Development and utilization of a macaque-based mammosphere culture technique for breast cancer research

Includes bibliographical references.2016 Fall.Human breast cancers are thought to commonly arise from progressive neoplastic changes to the adult stem cells within the normal mammary gland. Research in this area of breast carcinogenesis currently relies heavily on the acquisition of mammary gland stem cells from the tissues of rodents and humans. While a great deal of information has been gained utilizing these models, there remain large gaps in our knowledge of breast cancer due to certain limitations with these species. The relevance of rodents as models for human breast cancer has been brought into question by notable differences between rodents and humans with regard to genetics, biology and mammary gland carcinogenesis. In contrast, the utility of human-derived samples is limited by ethical concerns and by the restricted availability of mammary tissues from women. Macaque monkeys are closely related to humans phylogenetically and these animals develop mammary gland tumors that are comparable to human breast cancers. Furthermore, mammary gland tissues can be easily collected from any demographic of animal. Despite their potential, only minimal breast cancer work has been undertaken in the macaques to date and research techniques common to both rodents and human are lacking for these species. This dissertation describes the optimization of a commonly-used mammary gland stem cell isolation technique, mammosphere culture, for the rhesus macaque (Macaca mulatta) and provides validation as to the reliability, relevance, and usefulness of this assay for human studies. Data obtained from this research demonstrated that the mammosphere culture technique is highly reproducible between homologous macaque tissue samples. This work also found that mammary gland biopsies collected from different anatomical locations on the same monkey share comparable mammosphere-forming ability and mammosphere-differentiation ability (collectively, the mammosphere potential). Finally, these initial studies identified macaque mammospheres to have proliferative and differentiating properties that are nearly identical to those described for human mammospheres. This dissertation also describes a series of macaque studies performed using the optimized mammosphere culture technique. In the first study, mammary gland tissues were obtained from female macaques in different reproductive demographics and the mammosphere potential of these animals was compared. The results suggested that the mammosphere potential of nulliparous mammary glands is significantly greater than that of multiparous mammary glands and that this difference is likely due to greater ratios of mammary gland stem cells within the nulliparous mammary gland. These data also suggested that there are differences in the mammosphere potential of mammary glands collected from animals at different stages of the reproductive cycle. An additional study comparing the mammosphere potential of young-multiparous and multiparous macaques collected during the menses stage of the menstrual cycle supported the parity-related findings of the first study. Data from the second study also identified significantly larger ratios of senescent cells in the mammosphere cultures of multiparous macaques as compared to young-nulliparous macaques. Finally, a study comparing the effects of ionizing radiation on mammospheres derived from young-nulliparous and multiparous macaques was performed. This last study found that stem cell-like cells of the young-nulliparous mammary gland were more resistant to the lethal effects of ionizing radiation than were those of the multiparous gland. The findings of these three studies are notable in that young-nulliparous girls are known to have a higher susceptibility to radiation-induced breast cancer than are multiparous women and these studies provide the first direct evidence as to the potential mechanistic reasons behind this observation. Specifically, as macaques appear to be relevant models for the study of the human breast, these data suggest that the increased susceptibility of young-nulliparous girls to radiation-induced carcinogenesis could arise from: 1) higher number of mammary stem cells within the breast; 2) a decreased predilection of these stem cells to undergo senescence; and 3) a decreased sensitivity of these stem cells to the lethal effects of ionizing radiation. In summary, macaque mammospheres appear to be relevant models for the study of the human breast. Use of this model allows for the study of mammary gland tissues from some demographics of interest (e.g., prepubescent individuals) that are impossible to investigate utilizing human tissues. The mammosphere culture techniques and data described in this dissertation serve as a foundation toward the use of macaques in future breast cancer research projects and other study data from this dissertation has provided novel insight as to the increased risk of radiation-induced breast cancers in young women

