Date of Degree

5-2018

Document Type

Dissertation

Degree Name

Ph.D.

Program

Physics

Advisor(s)

Gregory S. Boutis

Committee Members

Keith T. Downing

Emmanuel J. Chang

Ruth E. Stark

Nicolas Biais

Subject Categories

Biological and Chemical Physics

Keywords

Elastin, Collagen, Desmosine, NMR, MALDI

Abstract

This thesis reports on structural and dynamical modifications of reproductive tract elastin and collagen as a function of parity. Pelvic floor dysfunction, including pelvic organ prolapse (POP) is a major concern affecting female health worldwide, leading to surgeries costing billions of dollars annually. Collagen, elastic fibers, and proteoglycans are major extracellular matrix (ECM) components found in connective tissues. Vaginal child birth, advancing age, and disruption or dysfunction of connective tissue are major risk factors of POP. In the female reproductive tract, the assembly of elastic fibers is crucial for the pelvic floor support. Any disturbance in the synthesis, assembly, and degradation of elastic fibers in the reproductive tract during parturition or aging may result in pelvic floor dysfunction. Various experimental methods were implemented to probe the structural and dynamical changes of these proteins, as a function of the number of pregnancies, including 13C nuclear magnetic resonance (NMR), 2H NMR, spectrophotometry, MALDI mass spectrometry, and histology. Results of this study provide the gynecological community with further understanding relating to how elastin and collagen of the reproductive tract are altered as a function of the number of births, and may contribute to the loss of tissue elasticity and the onset of pelvic floor dysfunction.

The concentration of elastin in the reproductive tract is approximately 1.5% by weight making quantification challenging. This thesis has provided an important step in understanding how pregnancy and/or parturition may lead to alterations of reproductive tract elastin. In the elastin study, experimental data unveils remarkably different 13C spectra for virgin rat cohorts may be due to higher concentration of MMP-2 and MMP-9, which degrade elastin, compared to elastin from the multiparous rat cohorts. Single pregnancy cohorts exhibit spectra similar to the virgin rat, whereas elastin from cohorts that have undergone three pregnancies resembled that from the multiparous rat cohorts. By applying solid state NMR relaxation measurements the dynamics of major backbone and side chain carbon nuclei in the major amino acids, (e.g., glycine, valine, proline, etc.) were found to vary significantly in virgin cohorts in comparison to multiparous cohorts. Furthermore, the concentration of desmosine was found to be highest in the multiparous cohorts and similar to that found in other mammalian elastins. While the microscopic structure of elastin in multiparous cohorts appears strikingly similar to elastin in other mammalian tissues, the elastic fibers are fragmented and highly tortuous. Our current model suggests that the elastic fiber fragmentation, which occurs over length scales of approximately 10-50 micrometers (the length of an elastic fiber), together with collagen content may play a crucial role in tissue scaffolding than alterations in microscopic structure (e.g. desmosine crosslinks).

Collagen, another major component of extracellular matrix protein in the reproductive tract, also undergoes significant remodeling and alterations during pregnancy. Interestingly, the structure of collagen was found to be intact despite tremendous remodeling and alterations during pregnancy. However, collagen content was observed to be highest in the virgin cohorts compared to multiparous cohorts, by histological methods. 2H NMR relaxation methods were used to characterize the dynamics and distribution of water in both collagen and elastin as well as only elastin of various cohorts. The redistribution of water due to pregnancy and/or parturition results in additional water reservoir in postpartum and multiparous cohorts in collagen and elastin samples. However, in only elastin from multiparous and virgin cohorts, dynamics of water were observed to vary significantly across cohorts due to fiber fragmentation, and microscopic alterations driven by MMP concentration.

Apart from reproductive tract collagen and elastin, this thesis has provided detailed measurements relating to the effects of ultraviolet (UV) irradiation on elastic fibers. UV-A (365 nm) exposure of elastic fibers was shown to result in distinct cracks and disruption in regular array of elastic fibers. Additionally, the concentration of desmosine in elastin is reduced in comparison to the nonirradiated elastic fibers after UV irradiation. This portion of the thesis has introduced a method for readily measuring the concentration of desmosine in tissues and other bodily fluids by MALDI mass spectrometry.

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