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Non-alcoholic fatty liver disease (NAFLD), described by the build-up of excess fat in the liver, is the most prevalent chronic liver condition globally. One of the essential metabolic functions of the liver is the production of ketone bodies, a process called, ketogenesis. Ketone bodies serve as alternative fat-derived sources of fuel for tissues under conditions of nutrient deficit (i.e., fasting). Interestingly, recent studies have found that ketogenesis is dysregulated in NAFLD patients. Similarly, we also found that high-fat diet-induced NAFLD mice exhibited diminished fasting-induced ketogenesis with reduced expression of liver Hmgcs2, the rate-limiting enzyme of ketogenesis. To understand the role of ketogenesis in NAFLD pathogenesis and treatment, we generated mouse models of ketogenic insufficiency and activation through Hmgcs2 loss- and gain-of-function, respectively. Notably, a change in dietary environment rescued the fatty liver phenotype of Hmgcs2 knockout mice and increased ketogenic function through HMGCS2 overexpression improved NAFLD-associated metabolic dysfunction and hepatosteatosis in adult mice. Furthermore, an untargeted metabolomics approach provided a comprehensive metabolic view underlying HMGCS2 overexpression-mediated NAFLD improvement, suggesting that hepatic ketogenesis impacts liver metabolism via regulation of other metabolic pathways. Together, our study adds new knowledge to the field of ketone body metabolism and suggests a viable therapeutic strategy involving ketogenesis activation in the prevention and treatment of NAFLD.

Background: The increased incidence of obesity and associated metabolic diseases has driven research focused on genetically or pharmacologically alleviating metabolic dysfunction. These studies employ a range of fasting-refeeding models including 4-24 h fasts, "overnight" fasts, or meal feeding. Still, we lack literature that describes the physiologically relevant adaptations that accompany changes in the duration of fasting and re-feeding. Since the liver is central to whole body metabolic homeostasis, we investigated the timing of the fast-induced shift toward glycogenolysis, gluconeogenesis, and ketogenesis and the meal-induced switch toward glycogenesis and away from ketogenesis. Methods: Twelve to fourteen week old male C57BL/6J mice were fasted for 0, 4, 8, 12, or 16 h and sacrificed 4 h after lights on. In a second study, designed to understand the response to a meal, we gave fasted mice access to feed for 1 or 2 h before sacrifice. We analyzed the data using mixed model analysis of variance. Results: Fasting initiated robust metabolic shifts, evidenced by changes in serum glucose, non-esterified fatty acids (NEFAs), triacylglycerol, and beta-OH butyrate, as well as, liver triacylglycerol, non-esterified fatty acid, and glycogen content. Glycogenolysis is the primary source to maintain serum glucose during the first 8 h of fasting, while de novo gluconeogenesis is the primary source thereafter. The increase in serum a-OH butyrate results from increased enzymatic capacity for fatty acid flux through beta-oxidation and shunting of acetyl-CoA toward ketone body synthesis (increased CPT1 (Carnitine Palmitoyltransferase 1) and HMGCS2 (3-Hydroxy-3-Methylglutaryl-CoA Synthase 2) expression, respectively). In opposition to the relatively slow metabolic adaptation to fasting, feeding of a meal results in rapid metabolic changes including full depression of serum a-OH butyrate and NEFAs within an hour. Conclusions: Herein, we provide a detailed description of timing of the metabolic adaptations in response to fasting and re-feeding to inform study design in experiments of metabolic homeostasis. Since fasting and obesity are both characterized by elevated adipose tissue lipolysis, hepatic lipid accumulation, ketogenesis, and gluconeogenesis, understanding the drivers behind the metabolic shift from the fasted to the fed state may provide targets to limit aberrant gluconeogenesis and ketogenesis in obesity.

