Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 108-111).

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 66-68).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Biological Sciences. / Includes bibliographical references (leaves 69-70).

Patients with dysfunctional ovaries, such as those with premature ovarian insufficiency and granulosa cell tumours, do not have normal follicle development and may not respond to traditional assisted reproductive techniques. Using the reaggregated ovary (RO) technique, these patients' oocytes may be reaggregated with functional supporting cells and cultured in vitro to develop fertilisable eggs. However, current research using ROs have only used murine ovaries as a somatic cell source. In this thesis, with the aim of moving towards a clinical treatment, we assessed follicle development in ROs in vitro and progressed to using the technique with human tissues. To assess whether an older murine somatic cell source resulted in advanced follicle development, and how follicle development differed between transplanted and cultured ROs, ROs were generated using postnatal day 2 (P2) and P6 mouse ovaries. To investigate theca cell development in follicles from cultured tissue, mouse ovaries were cultured with mouse serum or encapsulated in hyaluronan hydrogels. Prior to generating and culturing chimeric human-mouse ROs (HuMoROs), competent handling and digestion of bovine cortical tissue was required. Broadly, ROs generated from both P2 and P6 exhibited similar follicle development in vitro after 14 d of culture, and follicles from cultured ROs were more developed than those from transplanted ROs. Theca cell development observed in follicles from cultured ovaries was still poorer than those from in vivo ovaries, even when ovaries were cultured in mouse serum or encapsulated in a hyaluronan hydrogel. Finally, some follicles containing potential human oocytes developed within the generated HuMoROs after 7 d of culture. These results have highlighted the potential of the RO technique as a method to generate fertilisable eggs and identified further aspects which need to be targeted in order to improve the success of the technique.

MeCP2 was identified by its ability to bind DNA in a methylation-specific manner. Yet, how it interprets the DNA methylome remains unclear. Several mechanisms have been proposed, including a role in transcriptional repression. MeCP2 is highly abundant in the brain, and loss-of-function mutations result in a neurological disorder called Rett syndrome (RTT). Strikingly, RTT-causing missense mutations are almost all located in either the methyl-CpG-binding domain (MBD) or a region that has been shown to bind the NCoR/SMRT co-repressor complex (NID). This suggests that the MBD and the NID are the key functional domains in MeCP2, and that the role of MeCP2 is to form a ‘bridge’ between chromatin and the co-repressor complex to regulate gene expression. To test this ‘bridge’ hypothesis, I have made an allelic series of knock-in mice with truncated forms of MeCP2 to determine whether the other regions are dispensable for protein function. The three other regions of MeCP2 (the N-terminus before the MBD, the Intervening region between the MBD and the NID, and the C-terminus after the NID) were deleted in a step-wise manner to produce progressively smaller truncated proteins. Knock-in mice which lack just the N-terminus or both the N- and C-termini are phenotypically normal. Therefore, these regions, which together make up 46% of the protein sequence, are dispensable for MeCP2 function in vivo. Additional deletion of the Intervening region, retaining only 34% of the original sequence, results in mild RTT-like symptoms in the knock in mice. This is likely to be caused by this protein’s decreased stability and reduced ability to bind the NCoR/SMRT complex in the brain. The most severely truncated protein is nevertheless able to reverse the Mecp2-null phenotype when reactivated after the onset of symptoms. Together, these findings strongly support the ‘bridge’ hypothesis.

