophils (myeloid/histiocyte antigen/calprotectin-positive), antigen presenting cells (MHCII), interferon gamma (IFN-γ) expressing cells, PCNA-positive proliferating cells and apoptotic cells (cleaved caspase 3-positive) was analysed by immunohistology. In uninfected, control foetuses in early gestation (90 days gestation), haemolymphatic tissues were moderately developed and exhibited normal morphological features with low lymphocyte turn over and no evidence of IFN-γ production. Uninfected foetuses in late gestation had fully developed haemolymphatic tissues with high lymphocyte turnover, indicative of a mature immune system. In the infected foetuses in early gestation, extensive apoptosis of lymphocytes was observed in the thymus and spleen compared to controls (p<0.001, student’s t-test). No histological changes were observed in the haemolymphatic tissues of infected foetuses in late gestation. In non-haemolymphatic tissues, infected foetuses in early gestation exhibited extensive hepatocellular necrosis and apoptosis, glial cell necrosis and apoptosis in the CNS and high parasite loads in the liver, CNS and myocardium. There was no evidence of cell death in the heart despite the high parasite loads. In late gestation, histological lesions were restricted mainly to the CNS where non-suppurative inflammation and low parasite loads were observed. Other non-haemolymphatic tissues exhibited only mild mononuclear inflammatory infiltrates. The results suggest that in early gestation, tachyzoites replicate preferentially in foetal liver, brain and myocardium in the absence of an inflammatory response and cause extensive necrosis in the liver and brain. Unlike foetuses in early gestation, those in late gestation exhibited a mild to moderate mononuclear inflammatory infiltrate in various tissues dominated mainly by lymphocytes, plasma cells and smaller numbers of macrophages. In Chapter 3, the observation that N. caninum appeared to induce cellular degeneration in hepatocytes but not in the myocardium was investigated in more depth. An in vitro tissue culture system using the human HepG2 hepatoma cell line and the murine HL-1 cardiomyocyte cell line was used to establish the mechanism of cell death following N. caninum infection. The activation of the initiator and effector caspases (caspases 3, 8 and 9) was measured and the mitochondrial organisation in cells following N. caninum infection evaluated. Quantitative (caspase 3) and semi-quantitative (caspase 8 and 9) analyses were used to assess differences in N. caninum-infected and uninfected HepG2 and HL-1 cells. A significant difference was observed in the numbers of cleaved caspase 3-positive HepG2 cells at 20-36 hours post infection (p=0.029, Mann-Whitney U test) in infected cultures compared to controls. No significant difference was observed for caspase 8 and 9 expression. In HL-1 cultures, no significant difference was observed in the number of caspase 3, 8 and 9-positive cells between infected and control cultures. This suggests that N. caninum infection is not associated with activation of the caspase cascade in cardiomyocytes. Neospora caninum tachyzoites were detected within intact HepG2 and HL-1 cells with normal cellular morphology and which were not labelled with the caspase antibodies; whereas uninfected surrounding cells were caspase 3, 8 and 9-positive, indicating that the parasites are involved in the inhibition of the caspase pathways (intrinsic and extrinsic). The mitochondrial organisation in N. caninum-infected and uninfected cells was assessed in both cell lines using double immunofluorescence, which involved staining with a N. caninum specific polyclonal antibody and COX 1 mitochondrial marker. In the control cultures of both HepG2 and HL-1 cells, mitochondrial clumping with large aggregates of mitochondria exhibiting a punctate pattern was observed in high numbers of cells, mainly in the perinuclear region and this is suggestive of mitochondrial fragmentation, which is associated with apoptosis. Other cells within the control cultures revealed an unaltered reticular pattern of mitochondria that is consistent with the normal cellular morphology. In the infected cultures, there was mitochondrial clumping with aggregates of mitochondria detected surrounding parasitophorous vacuoles; while in neighbouring uninfected cells, large aggregates of mitochondria, exhibiting a punctate pattern were present, suggesting mitochondrial clumping and fragmentation associated with cytochrome c release and apoptosis. Other uninfected HepG2 and HL-1 cells exhibited a diffuse, homogenous distribution of mitochondria, often with an unaltered reticular pattern as was observed in the control cultures and is consistent with the normal cellular morphology. The results indicate that N. caninum inhibits apoptosis in infected cells and is associated with increased apoptosis in infected HepG2 cultures, while not having any effects on HL-1 cardiomyocytes. Chapter 4 investigated the seroprevalence of N. caninum infection in Jamaican dairy herds. Serum samples were analysed from 499 Holstein-Friesian and Holstein Friesian crossbreed dairy cattle from three different farms in Jamaica. A seroprevalence of approximately 26% was found with the majority of seropositive animals aged 0-2 years old (25%), while the lowest seroprevalence was recorded in animals over 13 years old (13.3%). Pregnancy status was shown to influence the seroprevalence of cows, but no significant relation of seropositivity to age was found, suggesting that vertical transmission is the principal route of transmission in Jamaica.

