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Many factors affect membrane composition in ectotherms, including allometry, temperature, toxins such as PCB-153, and osmotic stress. This thesis seeks to describe the relationship between membrane composition, size, and phylogeny in twelve species of cypriniform fish; to describe interactions between the homeoviscous responses to temperature and to PCB-153 in goldfish and rainbow trout; and to describe the membrane response to hypoosmotic stress in goldfish. Commonalities in these patterns provide insight into shared mechanisms of phospholipid modulation. In particular, such similarities indicate whether the membrane pacemaker theory of metabolism, which connects allometric relationships between body size, membrane phospholipids, and metabolic rate, can serve as a general framework for understanding membrane composition. Chapter 2 investigates how cypriniform membrane unsaturation decreases with mass through different fatty acid substitutions than in endotherms, but these fatty acids are in turn shown to be due to the species’ relatedness to one another rather than to purely physiological causes. In Chapter 3, PCB-153 is shown to increase cholesterol in liver and brain, while high temperature primarily reduces phospholipid unsaturation. In Chapter 4, these patterns are further explored in trout. As in goldfish, cholesterol modulation is the primary response to PCB-153, whereas temperature primarily reduces phospholipid unsaturation. Trout show more pervasive fatty acid changes than goldfish in all tissues except the liver, which does not respond to PCB exposure, suggesting that PCB-153 pushes trout’s homeoviscous response to a limit that similarly-exposed goldfish do not face. Chapter 5 shows that goldfish intestines decrease membrane saturation; kidneys decrease membrane cholesterol; gills decrease neither; and muscles decrease both in response to long-term exposure to hypoosmotic conditions. The intestine and kidney are both involved in recovering ions from body fluids, but gills suppress ion loss and muscle concentrates ions from the bloodstream. Temperature, osmotic stress, PCB-153, and increasing body size are all addressed via a similar set of membrane responses in fish, which fits with the membrane pacemaker theory’s predictions regarding membrane composition, metabolic rate, and size.

The focus of this dissertation is centered on the mass spectral analysis of lipids and changes occurring in keeping with the concept of homeoviscous adaptation [1]. Homeoviscous adaptation is the process of modification of membrane lipids in response to environmental stimuli [1]. Dissertation investigations applied this concept to prokaryotic and eukaryotic organisms, and expanded the perception of environmental factors from exogenous organic solvents to intracellular environment. The field of lipidomics deals with the analysis of phospholipid and fatty acid components of membranes the changes that occur due to environmental stimuli and their biological significance [2-6]. The high sensitivity of mass spectrometry (MS) is an ideal tool for lipidomics allowing detection, quantification and structural elucidation [6]. Coupling of a mass spectrometer to a chromatographic system, such as gas chromatograph (GC), allows the separation of fatty acid methyl esters analytes prior to analysis [7]. The research investigations that comprise this dissertation are divided into three interrelated projects. The first project involved the analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains in response to adaptation of cultures to growth in ethanol. The hypothesis being that adaptation of cultures to growth in ethanol would result in compensatory change to the membrane composition. Rat mitochondrial fatty acid profiles isolated from brain, liver, kidney and heart tissues were compared. The hypothesis being that differences in cellular environments found among various tissues would be reflected in the mitochondrial membrane composition. These data support the concept that variations to the lipid content of neurological mitochondria may increase susceptibility to the products of oxidative stress. Lastly, changes in neurological mitochondria as a function of Alzheimer’s disease progression were studied. The hypothesis being that changes to the mitochondrial lipidome would be significantly reflected during advanced stages of AD, in addition to being more prevalent in regions displaying greater pathology. The three interrelated projects increased our understanding of the boundaries established by the concept of homeoviscous adaptation. Project specific hypotheses were supported by data obtained from these investigations.