Rapid Evolution of BRCA1 and BRCA2 in Humans and Other Primates

The maintenance of chromosomal integrity is an essential task of every living organism and cellular repair mechanisms exist to guard against insults to DNA. Given the importance of this process, it is expected that DNA repair proteins would be evolutionarily conserved, exhibiting very minimal sequence change over time. However, BRCA1, an essential gene involved in DNA repair, has been reported to be evolving rapidly despite the fact that many protein-altering mutations within this gene convey a significantly elevated risk for breast and ovarian cancers. Results: To obtain a deeper understanding of the evolutionary trajectory of BRCA1, we analyzed complete BRCA1 gene sequences from 23 primate species. We show that specific amino acid sites have experienced repeated selection for amino acid replacement over primate evolution. This selection has been focused specifically on humans and our closest living relatives, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). After examining BRCA1 polymorphisms in 7 bonobo, 44 chimpanzee, and 44 rhesus macaque (Macaca mulatta) individuals, we find considerable variation within each of these species and evidence for recent selection in chimpanzee populations. Finally, we also sequenced and analyzed BRCA2 from 24 primate species and find that this gene has also evolved under positive selection. Conclusions: While mutations leading to truncated forms of BRCA1 are clearly linked to cancer phenotypes in humans, there is also an underlying selective pressure in favor of amino acid-altering substitutions in this gene. A hypothesis where viruses are the drivers of this natural selection is discussed.National Institutes of Health R01-GM-093086, 8U42OD011197-13National Science Foundation BCS-07115972Burroughs Wellcome FundMolecular Bioscience

The Chlamydomonas reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the outer dynein arm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737– 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83.4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme

Online psychoeducation with parent management training: Examining the contribution of peer support

Psychoeducation is an empirically based intervention that is increasingly delivered online to individuals and groups. Low participation has been a problem for online designs that include peer support. New technology designs have been called for, and in response, we developed a model that synchronized the delivery of individual and group-based psychoeducational activities for parent management training. We used a problem-based learning strategy delivered to caregivers of youth demonstrating oppositional behaviours to encourage the development of helping processes and peer support. This mixed methods intervention study had high rates of participant retention and positive measurable changes for two of its three psychoeducational outcome measures. When we merged the study data, we observed that mutual aid—a frequently sought goal of group-based interventions—contributed to participant outcomes

Essays on river mechanics

CER94-95-PYJ-3.Presented by the Graduate Students in CE 717 - River Mechanics (Spring, 1995).Instructor: P.Y. Julien.Includes bibliographical references.April 1995

Treatment Strategies and Mechanisms Associated with the Prevention of NASH-Associated HCC by a Toll-like Receptor 4 Inhibitor

UNLABELLED: We evaluated the cancer preventive efficacy of TAK-242, an inhibitor of Toll-like receptor 4 (TLR4), in a mouse model of hepatocellular carcinoma (HCC) occurring in the context of nonalcoholic steatohepatitis (NASH). We also assessed the cellular events associated with the preventive treatment efficacy. We tested oral administration of TAK-242, at clinically relevant but toxicity-reducing doses and scheduling, in mice with hepatocyte-specific deletion of Pten (HepPten-). The optimal dose and oral gavage formulation of TAK-242 were determined to be 30 mg/kg in 5% DMSO in 30% 2-hydroxypropyl-β-cyclodextrin. Daily oral administration of 30 mg/kg TAK-242 over 18 weeks was well tolerated and resulted in reduced development of tumors (lesions \u3e 7.5 mm3) in HepPten- mice. This effect was accompanied by reduced macrovesicular steatosis and serum levels of alanine aminotransferase. In addition, 30 mg/kg TAK-242 daily treatment of small preexisting adenomas (lesions \u3c 7.5 mm3) over 18 weeks, significantly reduced their progression to HCC. RNA sequencing identified 220 hepatic genes significantly altered upon TAK-242 treatment, that significantly correlated with tumor burden. Finally, cell deconvolution analysis revealed that TAK-242 treatment resulted in reduced hepatic populations of endothelial cells and myeloid-derived immune cells (Kupffer cells, Siglec-H high dendritic cells, and neutrophilic granule protein high neutrophils), while the proportion of mt-Nd4 high hepatocytes significantly increased, suggesting a decrease in hepatic inflammation and concomitant increase in mitochondrial function and oxidative phosphorylation upon TLR4 inhibition. In conclusion, this study identified treatment strategies and novel molecular and cellular mechanisms associated with the prevention of HCC in the context of NASH that merit further investigations. PREVENTION RELEVANCE: Means to prevent development of HCC or progression of small adenomas to HCC in patients with NASH are urgently needed to reduce the growing mortality due to HCC. We characterized the chemopreventive effect of oral administration of the TLR4 inhibitor TAK-242 in a model of NASH-associated HCC

