Doctoral Dissertation Template
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UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE METAGENOMICS AND SOCIAL INCLUSION IN PERU A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By ALEXANDRA J. OBREGON-TITO Norman, Oklahoma 2013 METAGENOMICS AND SOCIAL INCLUSION IN PERU A DISSERTATION APPROVED FOR THE DEPARTMENT OF ANTHROPOLOGY BY ______________________________ Dr. Cecil M. Lewis, Chair ______________________________ Dr. Paul Spicer, Co-Chair ______________________________ Dr. Tassie Hirschfeld ______________________________ Dr. Morris Foster ______________________________ Dr. Charles Kenney © Copyright by ALEXANDRA J. OBREGON-TITO 2013 All Rights Reserved. Acknowledgements I express my appreciation to a number of people who made this dissertation possible. First and foremost I offer my sincerest gratitude to my mentor, Dr. Cecil Lewis, I am greatly thankful for his encouragement, patience and for allowing me the room to work in my own way. I could not wish for a better supervisor. I was honored to work with Raul Tito in the laboratory; I am blessed to have such as talented person in my life. I owe deepest gratitude to him and Juan Jose for being my backbone during each of my new adventures. I share the credit for this dissertation with them. I am also thankful to my mother and brothers that make me believe no distance can keep us apart. I would like to thank the Comunidad Matses Anexo San Mateo, Omar Trujillo, Luis Marin, Emilio Guija, Luzmila Troncoso, Susan Polo and Maria Elena Medina for all their help in the implementation of this project. I thank the faculty of the Anthropology department for their guidance, in particular Paul Spicer, Tassie Hirschfeld and Morris Foster, and Charles Kenney for their dedication as members of my doctoral committee. I greatly appreciate the support and encouragement of Jeffrey Weitzel, Kathleen Blazer whose constant encouragement kept me going. Finally, I would like to thank Silvia Smith, Milagros Granda, Lori Williamson, Lauren Cleeland, Lauren O’Shea and Andrew Ozga who had helped me tremendously through this process. I feel privileged to have them as my friends. iv iv Table of Contents Acknowledgements……………………………………………………………… iv List of Tables……………………………………….…………………………..... viii List of Figures……………………………………………….…………………… ix Abstract…………………………………………………….…………………….. xi Chapter1 - Gut Microbiota Of A Peruvian Hunter-Gatherer Community Introduction…………………………………………………………………….... 1 Background………………………………………………………………………. 2 Materials And Methods Sample and data collection…………………………………………………...…… 9 Fecal sample……………………………………………………………………… 10 Anthropometry……………………………………………………………………. 11 Dietary information………………………………………………………………. 12 Parasite Analysis…………………………………………………………………. 12 DNA extraction…………………………………………………………………… 13 DNA amplification………………………………………………………………... 13 Data analysis……………………………………………………………………… 14 Results……………………………………………………………………………. 17 Discussion………………………………………………………………………… 37 Conclusions………………………………………………………………………. 45 Chapter 2 - Implementing Biomedical Research With Indigenous Peoples: Facing Issues Of Trust v Introduction ………………………………………………..…………………… 46 Materials And Methods Sample and data collection…………………………………………..…………… 54 Results…………………………………………………………………………..... 55 Discussion ………………………………………………………………….….… 67 Conclusions…………………………………………………………………….… 82 Chapter 3 - Research In Wonderland: Including Indigenous Populations In International Biomedical Research Introduction……………………………………………………………………… 84 Diversifying human populations in biomedical research………………………… 86 Overcoming issues of trust……………………………………….…..…………… 88 Working with vulnerable populations from the developing world: ethics of protection………………………………………………………………………… 94 International research collaborations………………………………………..… 101 Fair Share of Benefits and property rights……………………………………… 103 Ensuring relevance of research and avoiding exploitation………………………… 105 Property rights …………………………………………………………………………... 107 Implementing metagenomics research with indigenous populations in Peru Building a team…………………………………………………………..…….… 109 Obtaining ethical approval………………………………………………..……… 111 Engaging regional authorities……………………………………………..……... 114 Engaging communities……………………………………………………..……... 117 vi Incorporating communities’ interests………………………………………..…… 118 Providing benefits for the community…………………………………….…..…... 119 An intermediate research organization………………………………………… 121 Building an intermediate research organization…………………………………. 123 Researcher initiated projects……………………………………………..…..…... 125 Improving accountability through an intermediate research organization……….. 126 Community initiated research…………………………………………………….. 128 Managing biological materials……………………………………….