It is generally accepted that a decrease in temperature causes the rates of chemical and biochemical reactions to slow down, usually resulting in poorer performance of biological wastewater treatment systems. Despite this, early researchers repeatedly showed that excess biological phosphorus removal (EBPR) was more efficient at colder temperatures. Recent studies, however, have demonstrated that the reaction rates of EBPR processes decrease with temperature in accordance with Arrhenius' Law, resulting in an apparent contradiction in the literature. The objective of this study was to investigate the EBPR temperature controversy. The experimental systems used were two, lab-scale UCT configuration plants fed with acetate as the sole volatile fatty acid (VFA) source. The results showed that EBPR systems do perform more efficiently at colder temperatures, i.e., at 5°C compared to 20°C. The reason for better system performance was determined to be related to reduced competition for substrate in the non-oxic zones that results in an increased population of phosphate accumulating organisms (PAOs) relative to non-PAOs and, therefore, greater EBPR efficiency even though the reaction rates are slower. The proliferation of PAOs relative to non-PAOs at cold temperature indicates that some of the PAOs are psychrophilic, i.e., they have alternate biochemical pathways that give them a competitive advantage over bacteria dependent upon glycogen metabolism. The activated sludge acclimated to 20°C had relatively high polyhydroxyvalerate (PHV) and glycogen contents relative to sludge acclimated to 5°C. It was initially hypothized that there is a significant competition between PAO and glycogen accumulating organisms (GAOs) at 20°C and cold temperature (5°C) nearly eliminates this competition in favor of the PAOs. A series of batch test experiments revealed that despite similar acetate utilization by the sludges grown at the two temperatures nearly 30% less PHA was produced by the sludge taken from the 20°C reactor, indicating that GAOs were a small fraction of the population at 20°C. Transmission electron microscopy pictures showed that the biomass acclimated to 20°C had a much more diverse bacterial population than the biomass acclimated to 5°C. However, no GAO population was detected in electron microscopy samples under any temperature conditions. The decreased P removal efficiency at 20°C was then attributed to the presence of fermentative or other non poly-P bacteria that are capable of utilizing substrate under anaerobic conditions. PHA production greatly increased at 5°C, whereas glycogen metabolism substantially reduced. Even though glycogen is an essential requirement for EBPR mechanism, the EBPR microorganisms have the ability to adapt their metabolic pathways to environmental conditions and greatly reduce their need for glycogen. It is apperant that cold temperature inhibits some of the key enzymes in glycogen metabolism resulting in lower glycogen accumulation that in turn increases the EBPR performance. Therefore temperature not only exerts selective pressure on the dominant population but also alters the metabolic pathways of the EBPR process. Increased glycogen accumulation, as observed in this study at 20°C, may not be related to GAO proliferation as suggested by Filipe et al. (2001) instead it may be related to EBPR bacteria to efficiently use glycogen metabolism. Current models (Brdjanovic et al. 1997; Filipe et al. 2002) consider that GAO metabolism is an integral part of EBPR metabolism and the performance of EBPR processes depends on PAO/GAO fraction in the EBPR system. No GAO proliferation was observed even the A/O process was operated without P addition for more than 3 weeks at 10°C. Therefore such important concept should be further investigated before it is included in EBPR models. EBPR stoichiometry was presumed to be insensitive to temperatures (Brdjanovic et al. 1997). However, observed stoichiometric values of PHA storage per unit glycogen utilization and PHA utilization per unit glycogen rephlenishment were quite different at different temperatures. Temperature, therefore, not only affects the kinetics of EBPR systems but also affects the EBPR stoichiometry. Most prokaryotic cells have the ability to alter their cellular membrane fatty acid composition as temperature decreases to counteract the adverse effects of temperature on membrane fluidity (Becker et al., 1996). This unique ability is known as "homeoviscous adaptation". In this study, homeoviscous adaptation by EBPR activated sludge was investigated for a series of temperatures ranging from 20°C to 5°C using one of the lab scale EBPR systems. The fatty acid analysis results showed that the unsaturated to saturated fatty acid ratio increased from 1.40 to 3.61 as temperature dropped from 20 to 5°C. The increased cis-9-hexadecanoic acid (C16:1) at 5°C strongly indicated the presence of homeoviscous adaptation in the EBPR bacterial community. Thus the cell membranes of the EBPR community were still in a fluid state, and solute transport and proton motive force mechanisms were operable even at 5°C. It was concluded that loss of EBPR performance at low temperatures, as reported by McClintock et al. (1992) was not related to the physical state of the cellular membranes, but was probably caused by unsuitable operational conditions. Even though the transport of volatile fatty acids (e.g. acetate) is an integral part of EBPR biochemistry and stoichiometry, this important concept has been ignored. Fleet (1997) concluded that acetate entry into bacterial cells in EBPR sludge was simple passive diffusion based upon the results of a single study (Baronofsky et al. 1984). However, this study showed that neither acetate nor propionate can cross the cell membrane via simple passive diffusion. The existence of apparent saturation curves when the substrate uptake rates (acetate and propionate) were plotted against the substrate concentrations suggested that transport of volatile fatty acids obey facilitated or active transport. Following from the above results, an investigation of the impacts of operational conditions such as low solids retention time (SRT), presence of electron acceptors in the non-oxic zones, low anaerobic detention time, and lack of acclimation was performed. The results showed that the "critical, i.e., wash-out" SRT increased as temperature decreased, but if the biomass was permitted to acclimate to the lower temperature, a major population shift would occur which would increase the capacity of the system for phosphorus (P) removal. When the 5 °C sludge was allowed to acclimate at a relatively high SRT (18 d), the system's P-removal capacity greatly surpassed that of the 20 °C system. The decrease in EBPR performance because of the presence of nitrates in the non-oxic zones was determined to be greater than what would be predicted based on accepted stoichiometry. / Ph. D.

The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.