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The primary purpose of this study was to determine the relationship of self-reported resistance training to lipid profiles. The study consisted of 10 subjects between the ages of 19 and 35. Participants were assigned to an exercise group or control group based on self-reported resistance training and according to ACSM standards. After means were determined for the data gathered, the exercise group had lower averages of weight, percent body fat, total cholesterol, low-density lipoprotein and triglycerides, as well as, a higher average of high-density lipoproteins. Two sample t-tests were performed to determine significance. It was determined that no significant difference existed between serum lipid profile levels of the control group and the self-reported exercise group. This study is important because it expands our knowledge of the relationship between resistance training (RT) and lipid profiles, relying on self-reported data and focusing on an at-risk population.

Plants are resilient organisms that are continually evolving and continue to withstand an adverse and dynamic world. SABP2-interacting protein (SIP)-470 is a non-specific lipid transfer protein (nsLTP) that was identified in tobacco. SIP470 was discovered during a yeast two-hybrid screening with SABP2, which is an important methyl esterase enzyme which catalyzes the conversion of immobile MeSA into active salicylic acid (SA) during pathogenic challenge. SA activation and mobility allows for immunity to be carried to other, non-infected parts of the plant. This induced responses is called systemic acquired resistance (SAR) and it is a broad spectrum defense. Like many nsLTPs, SIP470 is small and has a predicted characteristic hydrophobic cavity. nsLTPs are found in higher plants and have repeatedly demonstrated protection in biotic stress including disease resistance, and greater resistance to both bacterial and fungal pathogens in overexpressed transgenic lines. This diverse class is also significantly involved in plant adaptation to environmental changes, namely drought, salinity, and freezing, but also in osmotic stress and wounding. Furthermore, nsLTPs are involved in wax metabolism and seed development. Subcellular localization of nsLTPs varies considerably during in vitro and in recent in vivo studies. SIP470 was originally identified in tobacco plants, and therefore, it is important to study its role directly in tobacco plants. SIP470 and eGFP fusion construct has been generated to study the subcellular localization of SIP470 in tobacco cells. SIP470 localization has shown a discontinuous, punctate arrangement around the membrane periphery which is being further verified by subcellular fractionation. Transgenic tobacco lines that are silenced in SIP470 via RNAi have been generated, and these plants are being screened. Overexpressor transgenic lines of SIP470 have been generated and are under the control of an estradiol-inducible promoter. These transgenic lines will be tested for their response in basal resistance and SAR.

Exposure of human erythrocytes to elevated intracellular calcium causes alterations in cell shape and stimulates shedding of the cell membrane in the form of microvesicles. We hypothesized that both the shape transition and microvesiculation are influenced by microscopic membrane physical properties such as lipid order. To test this hypothesis, membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the internal ionic environment. Changes in membrane order were assessed using steady-state fluorescence spectroscopy with an environment-sensitive probe, laurdan. Our observations led us to the following conclusions: 1) the modest temperature dependence of membrane structure observed with laurdan is shifted to lower temperatures and becomes more cooperative upon removal of membrane cholesterol, 2) the calcium-induced shape change observed in erythrocytes requires a decrease in membrane order, 3) the influence of membrane order is not limited to shape transitions induced only by calcium, and 4) decreased order is also a permissive factor for microvesicle shedding. Our data suggest that while the mechansims that regulate the shape transition and the release of microvesicles are different, they both require a state of membrane disorder.

Microalgae have been in the center of research for several years due to their high production rates. The use of fresh-water algae in the production of biofuels coupled with wastewater treatment has become a topic of modern research. While most algal farming is performed in warm and sunny climate, this project focused on naturally occurring microalgae in Northern Sweden and their ability to reclaim wastewater and produce lipids. The four microalgae Chlorococcum sp. (MC1), Scotiellopsis reticulata (UFA-2), Coelastrella sp. (3-4) and Chlorella vulgaris (13-1) were grown under mixotrophic conditions in municipal wastewater (MWW), pulp and paper wastewater (PnP) and mixtures of both. Except of UFA-2, I found the growth of the tested species to be limited in pure PnP, however, mixtures of PnP and MWW were suitable for algal growth. The removal rates of total nitrogen achieved the goals regulated by the Swedish government for wastewater reclamation. Phosphorus, of which maximal levels according the Swedish regulations need to be below 0.5 mg/L, was efficiently removed by the strain 3-4 in PnP and in MWW + PnP (ratio 3+1) as well as by the strain 13-1 in MWW + PnP (ratio 3+1). The tested microalgae are therefore appropriate candidates to be used in sustainable wastewater treatment. The algal biomass composition was determined with the help of Fourier-transform infrared spectroscopy and an increase in the spectra for biomass grown in wastewaters within the fingerprint region (800 – 1800 cm-1) was observed. From this, I conclude that the lipid content was elevated in the biomass received after growth in PnP and all mixtures of MWW and PnP compared to the in BG11 grown biomass, which acts as a control group. A quantitative lipid analysis performed on the biomass of the strains UFA-2 and 3-4 confirmed higher lipid amounts after growth in PnP wastewater compared to growth in BG11. These findings show that the examined microalgae might have the potential to be used as a potential feedstock for biofuel productions after cultivation in local wastewaters.

Based on results of its Aquatic Species Program (1978-1996), which sought to develop algae-to-liquid fuel technology, the U.S. Department of Energy has suggested that algal wastewater treatment may be incorporated into biodiesel production schemes to reduce the operating costs of both processes. the purpose of the current research was to evaluate the triglycerides produced by wastewater-grown algae for their suitability as a fuel feedstock and to investigate the effectiveness of several solvent mixtures and extraction procedures at recovering lipids from fresh algae. The research involved two separate experiments. The first determined the quantity and quality of lipids produced over the lifetime of a batch culture of algae grown in a small outdoor high-rate pond. Transesterification of the algal triglycerides yielded mostly saturated and monounsaturated 16 and 18-carbon fatty acid methyl esters, together comprising approximately 8 to 30% of the biomass in the pond. The average triglyceride production rate during the grwoth phase of the culture was 0.97 grams per square meter of pond surface per day. The second experiment compared several industrially practicable extraction procedures to the Bligh and Dyer laboratory extraction method. The Bligh and Dyer laboratory extraction procedure provides excellent lipid recovery efficiency, but several factors limit its potential on an industrial scale. The Bligh and Dyer method requires a larger volume of solvents than other methods, uses the probable carcinogenic chemical chloroform, and involves a complex series of steps that are difficult to automate. A simple, low-energy extraction process using relatively non-toxic solvents was found to have an extraction efficiency comparable to that of the laboratory method.