This study examined the phenomena of herbal contraceptives and abortives and their use among enslaved women in the United States and the Caribbean. The conclusions that can be drawn from the research are that some women did choose to use herbal birth control and abortives. There is evidence to suggest that this use may have been directly used as a uniquely female means of resistance to slavery. It is also indicated that the more African cultural retentions there were in other areas of the lives of these women, the more likely that this phenomena would be employed as well. The profession of healer as a means of gaining respect and authority in the plantation community and in reference to how they aided women seeking abortions is discussed as well. The paper uses many historical sources as well as many science texts to authenticate the availability and properties of the flora and fauna of the regions in which women were enslaved. The author also postulates that this phenomena was aided by African retentions of these methods as well as additions by Native Americans upon arriving in North America and the Caribbean. Birth and death rates from a plantation are also used with three reproductive case studies of the women who lived on the plantation. Many slave narratives as well as contemporary sources were used in the research and writing of this paper.

Leung Kwan-chi.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 1994.<br>Includes bibliographical references (leaves 215-223).<br>ACKNOWLEDGEMENTS --- p.i<br>ABSTRACT --- p.ii<br>ABBREVIATIONS --- p.iii<br>Chapter CHAPTER 1 --- INTRODUCTION --- p.1<br>Chapter 1.1 --- Brief description of Momordica charantia --- p.2<br>Chapter 1.2 --- Toxicity of RIPs and their potential uses in the treatment of AIDS --- p.3<br>Chapter 1.3 --- General mechanism of action of RIPs --- p.6<br>Chapter 1.4 --- Structure of αMMC --- p.7<br>Chapter 1.5 --- "Antigenicities of αMMC, BMMC and TCS" --- p.13<br>Chapter 1.6 --- "Immunosuppressive properties of the abortifacient proteins αMMC, BMMC and TCS" --- p.14<br>Chapter 1.7 --- Objectives of our study --- p.15<br>Chapter CHAPTER 2 --- EXPRESSION OF FULL-LENGTH αMMC cDNA --- p.20<br>Chapter 2.1 --- Expression of αMMC cDNA as a fusion protein --- p.22<br>Chapter 2.1.1 --- Materials and methods --- p.22<br>Chapter 2.1.1.1 --- Construction of fusion vector pRIT2T/MMC --- p.22<br>Chapter 2.1.1.2 --- Preparation of αMMC insert by PCR --- p.26<br>Chapter 2.1.1.3 --- Cloning of αMMC cDNA into fusion vector pRIT2T --- p.27<br>Chapter 2.1.1.4 --- Transformation --- p.28<br>Chapter 2.1.1.5 --- DNA sequencing --- p.29<br>Chapter 2.1.1.6 --- Expression of protein A-αMMC fusion cDNA --- p.30<br>Chapter 2.1.1.7 --- Preparation of fusion αMMC for affinity chromatography --- p.31<br>Chapter 2.1.1.8 --- Affinity chromatography of Protein A-αMMC fusion protein --- p.31<br>Chapter 2.1.1.9 --- Cleavage of protein A-αMMC fusion protein by factor Xa --- p.32<br>Chapter 2.1.1.10 --- SDS-PAGE analysis --- p.33<br>Chapter 2.1.1.11 --- Western blot analysis --- p.33<br>Chapter 2.1.1.12 --- Assay of biological activity --- p.35<br>Chapter 2.1.2 --- Results --- p.37<br>Chapter 2.1.2.1 --- Construction of pRIT2T/MMC --- p.37<br>Chapter 2.1.2.2 --- DNA sequencing --- p.40<br>Chapter 2.1.2.3 --- Expression of protein A-αMMC fusion cDNA --- p.42<br>Chapter 2.1.2.4 --- Purification of protein A-αMMC fusion protein --- p.45<br>Chapter 2.1.2.5 --- Cleavage of protein A-aMMC fusion protein --- p.49<br>Chapter 2.1.2.6 --- Assay of biological activity --- p.49<br>Chapter 2.1.3 --- Discussion --- p.51<br>Chapter 2.2 --- Expression of αMMC cDNA as an unfused protein --- p.52<br>Chapter 2.2.1 --- Materials and methods --- p.52<br>Chapter 2.2.1.1 --- Construction of the plasmid pET/MMC --- p.52<br>Chapter 2.2.1.2 --- Preparation of αMMC insert by PCR --- p.56<br>Chapter 2.2.1.3 --- Enzyme digestions --- p.57<br>Chapter 2.2.1.4 --- Ligation --- p.58<br>Chapter 2.2.1.5 --- Transformation --- p.59<br>Chapter 2.2.1.6 --- Screening for αMMC inserts --- p.59<br>Chapter 2.2.1.7 --- DNA sequencing --- p.60<br>Chapter 2.2.1.8 --- Expression of unfused aMMC cDNA --- p.60<br>Chapter 2.2.1.9 --- SDS-PAGE analysis --- p.61<br>Chapter 2.2.1.10 --- Western blot analysis --- p.62<br>Chapter 2.2.1.11 --- Purification of recombinant αMMC --- p.62<br>Chapter 2.2.1.12 --- Biological activity of recombinant αMMC --- p.63<br>Chapter 2.2.1.13 --- Radioimmunoassay --- p.63<br>Chapter 2.2.2 --- Results --- p.67<br>Chapter 2.2.2.1 --- Screening of pET/MMC --- p.67<br>Chapter 2.2.2.2 --- DNA sequencing --- p.69<br>Chapter 2.2.2.3 --- Expression of unfused αMMC cDNA --- p.69<br>Chapter 2.2.2.4 --- Radioimmunoassay --- p.72<br>Chapter 2.2.2.5 --- Purification of recombinant αMMC --- p.74<br>Chapter 2.2.2.6 --- Biological activity of recombinant αMMC --- p.74<br>Chapter 2.2.3 --- Discussion --- p.80<br>Chapter CHAPTER 3 --- EXPRESSION OF MODIFIED FORMS OF αMMC cDNA --- p.82<br>Chapter 3.1 --- Expression of deletion fragments of αMMC cDNA --- p.83<br>Chapter 3.1.1 --- Materials and methods --- p.83<br>Chapter 3.1.1.1. --- Construction of deletion mutants --- p.83<br>Chapter 3.1.1.1.1 --- Modification of pRIT2T/MMC --- p.86<br>Chapter 3.1.1.1.2 --- Preparation of closed circular DNA --- p.86<br>Chapter 3.1.1.1.3 --- Alpha-phosphorothioate nucleotide --- p.87<br>Chapter 3.1.1.1.4 --- Exo III digestion --- p.89<br>Chapter 3.1.1.1.5 --- Ligation --- p.89<br>Chapter 3.1.1.1.6 --- Transformation --- p.90<br>Chapter 3.1.1.1.7 --- Screening of deletion subclones --- p.91<br>Chapter 3.1.1.2 --- Confirmation of sequences --- p.91<br>Chapter 3.1.1.3 --- Expression of deletion mutants --- p.92<br>Chapter 3.1.1.4 --- Purification of deletion mutants --- p.92<br>Chapter 3.1.1.5 --- Cleavage of deletion mutants --- p.93<br>Chapter 3.1.1.6 --- Subcloning of the αMMC cDNA fragments --- p.94<br>Chapter 3.1.1.7 --- Expression of the unfused deletion --- p.96<br>Chapter 3.1.2 --- Results --- p.97<br>Chapter 3.1.2.1 --- Designation of the deletion mutants --- p.97<br>Chapter 3.1.2.2 --- Screening of deletion mutants --- p.98<br>Chapter 3.1.2.3 --- DNA sequencing --- p.100<br>Chapter 3.1.2.4 --- Expression of deletion mutants --- p.109<br>Chapter 3.1.2.5 --- Purification of the fusion fragments --- p.111<br>Chapter 3.1.2.6 --- Digestion of deletion mutants by factor Xa --- p.113<br>Chapter 3.1.2.7 --- Subcloning of αMMC deletion fragments --- p.115<br>Chapter 3.1.2.8 --- Expression of the unfused aMMC deletion --- p.117<br>Chapter 3.1.3 --- Discussion --- p.119<br>Chapter 3.2 --- Expression of a chimeric αMMC/TCS cDNA --- p.121<br>Chapter 3.2.1 --- Materials and methods --- p.122<br>Chapter 3.2.1.1 --- Construction of the MMC/TCS chimeric plasmid --- p.122<br>Chapter 3.2.1.1.1 --- Digestion of pfG104 - Preparation of GH1100 --- p.125<br>Chapter 3.2.1.1.2 --- Preparation of the GH405 fragment --- p.125<br>Chapter 3.2.1.1.3 --- Digestion of pACYC177 --- p.126<br>Chapter 3.2.1.1.4 --- "Dephosphorylation, ligation and transformation" --- p.126<br>Chapter 3.2.1.1.5 --- Confirmation of insert orientation --- p.127<br>Chapter 3.2.1.1.6 --- "Preparation of a fragment without PstI, ScaI" --- p.128<br>Chapter 3.2.1.1.7 --- Preparation of the 750-bp TCS fragment --- p.128<br>Chapter 3.2.1.1.8 --- Ligation of the TCS fragment --- p.129<br>Chapter 3.2.1.1.9 --- Cleavage of pACYC177/TCS with ScaI and PstI --- p.129<br>Chapter 3.2.1.1.10 --- Preparation of the PstI/HhaI-digested αMMC --- p.130<br>Chapter 3.2.1.1.11 --- Ligation of the 252-bp fragment --- p.131<br>Chapter 3.2.1.1.12 --- Cloning of MMC/TCS chimeric fragment --- p.131<br>Chapter 3.2.1.2 --- Expression of pET/MMC-TCS --- p.132<br>Chapter 3.2.1.3 --- SDS-PAGE analysis --- p.133<br>Chapter 3.2.1.4 --- Western blot analysis --- p.134<br>Chapter 3.2.1.5 --- Purification of MMC-TCS chimeric protein --- p.134<br>Chapter 3.2.2 --- Results --- p.135<br>Chapter 3.2.2.1 --- Construction of pET/MMC-TCS --- p.135<br>Chapter 3.2.2.2 --- Expression of TCS/MMC chimeric cDNA --- p.140<br>Chapter 3.2.2.3 --- Purification of MMC-TCS chimeric protein --- p.142<br>Chapter 3.2.2.4 --- Reactivity of MMC-TCS chimeric protein with various antisera --- p.145<br>Chapter 3.2.3 --- Discussion --- p.146<br>Chapter CHAPTER 4 --- SCREENING OF αMMC IMMUNO-REACTIVE FRAGMENTS FROM A RANDOM FRAGMENT LIBRARY --- p.148<br>Chapter 4.1 --- Materials and methods --- p.150<br>Chapter 4.1.1 --- Description of the pTOPE vector --- p.150<br>Chapter 4.1.2 --- Construction of an αMMC random fragment library --- p.152<br>Chapter 4.1.2.1 --- Preparation of the cDNA insert of αMMC --- p.155<br>Chapter 4.1.2.1.1 --- Large scale prearation of theE plasmid MMC18p8 --- p.155<br>Chapter 4.1.2.1.2 --- Digestion of the plasmid MMC18p8 with EcoRI --- p.156<br>Chapter 4.1.2.1.3 --- Electro-elution --- p.157<br>Chapter 4.1.2.2 --- DNase I digestion --- p.158<br>Chapter 4.1.2.3 --- Fractionation of DNA fragments --- p.159<br>Chapter 4.1.2.3.1 --- Electrophoresis --- p.159<br>Chapter 4.1.2.3.2 --- Electro-elution --- p.160<br>Chapter 4.1.2.4 --- Single dA Tailing --- p.161<br>Chapter 4.1.2.5 --- Ligation --- p.162<br>Chapter 4.1.2.6 --- Transformation --- p.162<br>Chapter 4.1.2.7 --- Controls --- p.163<br>Chapter 4.1.2.7.1 --- Full-length αMMC cDNA control --- p.163<br>Chapter 4.1.2.7.2 --- T-Vector ligation control --- p.164<br>Chapter 4.1.2.8 --- Storage of the fragment library --- p.164<br>Chapter 4.1.3 --- Immunoscreening of the random fragment library OF αMMC --- p.165<br>Chapter 4.1.3.1 --- Anti-αMMC sera --- p.165<br>Chapter 4.1.3.2 --- Purification of anti-αMMC sera --- p.165<br>Chapter 4.1.3.3 --- Colony lift --- p.167<br>Chapter 4.1.3.4 --- Induction of expression --- p.169<br>Chapter 4.1.3.5 --- Colony lysis --- p.169<br>Chapter 4.1.3.6 --- Immunoscreening --- p.170<br>Chapter 4.1.4 --- PCR screening of inserts --- p.170<br>Chapter 4.1.5 --- Amplification of positive signals --- p.172<br>Chapter 4.1.6 --- Dot blot --- p.173<br>Chapter 4.1.7 --- Confirmation of positive signals by Western blotting --- p.174<br>Chapter 4.1.8 --- Analysis of positive clones by DNA sequencing --- p.175<br>Chapter 4.1.9 --- Analysis of 3-dimensional structure of αMMC --- p.176<br>Chapter 4.1.10 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of aMMC --- p.176<br>Chapter 4.2 --- Results --- p.178<br>Chapter 4.2.1 --- Theoretical considerations --- p.178<br>Chapter 4.2.2 --- Construction of a random fragment library of αMMC cDNA --- p.180<br>Chapter 4.2.3 --- Screening for immuno-reactive fragments of αMMC --- p.183<br>Chapter 4.2.4 --- Confirmation of positive signals by Western blotting --- p.186<br>Chapter 4.2.5 --- Estimation of fragment sizes by PCR --- p.188<br>Chapter 4.2.6 --- Analysis of the fragment sequences --- p.190<br>Chapter 4.2.7 --- Cross-reactivity of the immuno-reactive fragments --- p.194<br>Chapter 4.2.8 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of αMMC --- p.196<br>Chapter 4.3 --- Discussion --- p.198<br>Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.200<br>Concluding remarks --- p.214<br>REFERENCES --- p.215<br>APPENDIXES GENERAL PROCEDURES --- p.224<br>Chapter A.l --- DNA sequencing --- p.224<br>Chapter A.2 --- Purification of DNA with Gene Clean --- p.229<br>Chapter A.3 --- Purification of primers after synthesis --- p.230<br>Chapter A.4 --- Purification of plasmid DNA by Magic Prep (Promega) --- p.232<br>Chapter A.5 --- Large-scale preparation of plasmid DNA by QIAGEN --- p.234<br>Chapter A.6 --- Lowry protein determination --- p.236<br>Chapter A.7 --- Preparation of acid phenol --- p.237<br>Chapter A.8 --- SDS-polyacrylamide gel electrophoresis --- p.238

Poon Yin-tat.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1994.<br>Includes bibliographical references (leaves 101-111).<br>ACKNOWLEDGEMENTS --- p.I<br>ABSTRACT --- p.II<br>LIST OF ABBREVIATIONS --- p.IV<br>TABLE OF CONTENTS --- p.V<br>Chapter CHAPTER 1: --- INTRODUCTION --- p.1<br>Chapter CHAPTER 2: --- PURIFICATION OF α- AND β-MOMORCHARINS --- p.26<br>Chapter CHAPTER 3: --- N-GLYCOSIDASE ACTIVITY OF α- AND β-MOMORCHARINS --- p.45<br>REFERENCES --- p.101

by Go Tong-Ming, Thomas.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1992.<br>Includes bibliographical references (leaves 86-95).<br>Chapter CHAPTER 1: --- INTRODUCTION --- p.1<br>Chapter CHAPTER 2: --- PURIFICATION OF α- AND β-MMCs --- p.23<br>Chapter CHAPTER 3: --- DEOXYRIBONUCLEASE ACTIVITY OF αAND β-MMCs --- p.44<br>REFERENCES --- p.86