Treatment Strategies and Mechanisms Associated with the Prevention of NASH-Associated HCC by a Toll-like Receptor 4 Inhibitor

UNLABELLED: We evaluated the cancer preventive efficacy of TAK-242, an inhibitor of Toll-like receptor 4 (TLR4), in a mouse model of hepatocellular carcinoma (HCC) occurring in the context of nonalcoholic steatohepatitis (NASH). We also assessed the cellular events associated with the preventive treatment efficacy. We tested oral administration of TAK-242, at clinically relevant but toxicity-reducing doses and scheduling, in mice with hepatocyte-specific deletion of Pten (HepPten-). The optimal dose and oral gavage formulation of TAK-242 were determined to be 30 mg/kg in 5% DMSO in 30% 2-hydroxypropyl-β-cyclodextrin. Daily oral administration of 30 mg/kg TAK-242 over 18 weeks was well tolerated and resulted in reduced development of tumors (lesions \u3e 7.5 mm3) in HepPten- mice. This effect was accompanied by reduced macrovesicular steatosis and serum levels of alanine aminotransferase. In addition, 30 mg/kg TAK-242 daily treatment of small preexisting adenomas (lesions \u3c 7.5 mm3) over 18 weeks, significantly reduced their progression to HCC. RNA sequencing identified 220 hepatic genes significantly altered upon TAK-242 treatment, that significantly correlated with tumor burden. Finally, cell deconvolution analysis revealed that TAK-242 treatment resulted in reduced hepatic populations of endothelial cells and myeloid-derived immune cells (Kupffer cells, Siglec-H high dendritic cells, and neutrophilic granule protein high neutrophils), while the proportion of mt-Nd4 high hepatocytes significantly increased, suggesting a decrease in hepatic inflammation and concomitant increase in mitochondrial function and oxidative phosphorylation upon TLR4 inhibition. In conclusion, this study identified treatment strategies and novel molecular and cellular mechanisms associated with the prevention of HCC in the context of NASH that merit further investigations. PREVENTION RELEVANCE: Means to prevent development of HCC or progression of small adenomas to HCC in patients with NASH are urgently needed to reduce the growing mortality due to HCC. We characterized the chemopreventive effect of oral administration of the TLR4 inhibitor TAK-242 in a model of NASH-associated HCC

Mammary Gland Cell Culture of Macaca fascicularis as a Reservoir for Stem Cells

The mammary gland contains adult stem cells that are capable of self-renewal and are likely target for neoplastic transformation leading to breast cancer. In this study, we developed a cell culture derived from the mammary glands of cynomolgus monkeys (Macaca fascicularis)(MfMC) and furthermore identified the expression of markers for stemness and estrogenreceptor-associated activities. We found that the primary culture can be successfully subcultured to at least 3 passages, primarily epithelial-like in morphology, the cultured cells remained heterogenous in phenotype as they expressed epithelial cell markers CD24, CK18, and marker for fibroblast S1004A. Importantly, the cell population also consistently expressed the markers of mammary stem cells (ITGB1 or CD29 and ITGA6 or CD49f), mesenchymal stem cells (CD73 and CD105) and pluripotency (NANOG, OCT4, SOX2). In addition to this, the cells were also positive for Estrogen Receptor (ER), and ER-activated marker Trefoil Factor 1, suggesting an estrogen responsiveness of the culture model. These results indicate that our cell culture model is a reliable model for acquiring a population of cells with mammary stem cell properties and that these cultures may also serve as a reservoir from which more purified populations of stem cell populations can be isolated in the future

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease