Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. Includes: (as Section I.A.): Studies of the enzyme fumarase : II. Isolation and physical properties of crystalline enzyme / By Carl Frieden, Robert M. Bock and Robert A. Alberty. Reprinted from Journal of the American Chemical Society, vol. 76 (1954), p. 2482-2484 -- (as Section II. A.): Studies of the enzyme fumarase : III. The dependence of the kinetic constants at 25⁰ upon the concentration and pH of phosphate buffers / By Robert A. Alberty, Vincent Massey, Carl Frieden, and Armin R. Fuhlbrigge. Reprinted from Journal of the American Chemical Society, vol. 76 (1954), p. 2485-2493 -- (as Section III. A.): The effect of pH on fumarase activity in acetate buffer / By Carl Frieden and Robert A. Alberty. Reprinted from Journal of biological chemistry, Vol. 212, no. 2 (Feb. 1955), p. 859-868. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 99-101).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 79-84).

The finely tuned regulation of haematopoiesis is crucial in order to maintain life-long haematopoiesis. The disruption of the balance among cell fates, can lead to malignant transformation. It has become increasingly evident that the metabolic regulation haematopoietic stem cells is critical for stem cell fate decisions. Haematopoietic stem cells reside in a hypoxic microenvironment within the bone marrow and are thought to mainly utilize glycolysis rather than oxidative phosphorylation in order to maintain their pool. However recent evidence suggests that oxidative phosphorylation is critical for quiescent HSCs and in several cases, for leukaemic stem cells (LSCs). One of the key parts of mitochondrial respiration is the tricarboxylic cycle (TCA), providing co-factors for its efficient activity. The TCA functions by catalysing the oxidation of pyruvate via key enzymatic activities. A key component of the TCA cycle is fumarate hydratase (Fh1) which catalyses the hydration of fumarate into malate within the mitochondria, but also catalyses the same reaction in the cytoplasm. FH is a tumour suppressor in human lyomeioma and renal kidney cancer (HLRCC). Previous work conducted by our team has shown that Fh1 is essential for foetal and adult haematopoiesis, as Fh1 deletion within the haematopoietic system is embryonic lethal. Furthermore, conditional deletion of Fh1 in donor cells of the Mx1-Cre system that were injected in lethally irradiated recipients, resulted in the complete reduction of their chimerism in the peripheral blood of recipient mice. Mechanistically, these phenotypes were mostly associated with supra-physiological levels of fumarate as a result of Fh1 deletion. Interestingly, by employing mice that ubiquitously express the human cytosolic isoform of FH (FHCyt, which lacks the mitochondrial targeting sequence and therefore is excluded from the mitochondria), we rescued the embryonic lethality that Fh1 causes, and reduced the levels of fumarate. Importantly, although FHCyt expression restored fumarate-associated lethality, it did not restore the mitochondrial defects, allowing us to study the importance of genetically intact TCA in the context of haematopoiesis. Here I investigated the impact that a genetic truncation of the TCA cycle (as a result of the lack of the mitochondrial isoform of Fh1) has on leukaemic transformation and on aged haematopoiesis. Fh1fl/fl; FHCyt; Vav-iCre mice of approximately 60 weeks old displayed and expansion in the pool of early stem and progenitor compartment (Lin- Sca-1+ c-Kit+), as well as in the early progenitors HPC-1 (LSK CD48+ CD150-) and HPC-2 (LSK CD48+ CD150+). Furthermore, the mice exhibited a drastic depletion of B cells (CD19+ B220+) and an expansion in the frequency of the myeloid compartment (Mac-1+ Gr1+). In order to assess the importance of the TCA cycle in malignant transformation, I isolated stem and progenitor cells from Fh1fl/fl; FHCyt; Vav-iCre (and control (Fh1fl/fl; FHCyt Vav-iCre negative or Fh1fl/fl Vav-iCre negative)) E 14.5 day old embryos and infected them with retroviruses expressing Meis1 and Hoxa9, and generated pre-leukaemic cells (pre-LCs). Genetically intact TCA was required for the efficient generation of leukaemia-initiating cells (LICs), as injection of pre-LCs lacking mitochondrial Fh1 into sub-lethally irradiated recipient mice, resulted in 76 % of leukaemia-free mice while injection of control pre-LCs resulted in 25 % of leukaemia-free mice. However, the genetic perturbation of the TCA did not exert and effect on the long-term self-renewal capacity of LICs. Inducible deletion of mitochondrial Fh1 in established LICs of the Mx1-Cre background using poly (I:C) did not affect their ability to generate AML in primary and secondary recipient mice. These data indicate that genetically intact TCA is required for the efficient generation of LICs in vivo but is dispensable for their long-term self-renewal capacity, highlighting the metabolic rewiring that occurs at different stages of leukaemic transformation. In an effort to understand whether, similarly to HLRCC, Fh1 plays a tumour-suppressive role in malignant haematopoiesis, I isolated LSK cells from the foetal liver of E 14.5 old embryos lacking both isoforms of Fh1. Fh1fl/fl; Vav-iCre cells transduced with Meis1/Hoxa9 or MLL-AF9, MLL-ENL, AML-ETO (chromosomal translocations involved in AML development) -expressing retroviruses, failed to generate colonies in methylcellulose, indicating that stem and progenitor cells require Fh1 to undergo in vitro transformation by these oncogenes. Furthermore, acute deletion of Fh1 (via the use of lentivirally-expressed Cre) in pre-LCs generated using the Meis1/Hoxa9 retroviruses, rendered them unable to generate colonies in methylcellulose, indicating that Fh1 is required for the self-renewal capacity of pre- LCs in vitro. Similarly, when LICs (Fh1fl/fl; Vav-iCre negative) isolated from primary recipient mice were infected with Cre to induce deletion of Fh1, they were unable to generate colonies indicating that Fh1 is required for the self-renewal capacity of LICs in vitro. Finally, in order to identify whether Fh1 is important for LIC self-renewal in vivo I generated Fh1fl/fl; Mx1-Cre pre-LCs by infecting stem and progenitor cells of E 14.5 embryos with Meis1/Hoxa9 retroviruses, and injected them into sub-lethally irradiated mice. After the mice developed AML, I induced the deletion of Fh1, by injecting the mice with poly (I:C). Interestingly, the percentage of LICs in the peripheral blood of recipient mice was drastically decreased, leaving recipient mice leukaemia-free for the remaining time they were monitored. Surprisingly however, approximately 50 % of the recipient mice exhibited a drastic increase in LIC chimerism after two weeks post poly (I:C). Assessment of LICs isolated from recipient mice indicated that Fh1 was fully deleted. These data indicate that while in some cases Fh1 is required for LIC self-renewal in vivo, in other cases it is dispensable. Therefore, the tumour-suppressive roles of Fh1 are likely tissue-specific and do not extend to haematopoietic cells. Overall, this study agrees with published work supporting the notion that intact mitochondrial respiration is important (in varying degrees), in both the contexts of normal and malignant haematopoiesis.

Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in cancer cells. However, the efficacy of OVs as single agents remains limited. We postulate that resistance to oncolytic virotherapy results in part from the failure of tumor cells to be sufficiently infected. In this study, we provide evidence that in the context of sarcoma, a highly heterogeneous malignancy, the infection of tumors by different oncolytic viruses varies greatly. Similarly, for a given oncolytic virus, productive infection of tumors across patient samples varies by many orders of magnitude. To overcome this issue, we hypothesize that the infection of resistant tumors can be achieved through the use of selected small molecules. Here, we have identified two novel drug classes with the ability to improve the efficacy of OV therapy: fumaric and maleic acid esters (FMAEs) and vanadium compounds. FMAEs are enhancing infection of cancer cells by several oncolytic viruses in cancer cell lines and human tumor biopsies. The ability of FMAEs to enhance viral spread is due to their ability to inhibit type I IFN production and response, which is associated with their ability to block nuclear translocation of transcription factor NF-κB. Vanadium-based phosphatase inhibitors enhance OV infection of RNA viruses in vitro and ex vivo, in resistant cancer cell lines. Mechanistically, this involves subverting the antiviral type I IFN response towards a death-inducing and proinflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced anti-tumor immune-stimulation. Both FMAEs and vanadium compounds improve therapeutic outcomes of OV treatment in syngeneic tumor models, leading to durable responses, even in models otherwise refractory to OV and drug alone. Overall, we showcased novel avenues for the development of improved immunotherapy strategies.

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.

<p>In eukaryotic cells, DNA is packaged into a structure named chromatin which contains DNA and proteins. Nucleosomes are building blocks of chromatin and contain DNA wrapped around a histone octamer. Chromatin modifications (histone post-translational modifications and histone variants) play central roles in various cellular processes including gene expression and DNA damage response. Chromatin modifying enzymes use metabolites as co-substrates and co-factors, and changes in metabolic pathways and metabolite availability affects chromatin modifications and chromatin-associated functions. Moreover, recent studies have uncovered direct roles of metabolic enzymes in chromatin-associated functions. Fumarase, a TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate in mitochondria (a hydration reaction), is an example of an enzyme with dual functions in metabolism and genome integrity. Cytoplasmic fraction of yeast fumarase, Fum1p, localizes to the nucleus and promotes growth upon DNA damage. Fum1p promotes homologous recombination by enhancing DNA end resection. Human fumarase is involved in DNA repair by non-homologous end joining. Here, we provide evidence that yeast Fum1p and the histone variant Htz1p are also involved in DNA replication stress response and DNA repair by non-homologous end joining (NHEJ). Using mutants lacking the histone variant <i>HTZ1</i>, we show that high cellular levels of fumarate, by deletion of <i>FUM1</i> or addition of exogenous fumarate, suppressed the sensitivity to DNA replication stress by modulation of activity of Jhd2p. This suppression required sensors and mediators of the intra-S phase checkpoint, but not factors involved in the processing of replication intermediates. These results imply that high cellular levels of fumarate can confer resistance to DNA replication stress by bypassing or complementing the defects caused by loss of <i>HTZ1</i> and replication fork processing factors. We also show that upon induction of DSBs, exogenous fumarate conferred resistance to mutants with defects in NHEJ, early steps of homologous recombination (DNA end resection pathway) or late steps of homologous recombination (strand invasion and exchange). Taken together, these results link the metabolic enzyme fumarase and the metabolite fumarate to DNA damage response and show that modulation of DNA damage response by regulating activity of chromatin modifying enzymes is a plausible pathway linking metabolism and nutrient availability to chromatin-associated functions like genome integrity.<br><a></a></p>

Antibiotics are a foundation of modern medicine, helping to save millions of lives since their discovery in 1928. But the improper and excessive use of these drugs over the last few decades has led to an alarming increase in antimicrobial resistance; coupled with the recent decrease in antibiotic discovery, it is widely thought that we are approaching a post-antibiotic era. A less well-understood problem is that of drug tolerance. Even at high doses, antibiotics often cannot kill all the bacteria in an infection because of cells that are able to tolerate antibiotic treatment. Evidence points to drug-tolerant cells, also called persisters, to be a major cause of treatment failure and chronic and recurring infections It is imperative that we develop insight and methods to prevent the spread of antimicrobial resistance and combat antimicrobial tolerance. One key effort is characterizing bacterial responses to antibiotic drug treatment to generate a more comprehensive understanding of the factors that contribute to cell death and to elucidate potential targets for new therapies. Quinolones are an important class of antibiotics that target DNA replication. They bind to topoisomerase II and IV, leading to eventual DNA fragmentation and death. However, the precise mechanism by which they work is not well understood. Because they inhibit DNA replication, quinolones lead to up-regulation of the SOS response, which allows for increased mutagenesis and the potential for increased antimicrobial resistance, thus making quinolones an interesting class of antibiotics to study. Although quinolones are one of the most effective classes of antibiotics, there are many conditions in which they do not kill, such as in stationary-phase cultures. Understanding the mechanism behind quinolone killing, quinolone-induced mutagenesis and tolerance to quinolones is important to improve quinolone efficacy. Here I have presented my work on understanding quinolones: sensitivity, mutagenesis and tolerance. In understanding quinolone sensitivity, I focus on DNA repair and its involvement in quinolone-mediated death. I then probe the field of stress-induced mutagenesis by quinolones, uncovering phenotypes of dose-dependent mutagenesis that have previously been uncharacterized. Finally, I focus on drug tolerance and how density-dependent tolerance to quinolones can be reversed by up-regulating cellular respiration through the addition of a carbon source and electron acceptor. / 2018-11-02T00:00:00Z

Multiple Sclerosis (MS) is a chronic and potentially disabling disease of the central nervous system (CNS) in which the immune system attacks the protective myelin layer that surrounds nerve cells. While the majority of individuals diagnosed with MS initially present with a non-progressive relapsing form of the disease, there is significant risk of eventually transitioning to a more progressive form for which there are few effective treatments. Consequently, early intervention with disease-modifying therapies (DMTs) is essential for effective disease management. Newly diagnosed patients are typically started on one of four first-line therapies (beta interferon, glatiramer acetate, teriflunomide, or dimethyl fumarate). Though there are distinct differences between these treatments in regard to efficacy and safety, there is no uniform standard for making decisions about which to initiate treatment with. This review gives an overview of current first-line MS therapies, and seeks to highlight the lack of comparison data and the gaps in the current understanding of disease management, as well as the need for more comprehensive research in these areas.

Hypoxia inducible factor-1&alpha; (HIF-1&alpha;) plays a critical role in the oxygen homeostasis of all metazoans. HIF-1&alpha; is a master transcriptional regulator which coordinates the adaptive response to low oxygen tension. Through activation of a plethora of downstream target genes, HIF-1&alpha; facilitates oxygenation by promoting angiogenesis and blood vessel dilation, in addition to modulating metabolic pathways to inhibit oxidative phosphorylation and promote glycolytic energy production. Given the critical roles of hypoxia, insufficient blood supply and perturbed energetics in the pathogenesis of cardiovascular disorders, notably ischaemic heart disease, therapeutic modulation of HIF-1&alpha; is of significant clinical interest. Previous studies have demonstrated an acute cardioprotective role for both endogenous and supraphysiological HIF-1&alpha; signalling in the context of myocardial ischaemia. In contrast, chronic supraphysiological HIF-1&alpha; activation in the unstressed heart has been shown to induce cardiac dysfunction. To address the effect of chronic endogenous HIF-1&alpha; activation post-myocardial infarction (MI), the present work employed a murine coronary artery ligation (CAL) model in conjunction with temporally-inducible, cardiac-specific deletion of Hif-1&alpha;. While CAL surgery successfully modelled myocardial infarction – eliciting substantial adverse cardiac remodelling and contractile dysfunction – there was no evidence of chronic HIF-1&alpha; activation by CAL in HIF knockout or control left ventricular samples. In keeping with this, chronic ablation of Hif-1&alpha; (from 2 weeks post-CAL) had no discernible additional effect upon cardiac function. Overall, these findings do not support a potential therapeutic role for inhibition of HIF-1&alpha; signalling in the chronic phase post-MI. The fundamental tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) converts fumarate to malate. FH deficiency is associated with smooth muscle and kidney tumours which exhibit normoxic HIF signalling due to fumarate accumulation. To investigate the potential for fumarate accumulation to elicit protective HIF signalling, a cardiac-specific Fh1 null mouse was developed through Cre-loxP recombination. Strikingly, despite interruption of the TCA cycle in a highly metabolically demanding organ, cardiac Fh1 null mice were viable, fertile and survived into adulthood, demonstrating the remarkable metabolic plasticity of the heart. However, by 3-4 months Fh1 null mice develop a lethal cardiomyopathy characterised by cardiac hypertrophy, ventricular dilatation and contractile dysfunction. Despite lack of a pseudohypoxic response, Fh1 null hearts did exhibit another phenomenon observed in FH-deficient cancers and also attributed to fumarate accumulation – activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant pathway. Heterozygous, but not homozygous, somatic deletion of Nrf2 extended the life expectancy of cardiac Fh1 null mice. Exploration of redox status revealed a more reductive environment in Fh1 null hearts than controls. As a corollary, inhibition of the rate limiting enzyme of the pentose phosphate pathway – a major source of cellular reducing equivalents – with dehydroepiandrosterone conferred striking amelioration of the Fh1 null cardiomyopathy, suggesting a possible pathogenic role for reductive stress. While loss of mitochondrial Fh1 activity and subsequent TCA cycle dysfunction likely contribute to the Fh1 null phenotype, the importance of cytosolic FH was unclear. To clarify this, FH was expressed specifically in the cytosol in vivo. This was sufficient to substantially rescue the Fh1 null cardiomyopathy, supporting a role for cytosolic FH disruption in its pathogenesis. Taken together, these findings highlight the potential for reductive stress to contribute to cardiac dysfunction and suggest a function for cytosolic FH in cardiac metabolic homeostasis.