Background: Tissue non-specific alkaline phosphatase (TNAP) is an enzyme which functions within the body to catalyze the hydrolysis of pyrophosphate to phosphate, and is a well-known mediator of bone mineralization. It has also been identified as a positive mediator of intracellular lipid accumulation (ICLA) in both murine and human preadipocytes as well as in the hepatocellular cell line HepG2. However, the mechanism through which TNAP functions to control ICLA is not known. Both osteoblasts and adipocytes are both of mesenchymal origin and thus may share conserved mechanisms through which TNAP functions. Within bone, TNAP converts pyrophosphate (which inhibits mineralization) to phosphate. This phosphate is essential to the mineralization process through binding to hydroxyapatite crystals, and it also activates the transcription of genes whose products function in osteoblast differentiation, including NRF2. This thesis therefore aimed to determine the role of both pyrophosphate and TNAP-generated phosphate in ICLA. In addition, it is possible that TNAP may interact with other proteins, as it is known that TNAP is able to dephosphorylate proteins such as tau. This thesis therefore aimed to determine whether TNAP binds to other proteins in the context of ICLA. Lipids are not only stored within hepatocytes and adipocytes, but are also found in cells of the adrenal cortex, and TNAP is known to be expressed within such cells. Therefore, this thesis also aimed to determine whether TNAP is involved in the accumulation of cholesterol esters within lipid droplets in the adrenal cortex. Methods: To determine the effect of high intracellular pyrophosphate levels on ICLA, 3T3-L1 cells (a preadipocyte cell line) were cultured in the presence and absence of probenecid, an inhibitor of the pyrophosphate transporter ANK, and induced to accumulate lipids. Lipid accumulation was monitored through Oil red O staining. The effect of probenecid treatment on TNAP activity and intracellular pyrophosphate levels was also analysed. To determine whether TNAP functions in ICLA by producing phosphate for gene induction, 3T3-L1 cells were stimulated to undergo ICLA in the presence and absence of the TNAP inhibitor levamisole, which in turn blocks ICLA. Levamisole treated cells were also incubated with phosphate to see if this would overcome the inhibitory effect of levamisole on ICLA. The ability of phosphate to induce gene expression of NRF2 was determined through real-time PCR. In addition, an NRF2 expressing plasmid was transfected into cells treated with the TNAP inhibitor levamisole to determine if this would also overcome the block on ICLA caused by TNAP inhibition. In silico analysis identified TRAF2 as a potential binder of TNAP. The expression of TRAF2 during ICLA was determined through real time PCR, and the effect of overexpression of TRAF2 on intracellular lipid accumulation was determined through the transfection of a TRAF2 expressing plasmid in cells induced to undergo ICLA. To determine whether TNAP modulates lipid accumulation in cells of the adrenal cortex, the Y1 murine adrenocortical cell line was cultured in the presence and absence of TNAP inhibitor levamisole, and ICLA measured by Oil Red O staining. The location of TNAP within Y1 cells was identified by histochemical staining. Results: Cells treated with probenecid showed increased pyrophosphate levels (expressed as a % of levels observed at baseline) when compared to untreated controls (155.5 ± 15.1 % vs 51.1 ± 18.9 %; p=0.001) after 24 hours of culture. Increased pyrophosphate levels resulted in ICLA within 3T3-L1 cells surpassing levels seen in untreated controls (507.4 ± 30.4 % vs 337.6 ± 16.17 %; p=0.004). This increase in pyrophosphate was coupled to an increase in TNAP activity within the initial 24 hours (291.5 ± 72.8 % vs baseline of 100%; p=0.038) compared to that seen in control experiments (103.43 ± 24.3 % vs baseline of 100%; p=0.848). Cells treated with levamisole showed minimal ICLA and when exogenous phosphate was added, lipid levels were reconstituted to levels similar to that seen in cells induced to accumulate lipids in the absence of levamisole (284.01 ± 62.52% vs 275.86 ± 35.52%; p= 0.83). In the presence of levamisole plus exogenous phosphate, NRF2 expression was upregulated within 1 hour of treatment to levels greater than that seen in the absence of phosphate but presence of levamisole (216.64 ± 19.24% vs 98.28 ± 3.79%; p=0.004). Expression of NRF2 (through transfection with an NRF2 expression plasmid) in cells deficient in TNAP activity (via levamisole treatment), and induced to accumulate lipids, was not able to completely reconstitute ICLA when compared to cells not treated with levamisole (193.72 ± 16.51 vs 326.46 ± 47.64; p = 0.019), but ICLA was still greater than that observed at baseline. In silico analysis predicted that TNAP would bind to TRAF2, yet neither band shift assays nor immune co-precipitation showed evidence of this. However, TRAF2 mRNA was down regulated within 3T3-L1 cells during adipogenesis, reaching levels of 15.27 ± 10.27% (p= 0.014) of baseline (levels prior to induction of intracellular lipid accumulation) by day 4 of lipid accumulation. Overexpression of TRAF2 during adipogenesis markedly reduced intracellular lipid accumulation (147.88 ± 11.28% vs 326.46 ± 47.64%; p=0.028 (after 8 days of culture)). In Y1 cells TNAP activity is upregulated during ICLA, reaching 233 ± 37.56% (p=0.019 vs. baseline) of baseline levels within the initial 24 hours. Inhibition of TNAP activity through levamisole treatment resulted in a decrease in ICLA when compared to cells not treated with levamisole. Histochemical analysis showed that TNAP activity was localised to the lipid droplet. Discussion and Conclusions: Within 3T3-L1 cells TNAP mediates intracellular lipid accumulation through the generation of phosphate. The phosphate is able to increase the expression of NRF2, however it is likely that NRF2 is not the only gene whose expression is regulated by TNAP-generated phosphate. It was found that TNAP and TRAF2 do not bind to each other in the context of ICLA; however TRAF2 is a negative mediator of ICLA through a TNAP-independent mechanism. Functional TNAP is necessary for the accumulation of cholesterol esters within the Y1 cell line, suggesting that TNAP is essential for lipid accumulation in cell types that store lipids in intracellular membrane-bound droplets in the form of triglycerides or cholesterol esters. / GR2018

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.

Algae are a diverse group of simple organisms that lack roots, stems or leaves and are able to use sunlight, carbon dioxide, and nutrients to produce complex compounds, such as carbohydrates, proteins and lipids. These compounds, especially lipids, are highly sought-after by agricultural, nutraceutical and energy interests. Although there is great potential for algae derived biofuels, there are technical and economic challenges associated with their cultivation. Relevant to this dissertation, the environmental impacts associated with algae cultivation can be reduced by using municipal and agricultural wastewaters as a water and nutrient source. This research was divided into three sections to address current challenges in the algal industry and science, technology, engineering and math (STEM) education. The sections were: 1) examination of the growth of indigenous algae on wastewater (centrate) produced from dewatering anaerobically digested municipal sludge, 2) examination of the effect of non-axenic conditions on the growth of three different algal cultures using wastewater from a recirculating aquaculture system (RAS), and 3) using wastewater treatment and algae to increase scientific inquiry in authentic science research with high school students. In the first section, indigenous algae were cultivated on centrate under natural light conditions in a semi-continuous photobioreactor. A non- linear bio-optical model was developed considering Michaelis-Menten photosynthesis-irradiance response. The bio-optical model was applied to fit the cumulative biomass data and had an R-squared value of 0.96. The second section examined the growth and accumulation of storage product. Higher calorific values were observed for all algae cultures when grown under non-axenic conditions, most likely due to significantly higher lipid contents. Significantly higher algal lipid contents under non-axenic conditions may be attributed to the stress of the presence of RAS microorganisms. Finally, having a university-based algal project with involvement of University of South Florida (USF) researchers, teachers and high school (HS) students facilitated increased scientific understanding and skills among HS students. Outcomes included graduate students gaining greater in-depth practical understanding as these students had to learn skills, such as designing a photobioreactor and then immediately had to teach HS students how to construct photobioreactors, design and conduct experiments, and gather scientific data. HS students gained a greater understanding of biological and chemical processes, such as photosynthesis. In addition, they learned important skills, such as calculating means and standard deviations using Excel, orally communicating scientific concepts and preparation of a PowerPoint presentation.

