Date of Award

5-23-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Ali Nawab

Abstract

Inositol polyphosphates (InsPs) are naturally occurring compounds that are widely distributed in both plant and animal cells. They regulate a variety of cellular processes including programmed cell death or apoptosis. Apoptosis maintains tissue homeostasis in higher organisms. Among inositol polyphosphates, inositolhexakisphosphate was found to be the most potent inducer of apoptosis in mammalian cells, however, the mechanism of apoptosis particularly mediated by InsPs is not well understood in simple eukaryotes such as Dictyostelium discoideum, despite InsPs having been well studied in this model organism. MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide, LDH, and trypan blue assays were used to investigate the cytotoxic effects of InsP6 on D. discoideum. To determine the apoptotic effect of InsP6, EB/AO and flow cytometry analysis were used. Further studies such as mitochondrial membrane potential (MMP) using JC-1 assay and reactive oxygen species (ROS) were undertaken. Polyacrylamide gel electrophoresis (PAGE) was used to detect the level of InsPs. Our results demonstrated that the administration of exogenous InsP6 induced apoptosis in D. discoideum. This study is directed towards understanding the signaling mechanism of apoptosis including any role of higher InsPs, such as InsP6, InsP7, and InsP8 in D. discoideum using flow cytometry and the apoptotic marker PARP. Furthermore, to establish the mechanism of action of InsPs, we examined how the manipulation of endogenous levels of InsPs affected apoptosis. Because cellular levels of inositol polyphosphates are regulated by inositol phosphate phosphatases and kinases, enzymes that metabolize them, we have questioned whether the levels also changed during apoptosis after using a certain metabolic inhibitor of endogenous levels such as NaF and Antimycin A. The level of InsP6 and InsP7 increased during apoptosis suggesting that higher inositol polyphosphates are indeed one of the regulators of apoptosis. To exploit InsPs mediated apoptotic process in therapeutic applications, particularly in combination with nanotechnology, we investigated any impact nanomaterials had on InsP mediated apoptotic mechanisms in D. discoideum. Our results showed that SWCNTs inhibited the accumulation of InsPs at lower concentrations and abolished them completely at higher concentrations of SWCNTs. In summary, our results provide, for the first time, the main role of higher InsPs inducing apoptosis in D. discoideum both exogenously and endogenously. Our hypothesis is that InsPs play a signaling role in mediating the cell death process in D. discoideum by utilizing the InsPs mediated apoptotic pathways present in higher animal cells. This research will provide further insight into the role of InsPs when developing therapeutic applications such as cancer treatment in conjunction with the use of nanoparticles to potentiate apoptosis in mammalian cells. This dissertation contains background information on the topics relevant to this study following a broad review of the literature. These in vitro studies may then lead to more translational work to assess their potential applications in the fields of pharmacology and medicine. Data provided by viability assays suggest a strong interaction between SWCNTs and cellular structures.

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