…………... 130 Conclusion………………………………………………………………………... 133 Epilogue ………………………………………………………………………….. 135 References………………………………………………………………………... 138 vii List of Tables Table 1. Barcode and primer sequences……………………………………………. 15 Table 2: Ages and body mass index (BMI) of the participants. …………………… 18 Table 3: Summary of the Matses diet……………………………………………… 20 Table 4. Results of parasite screening. ……………………………………..……… 24 Table 5. Independent sample t-test to compare microbial profile of individuals found positive or negative during the parasite screening…………………………… 25 Table 6. Sequencing yield……………………………………………………..…… 26 Table 7 Table of genus frequencies……………………………………….…..….… 29 Table 8. One way ANOVA to compare means across populations………………… 32 Table 9. One way ANOVA, pair-wise comparisons…………………….………… 33 Table 10. Demographics of survey participants. ………………………………..… 56 Table 11. A fair share of benefits between researchers and host communities…….. 106 Table 12. Comparing distinct models of research with respect to level of community participation ……………………………………………….….............. 110 viii List of Figures Figure 1. Geographical location of the Matses community anexo San Mateo…….. 9 Figure 2. Public meeting with the community……..……..……..……..………….. 10 Figure 3. Anaerobic sampling……..……..……..……..……..……..…….....…….. 11 Figure 4. Anthropometry……..……..……..……..……..……..……..……..…….. 12 Figure 5. Parasite screening……..……..……..……..……..……..……...…..…….. 13 Figure 6: Age distribution of samples obtained from the Matses community anexo San Mateo.…..……..……..……..……..……..……….……..……..……..……..… 17 Figure 7. Matses cooking..…………………………..……...……..………………. 23 Figure 8. Firmicutes is the dominant phylum in the gut microbiome of our Matses sample ……………………………………………………………………………... 28 Figure 9: Rarefaction curve of the Matses Samples……………………………….. 29 Figure 10: Distribution of most representative phyla across populations..………... 31 Figure 11. Primary Component Analysis...………………………………………... 36 Figure 12. Identification with authorities .……………………………………...…. 56 Figure 13. Monitoring of research activities in the community..…..……………… 57 Figure 14. Attitudes towards visitors………………………………………....……. 58 Figure 15. Attitudes towards national and international visitors and authorities….. 59 Figure 16. Interest to participate in research.…….……….……….………………. 60 Figure 17. Sample ownership..…………………………………………….....……. 62 Figure 18. Ownership of microorganisms…………………………………………. 63 Figure 19. Willingness to donate biological samples………………………...……. 65 Figure 20. Attitudes towards publication of research results..……………………. 66 ix Figure 21. Visitors in the community..……………………………………….……. 73 Figure 22. Attempts to divide the community..……………………………………. 75 Figure 23. Suspicious activities in the Matses community becoming public ……... 76 Figure 24. The trust process ..……………………………………………………... 81 Figure 25 Snapshot of research in the world ..…………………………………….. 104 Figure 26 Authorities with jurisdiction over research projects with indigenous peoples in Peru…………………………………………………………………….. 112 Figure 27. Ethics review process…………………………………………………... 115 Figure 28. Interactions among stakeholders in the research enterprise under the Intermediate Research Organization model..……………………………………… 122 Figure 29. Goals of the Intermediate Research Organization. ……………………. 124 Figure 30. Structuring an Intermediate Research Organization. ………………….. 129 Figure 31. Researcher initiated research..………………………………………….. 132 x Abstract Metagenomics is the study of all the genetic material found in an ecosystem. Each part of the human body is an ecosystem with a distinct bacterial profile or microbiota whose metagenomic signature is called a human microbiome. The study of human microbiomes is a promising new area of biomedical research. Certain bacterial species are directly associated to specific diseases, suggesting their potential as biomarkers or even as therapeutic alternatives. As it has happened with genetics and genomics applications, most microbiome research is focused on urban human populations, thus providing a very biased set of study populations, both human and bacterial. The developing world harbors biological phenomena absent in the developed world. The study of traditional communities from developing countries provides unique opportunities for scientific advancement. At the same time, the knowledge obtained might impact public health programs and help to build research capacity in these countries. However, including traditional communities from developing countries in research has proved to be a sensitive matter. Research with indigenous populations carries the stigma of being exploitative, neocolonialist, and imperialist. Engaging communities in research can reduce the risk of exploitation