The aim of his study was to investigate the role of Dctn2 in mouse preimplantation embryonic development, specifically its effect on the first cell fate decision, when the number of cells increases from eight to sixteen.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Rift Valley fever virus (RVFV) is an enveloped, negative-sense, ssRNA virus with a tripartite genome that causes morbidity and mortality in both livestock and humans. Although RVFV is mainly circulating in mainland Africa, this arthropod-borne virus is a potential threat to the other parts of the world. No fully licensed vaccines for human or animal use in the U.S., and effective antiviral drugs have not been identified. As virulent RVFV strains are only handled in biosafety level (BSL) 3 or higher level facilities in the U.S., few laboratories have access to RVFV which limits antiviral development. However, it is crucial to develop effective antivirals to protect public and animal health. Animal models that reproduce Rift Valley fever are vital to identifying and developing antiviral compounds. The currently available attenuated RVFV strain, MP12, provides a BSL-2 challenge model virus for preliminary investigations of RVFV prior to using the virulent RVFV strains. All strains of RVFV have a highly conserved genome, indicating that antivirals or vaccines effective against any RVFV strain will most likely be effective for all RVFV strains. Therefore, we hypothesize that the MP12 is a suitable model virus that can be used for identification and evaluation of effective RVF antivirals. The first objective of this project was to establish a mouse model susceptible to MP12 infection. Based on the literature, we selected and screened six different strains of mice to test their susceptibilities to MP12. We found the STAT-1 knockout mice are the most susceptible to MP12 infection based on clinical symptoms, mortality, viremia, virus replication, histopathological, and immunochemical analyses. Importantly, these mice displayed acute-onset hepatitis and delayed-onset encephalitis similar to severe cases of human RVFV infection. Our second objective was to identify potential antiviral drugs in vitro. We developed and employed a cell-based assay using the recombinant MP12 virus expressing Renilla luciferase to screen a library of 727 small compounds purchased from National Institutes of Health. Of the compounds, 23 were identified and further tested for their inhibitory activities on the recombinant MP12 virus expressing green fluorescent protein. Further plaque reduction assays confirmed that two compounds inhibited replication of parental RVFV MP12 strain with limited cytotoxic effects. The 50% inhibitory concentrations using an MP12 multiplicity of infection (MOI) of 2 were 211.4 µM and 139.5 µM, respectively. Our third objective was to evaluate these two candidates, 6-azauridine and mitoxantrone, in vivo using our mouse model. After one-hour post MP12 infection via an intranasal route, treatment was given intranasally twice daily. Mice treated with placebo and 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurological signs, while mice treated with ribavirin (a known antiviral drug) or mitoxantrone showed delayed onset of disease. This result indicates that the mitoxantrone can improve the outcome of RVFV infection in our mouse model. The underlying mechanism of mitoxantrone to inhibit RVFV replication remains to be investigated. Our studies build the foundation for identification and development of antivirals against RVFV in a BSL-2 environment.

Doctor of Philosophy / Department of Biology / Stephen Chapes / Antibodies are an essential part of the immune system. Each B cell, a type of white blood cell, produces a unique antibody. This antibody molecule is comprised of two identical light chains and two identical heavy chains. Each chain has a variable region, which is responsible for antigen binding, and a constant region, which is responsible for effector function in the host. The variable region in the heavy chain is composed of three gene segments, the variable (V), diversity (D), and joining (J) gene segments. The light chain is composed of only V- and J-gene segments. Each immunoglobulin locus contains multiple versions of each gene segment, ranging from over 130 possible V gene segments in the heavy chain to four possible J-gene segments in both the heavy and kappa light chain. The recombination of gene segments occurs in the germline DNA and results in the formation of the unique antibody. The diversity and binding abilities of the antibodies are important for a proper and robust immunological response. Of importance to binding and specificity is the complementary determining region three (CDR3) which plays a major role in determining specificity and antibody-antigen binding. Due to its uniqueness, is used as a measure of diversity in the repertoire. In this work, I used Illumina MiSeq 2x300nt high-throughput sequencing to assess the mouse splenic transcriptome. The work I present here shows the splenic immunoglobulin gene repertoire from unchallenged, unvaccinated conventionally housed mice, mice flown aboard the International Space Station (ISS), and mice challenged with tetanus toxoid (TT) and/or adjuvant (CpG) and subjected to skeletal unloading by antiorthostatic suspension (AOS). AOS is used to induce some of the physiological changes that parallel those that occur during space flight. The characterization of the repertoire includes analysis of V-, D-, and J-gene segment usage, constant region usage, V- and J-gene segment pairing, and CDR3 length and usage. The work included validation of the methodology needed for tissue preparation and storage aboard the ISS, showing that the data obtained was similar to those used in standard ground-based methodologies (Chapter 2). I further validated our nonamplified sequencing methodology with comparisons to methods that use amplification as part of the process (Chapter 3). My work characterized the antibody repertoire of the conventionally housed C57BL/6J mouse (Chapter 4), an important mouse strain in the field of immunology, and demonstrated the homogeneity of gene segment usage in unchallenged animals. We also demonstrated that short duration (~21 days) space flight does not significantly alter the antibody repertoire (Chapter 5). The work culminates in an AOS study to assess changes to the B-cell immunoglobulin repertoire after vaccination with TT and/or CpG. The results show that changes to V-, D-, and J-gene segment usage occur after antigen challenge with AOS causing decreased class switching and frequency of plasma cells. Tetanus toxoid challenge decreased multiple gene segment usage and CpG administration increased isotype switching to the IgA constant region (Chapter 6).

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate