Dissertations / Theses on the topic 'Biochemistry and cell biology not elsewhere classified'

This thesis describes the development of a computer controlled on-line system for fermentation monitoring and control. The entire system consists of a laboratory fermenter, flow injection system (four channels), a newly designed on-line filter, biomass analysis channel, pH and oxygen controllers as well as a spectrophotometer. A new design of gas driven flow injection analysis (FIA) allows a large number of reagents to be handled. The computer-controlled four channel PIA system is well suited for sequential analysis, which is important for fermentation on-line mOnitoring. The system can change the wavelength of the spectrophotometer automatically for each PIA channel, which makes the system powerful and flexible. A high frequency, low energy ultrasonic filter was modified and applied to the system for on-line mammalian cell culture sampling without breaking the sterile barrier. The results show that this novel application of ultrasonic filter technology results in higher efficiency and reliability and a longer life cycle than other types of filter. All the operations of the analytical system are controlled by a Macintosh computer (Quadra 950). The control program was written in LabVIEW which is a graphical programming language and well applicable to fermentation control. The software communicates with detectors, data acquisition, data analysis and presentation. The system can programmatically control up to 50 devices. Mammalian cell batch culture was used as an example of the application of the system. The system consists of a laboratory fermenter with a continuous sample withdrawal filter and an analysis system where glucose, lactate and ammonia, lactate dehydrogenase and biomass were measured. Cell viability was estimated by microscopic assay with trypan blue. pH and Oxygen were also measured. The system response was fast and yields a large number of reliable and precise analytical results which can be of great importance in the monitoring and control of mammalian cell culture conditions.

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

As far back as the 1920s, Otto Warburg observed that cancerous cells display an altered state of metabolism surrounding lipid biosynthesis. However, only until recently has metabolic reprogramming been a recognised hallmark of the disease. The number of cancer cases diagnosed is set to triple by 2030, demonstrating the need for disease prevention, improved diagnostic testing and personalised treatment therapies. However, with some cancers occurring in the brain and spinal cord, the type of treatment available can become challenging due to their locality. Such cancer types include meningioma and glioma which are the most common brain tumours diagnosed. An initial study involving human meningioma tissue revealed unusually high levels of the phosphatidylserine enriched with docosahexaenoic acid (DHA). In this study, the metabolism surrounding lipid biosynthesis was examined to establish if such alterations in lipid profiles were related to an altered state of metabolism. From the results gained, it can be suggested that meningioma does have an altered state of metabolism, evolving around serine as opposed to DHA. From the grade I and grade II meningioma tissues immunochemically examined, positive expressions of pyruvate kinase isoform 2 (PKM2) and phosphoglycerate dehydrogenase (PHGDH) were shown. Therefore, the results demonstrated that within meningioma tissues, serine can allosterically regulate the flux through glycolysis. The association that serine presence alone can alter the metabolic flux was demonstrated in the model organism, Lipomyces starkeyi. Those L. starkeyi cells supplemented with serine, displayed a 50% reduction in the amount of radiolabelled acetate taken up during exponential and stationary growth phases. The radiolabelled study also highlighted that with serine presence, de novo lipid biosynthesis was altered. Once synthesised, these neutral lipids go on to be 4 stored in membrane bound organelles. Within the phenotype of cancerous cells, such storage of neutral lipids into lipid droplets prevent lipotoxicity. The light microscopy study of L. starkeyi cells supplemented with serine demonstrated that the formation of such lipid droplets was enhanced during lipid accumulation. These findings suggest that the production, storage and mobilisation of lipids within serine supplemented cells are adapted to cellular requirements, promoting a cancerous phenotype. In order to gain an insight into the potential impact that an altered metabolic state may give to meningioma, a liposomal study was developed. Supplementation of both phosphatidylserine-consisting liposomes, as well as tumour-derived liposomes, enhanced the cellular viability of the non-cancerous cell line, SVG, during exponential phase. The supplementation of meningioma-derived liposomes also increased the viability of the non-cancerous human fetal glial SVG cell line, similar to that observed with phosphatidylserine containing liposomal preparations. Therefore, the data suggest that in fact, the phospholipid (phosphatidylserine), rather than the fatty acid (DHA) plays a role in cellular viability. It is concluded that the results gained from this study can be used clinically in the diagnosis and management of meningioma as well as other diseased cells displaying ectopic lipid accumulation. The observation that meningioma has an altered biochemistry may provide guidance when histologically grading meningioma tumours. For those tumours expressing the enzymes involved in serine biosynthesis, such as PKM2 and PHGDH, a targeted treatment therapy surrounding enzyme inhibitors can be examined. By targeting serine biosynthesis, the resources needed to enable a cancerous phenotype are depleted. Future research can examine such targeted therapies utilizing either the developed model organism, L. starkeyi or the conventional SVG and U87 cell lines.

A low technology fermentation system for biodegradation of cellulose based waste material is described. Using a substrate of thermally bonded paper plastic laminate in a defined mineral salts medium the cellulolytic nicrofungus Trichoderma viride XXI 2% 458 was shown to effect the separation of the laminate into its components by enzymic degradation of the paper. This aerobically induced separation occurred within 14 days compared to upto 90 days by purely physical means. Growth of the fungus within the fermentation system was found to provide conditions that control the growth of contaminating microorganisms. Carbonolytic enzyme activity of Trichoderma viride was investigated by measurenent of substrate weight loss and by reduction in the viscosity of soluble substrate analogues when acted upon by filtrate front-the fermentation systais. An investigation of the effect of changes in carbon to nitrogen ratios during fermentation indicated that maximum carbonolytic enzyme activity occurred at a carbon to nitrogen ratio of 0.6:1 within this system. An examination of three other cellulolytic fungi in order to assess their potential as biodegradative organisms within the fermentation system showed that under microthermophilic conditions only Acrenonium stricturn approached the biodegradative activity of Trichoderma viride at mesophilic temperatures. The products of fermentation by Trichoderma viride were examined; protein levels in the residual cellulolytic substrate following biodegradation of paper plastic laminate were determined and found to reach levels of up to 9% following 35 days fermentation. The amino acid composition of protein produced by Trichoderma viride when grown on a range of substrates was determined and found to contain a wide range of essential and non essential amino acids. An investigation of mycotoxins both in the culture medium and residual paper component was undertaken. Comparisons with eleven common mycotoxin standards revealed no positive identifications under the conditions investigated.

Methods for the preparation of concentrates of factor VIII, factor IX, high purity factor IX, Cl esterase inhibitor, specific immunoglobulin and platelet factor XIII are described. These procedures were developed or modified with the aim of integration into an automated process that would allow sequential recovery of all the clinically significant trace proteins from a single plasma pool. Concomitant recovery of important proteins such as transferrin, alpha-1-antitrypsin and platelet-derived growth factor was considered. A high-purity factor VIII concentrate heat-treated at 80°C for 96 h was prepared by a process that incorporated heparin fractionation. This method was shown to be suitable for assimilation into an existing regional blood processing laboratory. Several ion-exchange procedures for the recovery of factor IX were evaluated and higher purification of a factor IX concentrate was achieved on a new cellulose-based chromatographic medium. A chromatographic procedure for the preparation of a heat-treated high-purity Cl esterase inhibitor concentrate was described and the performance of a new cellulose-based desalting medium was evaluated in comparison with ultrafiltration. A heat-treated specific immunoglobulin concentrate was prepared from side-stream fractions of an automated chromatographic process for the production of albumin concentrate, and a pilot study for the fractionation of outdated platelet concentrates was carried out with the aim preparing components of potential therapeutic value. See summary flow diagrams of fractionation processes included with references in the back of this thesis.

External representations (ERs) used in science education are multimodal ensembles consisting of design elements to convey educational meanings to the audience. As an example of a dynamic ER, an animation presenting its content features (i.e., scientific concepts) via varying the feature’s depiction over time. A production team invited the dissertation author to inspect their creation of a biochemistry animation about the role of the actin cytoskeleton in cell motility and the animation’s implication on learning. To address this, the author developed a four-step methodology entitled the Multimodal Variation Analysis of Dynamic External Representations (MVADER) that deconstructs the animation’s content and design to inspect how each content feature is conveyed via the animation’s design elements.

This dissertation research investigated the actin animation’s educational value and the MVADER’s utility in animation evaluation. The research design was guided by descriptive case study methodology and an integrated framework consisting of the variation theory, multimodal analysis, and visual analytics. As stated above, the animation was analyzed using MVADER. The development of the actin animation and the content features the production team members intended to convey via the animation were studied by analyzing the communication records between the members, observing the team meetings, and interviewing the members individually. Furthermore, students’ learning experiences from watching the animation were examined via semi-structured interviews coupled with post- storyboarding. Moreover, the instructions of MVADER and its applications in studying the actin animation were reviewed to determine the MVADER’s usefulness as an animation evaluation tool.

Findings of this research indicate that the three educators in the production team intended the actin animation to convey forty-three content features to the undergraduate biology students. At least 50% of the student who participated in this thesis learned thirty-five of these forty-three (> 80%) features. Evidence suggests that the animation’s effectiveness to convey its features was associated with the features’ depiction time, the number of identified design elements applied to depict the features, and the features’ variation of depiction over time.

Additionally, one-third of the student participants made similar mistakes regarding two content features after watching the actin animation: the F-actin elongation and the F-actin crosslink structure in lamellipodia. The analysis reveals the animation’s potential design flaws that might have contributed to these common misconceptions. Furthermore, two disruptors to the creation process and the educational value of the actin animation were identified: the vagueness of the learning goals and the designer’s placement of the animation’s beauty over its reach to the learning goals. The vagueness of the learning goals hampered the narration scripting process. On the other hand, the designer’s prioritization of the animation’s aesthetic led to the inclusion of a “beauty shot” in the animation that caused students’ confusion.

MVADER was used to examine the content, design, and their relationships in the actin animation at multiple aspects and granularities. The result of MVADER was compared with the students’ learning outcomes from watching the animation to identify the characteristics of content’s depiction that were constructive and disruptive to learning. These findings led to several practical recommendations to teach using the actin animation and create educational ERs.

To conclude, this dissertation discloses the connections between the creation process, the content and design, and the educational implication of a biochemistry animation. It also introduces MVADER as a novel ER analysis tool to the education research and visualization communities. MVADER can be applied in various formats of static and dynamic ERs and beyond the disciplines of biology and chemistry.

Prostate cancer is (PCa) the second leading cause of cancer death in males in the United State, with 174,650 new cases and 31,620 deaths estimated in 2019. Polo-like kinase 1 (PLK1) has been postulated to have a pro-tumorigenesis function, besides its critical role in regulation of cell cycle, and to be overexpressed in various types of human cancer, including prostate cancer (PCa). However, our understanding remains unclear regarding the pro-tumor properties of PLK1 partially due to a lack of proper animal model. Integrating our recently generated prostate-specific PLK1 knock-in genetically engineered mouse model (GEM) and the transcriptome data of human PCa patients, we identify an oncogenic role of PLK1 in the prostate adenocarcinoma progression, castration resistance and metastatic dissemination. To elucidate the underlying mechanism, we investigate the link between PLK1 and tumor microenvironment in PCa using the transgenic mouse model, and find that PLK1overexpression enable the macrophages polarization towards M2 phenotype via driving the activation of IL4/IL13/STAT6 pathway. These findings first validates PLK1 as a critical oncogene closely associated with PCa progression in vivo, and uncover a novel function of PLK1 to facilitate IL4/STAT6 signaling and M2 macrophage polarization. Importantly, these findings suggest an efficient therapeutic strategy targeting STAT6 for treatment of advanced PCa which usually possessing a high level of PLK1 expression. To further explore the molecular mechanism underlying PLK1-induced PCa progression and resistance to therapy, we turned our eyes to epigenetic modifications. It has been documented that epigenetic deregulation such as histone modification and DNA methylation contributes to PCa initiation and progression. Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb-repressive complex 2 (PRC2), plays a critical role in repressing gene expression by tri-methylation of histone 3 at lysine 27 (H3K27me3). Emerging data have demonstrated that there is a link between EZH2 and oncogenesis as EZH2-mediated methylation acts as an important factor in epigenetic silencing of tumor suppressor genes in cancer. Expression of EZH2 is often upregulated in castration-resistant prestate cancer (CRPC), thus EZH2 has been proposed as a target for CRPC. Importantly, it has been demonstrated that EZH2 becomes hyperphosphorylated in CPRC cells. Further, it has been shown that the oncogenic function of EZH2 is usually regulated by the post-translational modifications. PLK1 acting as a serine/threonine kinase to regulate multiple signaling pathways in human cancer, however, whether PLK1 is involved in EZH2 phosphorylation is not known. Herein, we show that Plk1 physically interacts with EZH2 and negatively regulates H3K27 trimethylation (H3K27me3). Furthermore, Plk1 can phosphorylate EZH2 at T144, and Plk1-mediated phosphorylation of EZH2 is involved in inhibiting EZH2 activity toward H3K27me3. More importantly, EZH2 phosphorylation by Plk1 is inhibitory for PRC2-mediated gene repression but required for transcriptional activation toward oncogenesis. Finally, by combination with Plk1 inhibitor BI2536, we show a robust sensitization of EZH2 inhibitors in CRPC cell lines, as well as in CRPC xenograft tumors. Our findings provide a new mechanism to define the oncogenic activity of EZH2 and suggest that inhibition of Plk1-mediated EZH2 activity may provide a promising therapeutic approach for CRPC.

The phenylpropanoid pathway gives rise to a wide variety of specialized metabolites, but the majority of carbon flux going through this pathway is directed towards the synthesis of the lignin monomers: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Lignin is a major impediment in biomass saccharification, which negatively affects animal feed and biofuel production. In an effort to improve biomass for the latter purposes, researchers have altered the polymer through genetic manipulations and generated biomass with lower recalcitrance to saccharification; however, in many cases these efforts have resulted in plant dwarfism. To date, we do not have a full understanding of the extent of lignin modifications a plant is able to tolerate without affecting its growth. More importantly, the mechanism that links dwarfism and modifications in lignin content and composition remains unknown. To contribute to answering these questions, we designed a strategy to incorporate a novel monomer into the lignin of Arabidopsis thaliana. We used mutants in genes that code for enzymes and regulators of the phenylpropanoid pathway to redirect the pathway’s flux towards the synthesis of p-coumaraldehyde and prevent the incorporation of p-coumaryl alcohol. Despite being mutated for the genes typically considered to be required for monolignol biosynthesis, the plants we generated continue to incorporate p-coumaryl alcohol into their lignin. This result suggests that the pathway’s architecture has not been completely elucidated and that there are more enzymes involved in lignification than previously thought. Additionally, we explored the connection between perturbations in phenylpropanoid metabolism and plant growth, by using an inducible system to track the changes in gene expression and metabolism that occur when phenylpropanoid metabolism is restored in a lignin biosynthetic mutant. The use of an inducible system allowed us to not only determine the metabolic processes affected in this mutant, but the proximal sequence of events that lead to restored growth when a functional copy of the mutant gene is induced. Finally, we redirected the flux through the pathway to assess the effects of simultaneously modulating lignin content and composition. Through this project we discovered that redirecting phenylpropanoid flux towards the synthesis of sinapyl alcohol in lignin-deficient mutant backgrounds, results in plant dwarfism. The growth impairment of these mutants can be overcome by providing exogenous coniferyl alcohol, suggesting that dwarfism in these mutants is caused by deficiency in coniferyl alcohol and/or derivatives thereof and not lignin alone. Altogether these projects allowed us to define the cellular processes affected by perturbations in phenylpropanoid homeostasis and the role of other phenylpropanoids besides lignin in this process.

Malonyl-thioesters are reactive at the thioester carbonyl and the carboxylate moieties, as seen in acyl transfer or hydrolysis and decarboxylation. Enzymes use these reactive centers to perform different enzyme chemistry throughout metabolism. This enzyme chemistry coupled with the inherent reactivity of malonyl-thioesters makes structure-function studies difficult. When malonyl-thioesters are used for structure-function studies, it usually results in a hydrolyzed or decarboxylated product. There are examples, however, where this is overcome, many of which are discussed throughout this thesis. To overcome the inherent reactivity of malonyl-thioesters and enzymes, analogs have been synthesized to perform structure-function studies. Initial studies focused on altering the thioester carbonyl to limit hydrolysis and decarboxylation; however, these studies revealed the importance of retaining the thioester carbonyl to be positioned in the oxyanion hole. My thesis work focused on the synthesis, characterization, and use in structure-function studies of malonyl-thioester analogs that either preserve the thioester carbonyl or alter it to an ester or amide, and alter the carboxylate to a sulfonate or nitro group. After synthesizing the methylmalonyl-CoA analogs, we performed structure-function studies with methylmalonyl-CoA decarboxylase. This case study revealed the potential of these analogs to both inhibit decarboxylase activity and their use in structure-function studies to gain mechanistic insights. This successful study prompted us to continue these structure-function studies in enzymes with different chemistries such as an epimerase or bi-functional acyltransferase/decarboxylase. The widespread use of these methylmalonyl-CoA analogs also motivated us to add more malonyl-thioester analogs to our toolbox. I have preliminary data that these malonyl-thioester analogs inhibit β-keto-acyl-synthase III, an enzyme involved in fatty acid production in E. coli. This inhibition gives us confidence that these analogs will be useful in structure-function studies that will reveal answers to long standing mechanism and protein-protein interaction questions in the polyketide and fatty acid synthase field.

Viruses are pathogenic agents which affect all varieties of organisms, including plants, animals and humans. These microscopic particles are genetically simple organisms which encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope coat that surrounds them. Lipid enveloped viruses comprise a diverse range of pathogenic viruses, known to cause disease in both animals and human which often leads to high fatality rates, many of which lack effective and approved therapeutics. This report focuses on learning how a multifunctional protein within lipid enveloped viruses, the matrix protein, interacts with the plasma membrane of cells to enter and exit cells. Specifically, four viruses are investigated, Measles virus and Nipah virus (within the Paramyxoviridae family) and Ebola virus and Marburg virus (within the Filoviridae family). Through numerous in vitro experiments, functional cellular assays, a myriad of microscopy techniques, and experiments in high containment bio-safety level 4 settings, this report identifies specific lipids at play during the viral assembly process for each virus. Moreover, mechanistic insight is presented as to how each matrix protein interacts with the plasma membrane to facilitate: membrane association, viral matrix protein oligomerization and assembly, the rearrangement of lipids within the plasma membrane, and viral production. Lastly, numerous small molecule inhibitors targeting specific lipids, (e.g. phosphatidylserine and phosphatidylinositol 4,5 bisphosphate) within the cell were investigated for their efficacy in inhibiting matrix protein-dependent viral like particle production and viral spread in cells. As a whole, these projects lend credence to the significant role that lipids and the plasma membrane play throughout lipid enveloped viral life cycles, and provide compelling evidence for the merit of future drug-development research geared at targeting the matrix protein-plasma membrane interaction.

The B3-adrenoceptor (B-AR) was first classified in 1984 in rat brown adipose tissue. The classification of this receptor in other tissues and species is hampered by the lack of selective B3-antagonists which, despite the identification of further classes of B3-agonist ligands, remains elusive. In this study, a series of novel B3-AR antagonist drugs were synthesised and their pharmacological profile in rat ileum investigated with the aim of increasing our understanding of the structural requirements of drug-receptor binding in B3-ARs. Analogues of iodocyanopindolol (ICYP) (17) and conformationally impaired analogues of BRL 37344 were identified as key synthetic targets.

ICYP and eleven analogues were synthesised from epoxide precursor (38). The pharmacological activity of these compounds was determined in a rat ileum preparation with tissue contraction solely due to the B3-AR. All ICYP analogues were active at the B3-AR. The pharmacological data revealed: (i) of the analogues tested, ICYP (17) and CYP (18) had the highest affinities at the B3-AR. This is in sharp contrast to the binding of ICYP (17) and CYP (18) at B1- and B2-ARs where ICYP (17) has a hundred fold higher affinity than CYP (18); and (ii) two pharmacological characteristics determined for the drugs, namely pD2 and the pKb, were significantly different for eight of the analogues studied. These drugs were partial agonists, and the discrepancy between pD2 and pKb values indicated binding to more than one receptor population.

Three hypotheses were proposed to explain this observation; (i) two different B3-ARs are present; (ii) the result is an enantiomeric effect; and (iii) the difference is a non-specific lipophilic effect.

Log P values for the series were determined using HPLC, and no correlation was found between Log P and pD2 or pKb values.

To examine the second hypothesis, both enantiomers of CYP (18) and bupranolol (8) were synthesised and their pharmacological activity investigated in rat ileum. All four enantiomers tested were antagonists at the B3-AR, with the receptor displaying stereoselectivity for the (S)-enantiomers, with (S)-CYP (18b) being the most potent B3-antagonist drug identified. Examination of the partial agonist activity of CYP and bupranolol enantiomers suggested that this effect was independent of the mechanism of B3-antagonism. The partial agonist effect was selective for (R)-CYP (18a) and non-selective for bupranolol. This result clearly defines (S)-CYP (18b) as a potent B3-AR antagonist and highlights structure-activity studies of CYP analogues as an important new source of information for the design of new classes of B3-antagonist drugs.

To further investigate the structure-function relationship being developed for B3-AR selective drugs the "extended conformation" hypothesis of Blin and co-workers was examined. These researchers proposed that the discrepancies observed between the B3-AR activity, and the B1/B2-AR activity of known agonist and antagonist drugs resulted from the ability of B3-agonist drugs to adopt an extended conformation at the B3-AR. To investigate this postulate, conformationally impaired analogues of the B3-AR agonist BRL 37344 (6) were targeted for synthesis. Allylic amine (112) was identified as a key precursor for conformationally impaired BRL 37344 analogues and was synthesised with solely (E) geometry from (d)-Bocalinal (109) and 4-methoxy benzylphosphonium chloride (111). Progress in the synthesis of other subtargets is described.

Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol a), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase a tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol a tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

The Mediator (Med) complex comprises about 30 subunits and is a transcriptional co-regulator in eukaryotic systems. The core Mediator complex, consisting of the head, middle and tail modules, functions as a bridge between transcription factors and basal transcription machinery, whereas the CDK8 kinase module can attenuate Mediator’s ability to function as either a co-activator or co-repressor. Many Arabidopsis Mediator subunit has been functionally characterized, which reveals critical roles of Mediator in many aspects of plant growth and development, responses to biotic and abiotic stimuli, and metabolic homeostasis. Traditional genetic and biochemical approaches laid the foundation for our understanding of Mediator function, but recent transcriptomic and metabolomic studies have provided deeper insights into how specific subunits cooperate in the regulation of plant metabolism. In Chapter 1, we highlight recent developments in the investigation of Mediator and plant metabolism, with emphasis on the large-scale biology studies of med mutants.

We previously found that MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi-dominant mutation (reduced epidermal fluorescence 4-3, ref4-3) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. In contrast, loss of MED5a and MED5b (med5) results in increased levels of phenylpropanoids. In Chapter 2, we present our finding that ref4-3 requires CDK8, a Mediator kinase module subunit, to repress plant growth even though the repression of phenylpropanoid metabolism in ref4-3 is CDK8-independent. Transcriptome profiling revealed that salicylic acid (SA) biosynthesis genes are up-regulated in a CDK8-dependent manner in ref4-3, resulting in hyper-accumulation of SA and up-regulation of SA response genes. Both growth repression and hyper-accumulation of SA in ref4-3 require CDK8 with intact kinase activity, but these SA phenotypes are not connected with dwarfing. In contrast, mRNA-sequencing (RNA-seq) analysis revealed the up-regulation of a DNA J protein-encoding gene in ref4-3, the elimination of which partially suppresses dwarfing. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.

In Chapter 3, we characterize other phenotypes of med5 and ref4-3, and find that in addition to the up-regulated phenylpropanoid metabolism, med5 show other interesting phenotypes including hypocotyl and petiole elongation as well as accelerated flowering, all of which are known collectively as the shade avoidance syndrome (SAS), suggesting that MED5 antagonize shade avoidance in wild-type plants. In contrast, the constitutive ref4-3 mutant protein inhibits the process, and the stunted growth of ref4-3 mutants is substantially alleviated by the light treatment that triggers SAS. Moreover, ref4-3 mimics the loss-of-function med5 mutants in maintaining abscisic acid (ABA) levels under both normal and drought growth conditions. The phenotypic characterization of med5 mutants extend our understanding of the role of Mediator in SAS and ABA signaling, providing further insight into the physiological and metabolic responses that require MED5.

In Chapter 4, we explore the function of MED5 and CDK8 in gene expression regulation by investigating the effect of mutations in Mediator including med5, ref4-3, cdk8-1 and ref4-3 cdk8-1 on genome-wide Pol II distribution. We find that loss of MED5 results in loss of Pol II occupancy at many target genes. In contrast, many genes show enriched Pol II levels in ref4-3, some of which overlap with those showing reduced Pol II occupancy in med5. In addition, Pol II occupancy is significantly reduced when CDK8 is disrupted in ref4-3. Our results help to narrow down the direct gene targets of MED5 and identify genes that may be closely related to the growth deficiency observed in ref4-3 plants, providing a critical foundation to elucidate the molecular function of Mediator in transcription regulation.

This thesis attempts to broaden what is known about bacterial symbionts within amoebae by the use of a number of different molecular methods. Initially a number of different amoeba strains were screened for bacterial symbionts by 16S rRNA gene PCR, then the symbionts were identified by comparative sequence analysis and phylogenetic analysis. The amoeba strains containing bacterial symbionts were characterised by cell morphology, 18S rRNA gene sequencing, internal transcribed spacer sequencing and allozyme electrophoresis. Amoebae belonging to the genera Acanthamoeba, Naegleria, Ripidomyxa and Saccamoeba were identified as containing symbionts that belonged to a wide range of different bacterial genera. Relationships between bacterial symbionts and their host amoebae were analysed by the use of transmission electron microscopy and fluorescent in situ hybridisation using symbiont specific probes. Also described are attempts that were made to isolate and grow the bacterial symbionts outside of their host amoebae as well as experiments to try to transfer bacterial symbionts from one amoeba strain to another. Lastly the results from this study are discussed as a whole to put into perspective how they contribute to the body of knowledge of symbionts within protozoa.

DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While dnmt3a and dnmt3b genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme.

DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.

Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B.

T-cells are present in the immune system to fight against invaders. Once their job is done, suppressing their activity is an important step in maintaining a proper immune response. Myeloid derived suppressor cells (MDSCs) are immune cells that suppress T-cell activity. Currently, MDSCs are defined as a heterogeneous population of immature cells that are derived in the bone marrow and travel to the site of inflammation or cancer. Two major subtypes of MDSCs have been identified in mice and humans, monocyte-like MDSCs (M-MDSC) and granulocyte MDSCs (G-MDSC). G-MDSCs typically make up the majority of the total population of MDSCs but are less T-cell suppressive than M-MDSCs. One of the major problems in the study of MDSCs is that the current marker system for subtypes does not differentiate between precursor MDSCs (lacking suppressive ability) and functional MDSCs (those with suppressive ability). Therefore, using cancer models in mice, we investigated the development and potential to classify precursor MDSCs from functional MDSCs. While MDSCs have been highlighted as a target cell to inhibit in cancer, in other conditions, such as pregnancy, MDSCs have been shown to be beneficial in maintaining a normal pregnancy. Therefore, targeting the increase of MDSCs in abnormal pregnancy conditions like pre-eclampsia may act as a prevention or therapeutic strategy. Finally, it is known that many dietary components can act as modulators of immune cells. Specifically, the polyphenol like phytochemical, curcumin has been shown to act as an anti-inflammatory agent with the potential to modulate multiple immune cells. Therefore, we propose two different studies to investigate the potential of curcumin as either an inhibitor and/or promotor of MDSCs in a disease-specific context. Together the role of phytochemicals as immunomodulators of MDSCs is still very young, in part due to the complexity of phytochemicals themselves, but the studies cited here provide evidence that the field is ripe for additional questions to be asked.

Breast cancer is the predominant cancer diagnosed among women, and the second most deadly cancer. The vast majority of cancer-related deaths is caused by the metastatic spread of cancer from the primary tumor to a distant site in the body. Therefore, new strategies which minimize breast cancer metastasis are imperative to improve patient survival. Cancer cells which acquire anchorage independence, or the ability to survive without extracellular matrix attachment, and metabolic flexibility have increased potential to metastasize. In the present studies, the ability to survive detachment and subsequent metabolic changes were determined in human Harvey-ras transformed MCF10A-ras breast cancer cells. Detachment resulted in reduced viability in a time-dependent manner with the lowest cell viability observed at forty hours. In addition, decreased cell viability was observed in both glutamine and glucose depleted detached conditions, suggesting a dependence on both nutrients for detached survival. Compared to attached cells, detached cells had reduced total pool sizes of pyruvate, lactate, α-ketoglutarate, fumarate, malate, alanine, serine, and glutamate, suggesting the metabolic stress which occurs under detached conditions. However, intracellular citrate and aspartate pools were unchanged, demonstrating a preference to maintain these pools in detached conditions. Compared to attached cells, detached cells had suppressed glutamine metabolism, as determined by decreased glutamine flux into the TCA cycle and reduced mRNA abundance of glutamine metabolizing enzymes. Further, detached glucose anaplerosis through pyruvate dehydrogenase activity was decreased, while pyruvate carboxylase (PC) expression and activity were increased. A switch in metabolism was observed away from glutamine anaplerosis to a preferential utilization of PC activity to replenish the TCA cycle, determined by reduced PC mRNA abundance in detached cells treated with a cell-permeable analog of α-ketoglutarate, the downstream metabolite of glutamine which enters the TCA cycle. These results suggest that detached cells elevate PC to increase flux of carbons into the TCA cycle when glutamine metabolism is reduced.

Vitamin D is recognized for its role in preventing breast cancer progression, and recent studies suggest that regulation of energy metabolism may contribute to its anticancer effects. Vitamin D primarily acts on target tissue through its most active metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)₂D). The present work investigated 1,25(OH)₂D’s effects on viability of detached cells through regulation of energy metabolism. Treatment of MCF10A-ras cells with 1,25(OH)₂D resulted in decreased viability of detached cells. While 1,25(OH)₂D treatment did not affect many of the glucose metabolism outcomes measured, including intracellular pyruvate and lactate pool sizes, glucose flux to pyruvate and lactate, and mRNA abundance of enzymes involved in glucose metabolism, 1,25(OH)₂D treatment reduced detached PC expression and glucose flux through PC. A reduction in glutamine metabolism was observed with 1,25(OH)₂D treatment, although no 1,25(OH)₂D target genes were identified. Further, PC depletion by shRNA decreased cell viability in detached conditions with no additional effect with 1,25(OH)₂D treatment. Moreover, PC overexpression resulted in increased detached cell viability and inhibited 1,25(OH)₂D’s negative effects on viability. These results suggest that 1,25(OH)₂D reduces detached cell viability through regulation of PC. Collectively this work identifies a key metabolic adaptation where detached cells increase PC expression and activity to compensate for reduced glutamine metabolism and that 1,25(OH)₂D may be utilized to reverse this effect and decrease detached cell viability. These results contribute to an increased understanding of metastatic processes and the regulation of these processes by vitamin D, which may be effective in preventing metastasis and improve breast cancer patient survival.

Approximately 6.3 million bone fractures occur annually in the USA, resulting in considerable morbidity, deterioration in quality of life, loss of productivity and wages, and sometimes death (e.g. hip fractures). Although anabolic and antiresorptive agents have been introduced for treatment of osteoporosis, no systemically-administered drug has been developed to accelerate the fracture healing process. To address this need, we have undertaken to target a bone anabolic agent selectively to fracture surfaces in order to concentrate the drug’s healing power directly on the fracture site. We report here that conjugation of dasatinib to a bone fracture-homing oligopeptide via a releasable linker reduces fractured femur healing times in mice by ~60% without causing overt off-target toxicity or remodeling of nontraumatized bones. Thus, achievement of healthy bone density, normal bone volume, and healthy bone mechanical properties at the fracture site is realized after only 3-4 weeks in dasatinib-targeted mice, but requires ~8 weeks in PBS-treated controls. Moreover, optimizations have been implemented to the dosing regimen and releasing mechanisms of this targeted-dasatinib therapy, which has enabled us to cut the total doses by half, reduce the risk of premature release in circulation, and still improve upon the therapeutic efficacy. These efforts might reduce the burden associated with frequent doses on patients with broken bones and lower potential toxicity brought by drug degradation in the blood stream. In addition to dasatinib, a few other small molecules have also been targeted to fracture surfaces and identified as prospective therapeutic agents for the acceleration of fracture repair. In conclusion, in this dissertation, we have successfully targeted dasatinib to bone fracture surfaces, which can significantly accelerate the healing process at dasatinib concentrations that are known to be safe in oncological applications. A modular synthetic method has also been developed to allow for easy conversion of a bone-anabolic warhead into a fracture-targeted version for improved fracture repair.

The maternal liver exhibits robust adaptations to pregnancy to accommodate the metabolic needs of developing and growing placenta and fetus by largely unknown mechanisms. We found that achaete-scute homolog 1 (Ascl1), a basic helix-loop-helix transcription factor essential for neuronal development, is highly activated in maternal hepatocytes during the second half of gestation in mice. Our aim is to investigate whether and how Ascl1 plays a pregnancy-dependent role. We deleted the Ascl1 gene in the maternal liver using three independent mouse models from mid-gestation until term and identified multiple Ascl1-dependent phenotypes. When Ascl1 was deficient in maternal hepatocytes, maternal livers exhibited aberrant hepatocyte histology, fat accumulation, increased hepatocyte cell cycle, and enlarged size, accompanied by reduced albumin production and elevated levels of free fatty acids, ALT, and AST in the maternal blood, indicating maternal liver dysfunction. In the same situation, maternal spleen and pancreas displayed marked enlargement without an overt structural change; the placenta exhibited striking overgrowth with increased ALP production; and the cecal microbiome showed alterations in the relative abundance of several bacterial subpopulations. Moreover, litters born from maternal hepatic Ascl1 null mutated dam experienced abnormal postnatal growth after weaning. RNA-seq analysis revealed Ascl1-regulated genes in the maternal liver associated with Ascl1-dependent phenotypes. Of particular interest, we found that, in maternal hepatocytes, Ascl1 loss-of-function caused the activation of paternally imprinted gene insulin-like growth factor 2 (Igf2) encoding a major placental and fetal growth factor. IGF2 is also a known mitogen for hepatocytes and several hematopoietic lineages. Thus, IGF2 is a potential inducer of Ascl1-dependent phenotypes including placental overgrowth and maternal organ enlargement. Our studies revealed Ascl1 as a novel regulator of maternal liver physiology during pregnancy. Ascl1 activation in maternal hepatocytes is essential for normal placental growth and appropriate maternal organ adaptations, ensuring the health of both the mother and the fetus.

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is an FDA-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period to enzalutamide, tumors will develop drug resistance. In these studies, we uncovered that NOTCH signaling and DNA methylation are a deregulated in enzalutamide-resistant cells. NOTCH2 and c-MYC gene expression positively correlated with AR expression in samples from patients with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide-resistance. The expression of Notch signaling components was upregulated in enzalutamide-resistant cells suggesting the activation of the pathway. Inhibition of this pathway in vitro and in vivo promoted an increase in the sensitivity to enzalutamide with an impact on AR expression. On the other hand, DNMT activity and DNMT3B expression were upregulated in resistant lines. Enzalutamide induced the expression of DNMT3A and DNMT3B in prostate cancer cells with a potential role for p53 and pRB in this process. The overexpression of DNMT3B3, a DNMT3B variant, promoted an enzalutamide-resistant phenotype in C4-2 cells. DNA methylation inhibition, using low-concentration decitabine, and DNMT3B knockdown induced a re-sensitization of resistant prostate cancer cells and tumors to enzalutamide. Decitabine treatment in enzalutamide-resistant induced a decrease in the expression of AR-V7 and changes in genes from the apoptosis, DNA repair and mRNA splicing pathways. Decitabine plus enzalutamide treatment of 22RV1 xenografts induced a decrease in tumor weight, KI-67 and AR-V7 expression and an increase in Cleaved-Caspase3 levels. All the above suggest that Notch signaling and DNA methylation pathways are deregulated after enzalutamide resistance onset, and targeting these pathways restores the sensitivity to enzalutamide.