Disabled-2 (Dab2) is an adaptor protein that plays critical roles in various biological processes, including protein endocytosis, platelet activation and aggregation, tumor growth, and development. In platelets, Dab2 associates with membrane sulfatide at the platelet surface, modulating platelet inside-out and outside-in signaling pathways. A Dab2-derived peptide, named the sulfatide-binding peptide (SBP), is the minimal unit of Dab2 to exert its function as a negative regulator of platelet activation and aggregation. The work of this thesis refines the model of Dab2 SBP binding to sulfatide and provides structural and functional insights into the mechanism by which Dab2 SBP modulates platelet activation. Using molecular docking, lipid-protein overlay assay, nuclear magnetic resonance, and surface plasma resonance tools, this work identifies the critical residues within two major regions responsible for sulfatide interaction. First, docking a sulfatide to Dab2 SBP, a hydrophilic region, primarily mediated by Arg42, is thought to be responsible for the association with the sulfatide headgroup. We observed that Arg 42 could directly interact with sulfatide by forming hydrogen bonds with the OS atoms in the sulfatide head group. Further lipid-protein overlay assay and surface plasma resonance experiments confirmed that both the positive charge and stereochemistry of the side chain of Dab2 SBP Arg42 are required for the sulfatide binding. Moreover, Arg42 is found to be critical in the inhibition of P-selectin expression on activated platelets. The residues nearby Arg42 (i.e., Glu33, Ty38, and Lys 44) also contribute to sulfatide interaction. Second, the second polybasic motif located at the C-terminal -helix 2 is considered to interact with the acyl chain through hydrophobic interactions rather than direct binding to the charged sulfatide head group. Lysine residues in this region are suggested to exert a dual role in sulfatide association, that is, by favoring electrostatic interactions with the negatively-charged sulfatide and/or by employing their flexible hydrocarbon spacers for hydrophobic interactions with membrane lipids. Consistent with this suggestion, we found a hydrophobic patch in the wild type Dab2 SBP structure surrounded by Lys49, Lys51, and Lys53. Furthermore, the role of the second sulfatide binding motif in sulfatide binding is confirmed by mutagenesis analysis and lipid-protein overlay assays, highlighting the ability of molecular docking to accurately predict critical residues responsible for sulfatide binding. In summary, this work provides a detailed structural basis for Dab2 recognition by sulfatide through multiple biophysical methods. The corresponding biological implications in the inhibition of platelet activation are also evaluated by flow cytometry. By elucidating the underlying mechanisms of Dab2 mediating platelet activation through sulfatide binding, we provided structural and functional insights for designing a Dab2-derived peptide with altered sulfatide recognition features in platelets, which can be further employed in antiplatelet therapy. / Doctor of Philosophy / Platelets are blood cells that are fundamentally intended to help form clots to stop bleeding. They do so by being activated after getting signals from damaged blood vessels and reaching the injury site. Consequently, they form aggregates by attracting more platelets to clump on the clot. However, platelet activation induced by a tumor cell can, in turn, protect the tumor cell from immune system elimination and facilitates their growth and spread. This platelet-tumor complex formation suggests platelets as a therapeutic target for reducing tumor migration out of the bloodstream. Our study investigates the mechanism of a Disabled-2-derived peptide, named Dab2 SBP, which upon binding to a sulfatide lipid, can reduce the platelet activation extent, using molecular and cellular approaches. The results of this study may be instrumental in the generation of Dab2 SBP-derived peptides with altered sulfatide binding ability and selectivity, which may lead to a design of an antiplatelet drug that can limit the ability of tumor cells to invade other tissues.

Target of Myb 1 (TOM1) is an endosomal adaptor protein that plays a role in cargo membrane trafficking for degradation by serving as an alternative endosomal sorting complex required for transport component. TOM1 has also been shown to serve as a novel phosphatidylinositol 5-phosphate (PtdIns5P) effector at signaling endosomes through its VHS domain, delaying cargo degradation in a bacterial infection model. The aim of this thesis is to clarify the structural and functional basis of the autoregulation mechanism of TOM1 to switch from endosomal protein trafficking to the bacterial survival signaling pathway. Our thermal denaturation and spectroscopic studies demonstrate that PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of TOM1 VHS. The thermodynamic studies indicate that TOM1 VHS endothermically binds to PtdIns5P through two potential noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. These findings suggest that, under Shigella flexneri infection, TOM1 may interact with downstream effectors in a different VHS domain conformational state, thus involving the protein in bacterial survival signaling pathways. In order to obtain molecular details for the interaction of the TOM1 VHS domain for PtdIns5P and Ubiquitin (Ub), the backbone assignment information was obtained by performing NMR experiments, which assigned backbone 1H, 13C, and 15N resonances of the TOM1 VHS domain. With this structural information, our heteronuclear single quantum coherence and molecular dynamics simulations data revealed that TOM1 VHS interacts with PtdIns5P following a fast-exchange regime, with the PtdIns5P binding site predicted to be at a region spanning α-helices 6 to 8. Further mutagenesis and lipid-protein overlay assay studies indicated that K147 plays a critical role in the binding of TOM1 VHS domain to PtdIns5P. TOM1, unexpectedly, did not bind PtdIns5P. Using truncated forms of TOM1 protein, we discovered that neither TOM1 GAT domain nor the C-terminal domain modulated TOM1 VHS's PtdIns5P binding; however, surprisingly, a linker sequence between the TOM1 VHS and GAT domains exhibited an autoinhibition role for TOM1 binding to PtdIns5P. This linker region was observed to induce local conformational changes on the structure of TOM1 VHS domain, especially around α-helices 6 and 8, which are proposed to build up the binding pocket for PtdIns5P. In order to investigate whether the linker region between TOM1 VHS and GAT domain can also regulate the Ub association of TOM1 VHS domain, the binding properties of TOM1 and its domains to Ub were explored. Unexpectedly, the binding affinity of TOM1 VHS-linker for Ub was increased about 10-fold when compared with that for the TOM1 VHS domain, suggesting that the linker enhances the avidity of TOM1 for ubiquitinated cargo. Structural analysis indicated that the linker region may cap the conventional Ub-binding site of TOM1 VHS, thus forming a more compact structure. In summary, this study uncovered a novel intramolecular modulatory mechanism in TOM1 that regulates ligand recognition by its VHS domain. By providing the molecular basis of the TOM1 interactions, we may provide cargo sorting mechanistic insights, create functionally specific mutations, and precisely manipulate TOM1 function under bacterial infection conditions, and other yet-to-be-discovered PtdIns5P-dependent signaling pathways. / Doctor of Philosophy / Membrane trafficking is a delivery network established in a cell to transport proteins (cargoes) from one intracellular place to another one to control their activity. TOM1 is a protein involved in this process, which plays a role in transporting cargoes for degradation. Defects in this trafficking pathway lead to human diseases, such as immunodeficiency and neurodegeneration diseases. TOM1 has also been shown to be beneficial for bacterial survival in human cells. However, how TOM1 switches its role form protein trafficking to bacterial pathogenesis is still unclear. In our study, we discovered an internal region of TOM1 may serve as a switch to shift the role of TOM1 in human cells. In an "on" status, TOM1 favors to transport cargoes, while in an "off" status, TOM1 is used for bacteria survival. This study provides insights in the function of TOM1 which is beneficial for the design of novel therapeutic strategies against TOM1, which will prevent the progress of bacterial infections.