Diacylglycerol acyltransferase (DGAT) is a key for the synthesis of triacylglycerol (TAG) from diacylglycerol in the unicellular green alga Chlamydomonas reinhardtii.Acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS) are responsible for the synthesis of fatty acids. We found the TAG and fatty acid synthesis related genes in C. reinhardtii, including five DGAT (DGAT1 (JGI 184281), DGAT2 (JGI 400751), DGAT3 (JGI 285889), DGAT4 (JGI 141301), and DGAT5 (JGI 190539)), three £] ketoacyl-ACP reductase isoforms ( (JGI 153976), (JGI 153976), and (JGI 194728)) and two £] ketoacyl-ACP synthase isofroms ( (JGI 139619) and (JGI 205887)) for FAS, and ACC £\ (NCBI XP_001696945.1), ACC £] (NCBI XP_001703187.1) and ACC biotin carboxylase ( NCBI XP_001702319.1)) for ACCase in C. reinhardtii. This investigation designed the primers of the above genes to determine whether acetate influences their mRNA expression levels in cell-wall less strain CC400 in the nitrogen starvation condition. The results showed that the absence of nitrogen in the medium triggered the lipid accumulation for the strains of CC400 in the condition of 50 £gE light. DGAT3 mRNA levels were increased by nitrogen starvation. For the FAS genes, in the strain of CC400 showed no increased mRNA levels upon exposure to nitrogen starvation. The mRNA levels of ACC£\, ACC £] and ACC biotin carboxylase were more or less decreased by nitrogen starvation in CC400 strains. Thus, the responses of DGAT gene expression to acetate supplement were checked. The absence of acetate from the medium partly inhibited the nitrogen starvation induced increases in lipid and DGAT3 mRNA levels, and the mRNA levels of DGAT1 and DGAT2 in the nitrogen starvation condition. However, DGAT4 mRNA levels were significantly induced by the absence of acetate from the medium. In conclusion, the present study demonstrate that acetate is required for the nitrogen starvation induced DGAT3 gene expression (mRNA levels) and lipid accumulation in C. reinhardtii.

Monocyte recruitment promotes the accumulation of myeloid foam cells in early atherosclerotic plaques. However, initial foam cells form prior to increased monocyte recruitment in hypercholesterolemic Ldlr-/- mice. These initial foam cells are derived from myeloid cells residing in the normal intima, and express integrin alphaX (CD11c). The goal of this thesis was to assess the role of initial foam cells in atherogenesis. The approach was to delete these cells by diphtheria toxin-induced apoptosis in Ldlr-/- bone marrow chimeras. Depletion of CD11c+ leukocytes resulted in significant reductions of intimal lipid accumulation, monocyte recruitment, intimal chemokine expression, but not endothelial cell adhesion molecule expression, at 10 and 21 days of hypercholesterolemia. These data suggest that lipid uptake by resident intimal CD11c-expressing myeloid cells during the earliest stages of atherosclerosis promotes chemokine production that is required for increased monocyte recruitment.

Monocyte recruitment promotes the accumulation of myeloid foam cells in early atherosclerotic plaques. However, initial foam cells form prior to increased monocyte recruitment in hypercholesterolemic Ldlr-/- mice. These initial foam cells are derived from myeloid cells residing in the normal intima, and express integrin alphaX (CD11c). The goal of this thesis was to assess the role of initial foam cells in atherogenesis. The approach was to delete these cells by diphtheria toxin-induced apoptosis in Ldlr-/- bone marrow chimeras. Depletion of CD11c+ leukocytes resulted in significant reductions of intimal lipid accumulation, monocyte recruitment, intimal chemokine expression, but not endothelial cell adhesion molecule expression, at 10 and 21 days of hypercholesterolemia. These data suggest that lipid uptake by resident intimal CD11c-expressing myeloid cells during the earliest stages of atherosclerosis promotes chemokine production that is required for increased monocyte recruitment.

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in beta-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of corp plants.