Date

8-2015

Document Type

Dissertation

Degree Name

Ph.D.

Department

Department of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

Leishmania are the causative agent of leishmaniasis, a disease with many forms, the most severe of which is ultimately fatal if left untreated. Unfortunately, no vaccine is available, and resistance is emerging to currently employed therapeutics, many of which are costly and difficult to administer. Because the parasite must respond and adapt to a number of environmental insults throughout its lifecycle, including nutrient scarcity, inhibiting its mechanisms of adaptation is an attractive area for chemotherapeutic intervention. My thesis research focuses on the molecular mechanisms that allow Leishmania to persist in a non-dividing, but metabolically active, quiescent-like state for months in the absence of an extracellular purine—a nutrient essential for growth.

The introduction begins with a brief review of the unique biology of Leishmania as well as the current knowledge of nutrient sensing and adaptation in these parasites. Chapter 2 describes the global investigation into changes of both the proteome and transcriptome in response to the removal of purines from the parasite’s extracellular environment. These data reveal an extensive remodeling at both the protein and transcript level, highlighting how a few subtle increases or decreases of enzymes in a common pathway can lead to substantial metabolite changes, for example, increased intracellular proline. The data reveal a frequent discordant relationship between mRNA expression and protein abundance, thereby emphasizing the role of translation and posttranslational regulatory mechanisms.

The studies in Chapter 2 lead to the question of how the parasite senses these nutrient fluctuations, especially in the absence of canonical sensing and signaling mechanisms. My work in Chapter 3 details the investigation of internal versus external nutrient sensing mechanisms, showing for the first time that extracellular purine sensing occurs through perturbations in intracellular pools. Furthermore, by employing a cell line defective in purine interconversion, my thesis research determined that perturbation of the adenine-containing nucleotides alone is both necessary and sufficient for the adaptation of the parasite to purine stress—the first mechanistic insight for nutrient sensing in these parasites.

Chapter 4 concludes this dissertation by showcasing the utility of the mutant cell line described in Chapter 3 as a model for investigating molecular regulatory mechanisms. Preliminary studies into purine-pool specific protein abundance and phosphorylation are described. The long-term implications of such a robust model system for nutrient adaptation are discussed. In closing, it is my belief that the data generated by the studies described in this thesis will be useful many times over, not only in the understanding of the basic biology of the parasite, but also in combating a debilitating disease.

Identifier

doi:10.6083/M4348JCQ

School

School of Medicine

Table A.1.xlsx (17800 kB)
Table A.2.xls (14682 kB)
Table A.3.xlsx (149 kB)
Table A.4.xls (93 kB)
Table A.5.xlsx (987 kB)
Table A.6.xlsx (82 kB)
Table A.7.xlsx (46 kB)
Table A.8.xlsx (41 kB)
Table B.1.xlsx (36 kB)

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.