Ferroptosis is a recently characterized cell death pathway associated with the iron-dependent accumulation of lipid hydroperoxides in phospholipid bilayers. The origin of these hydroperoxides has been an ongoing topic of debate and many researchers argue for a lipoxygenase (LOX) enzyme-controlled mechanism of initiation, given their known role as dioxygenases of polyunsaturated fatty acids (PUFAs). In response to this, our lab investigated the induction and inhibition of ferroptosis in human embryonic kidney (HEK-293) cells transfected to overexpress the three most prevalent LOX isoforms, 5-LOX, p12-LOX, and 15-LOX-1. These studies did not support a role for LOX in the execution of ferroptosis; LOX inhibition was not associated with ferroptosis suppression and in fact, anti-ferroptotic activity was directly tied to purported LOX inhibitors’ ability to act as radical-trapping antioxidants (RTAs). We have investigated the effects of LOX inhibitors on ferroptosis in human fibrosarcoma (HT-1080) cells, the cell line in which ferroptosis was initially characterized, and mouse hippocampal neuronal (HT-22) cells, the cell line in which the closely related cell death modality oxytosis was characterized. In sum, our findings mirror those obtained in HEK-293 cells, and the effectiveness of an inhibitor is tied to its off-target RTA activity, not inhibition of LOX. Moreover, we observed suppression of ferroptosis via necrostatin-1 (Nec-1), a known receptor-interacting serine/threonine-protein kinase 1 (RIPK1) (and necroptosis) inhibitor. Herein, we show that Nec-1 is not an RTA and exerts its effects by a yet unknown mechanism which we investigate in a series of exploratory experiments. Conjugated fatty acids – particularly α-ESA – have recently been reported to induce ferroptosis by an unclear mechanism. Theorizing this phenomenon was tied to the autoxidation of α-ESA’s conjugated trienic unit, we aimed to investigate the kinetic and biological properties of natural α-ESA alongside a deuterated isotopologue. Herein, we report preliminary work to derive biologically relevant rate constants for addition and hydrogen-atom transfer (HAT) of α-ESA. Moreover, we report our progress towards the synthesis of a deuterated α-ESA which will facilitate future study alongside its natural counterpart.