Hoertz, Amanda J.
<p>The natural progression from the introduction of an antibiotic into the market to the emergence of resistant strains demands the constant influx of new drugs to treat infection. Ramoplanin A2, a drug with demonstrated resistance against antibiotic-resistant Gram-positive pathogens, is currently in clinical trials. Ramoplanin is composed of seventeen amino acids condensed with a N-acylated fatty acid tail and a sugar moiety. The biosynthesis of this important metabolite is performed by a host of enzymes including non-ribosomal peptides synthetases, fatty acid biosynthetic enzymes, glycosyltransferases, and other tailoring enzymes. </p><p> In the introductory chapter, the significance and mechanism of the family of ramoplanin antibiotics will be discussed. The biosynthetic cluster of the lipoglycodepsipeptide ramoplanin and its sister antibiotic enduracidin will be outlined and the previous work on this cluster summarized. The proposed formation of the critical N-acylated tail and the parallels to fatty acid biosynthesis will also be described in detail. Chapter two describes the development of an expression system to successfully isolate proteins of interest from the ramoplanin biosynthetic cluster. In addition to increasing the yields of proteins already expressing in heterologous hosts, this technique facilitated the isolation of previously unattainable proteins. This chapter also details the demonstration of their activity through a variety of biochemical techniques. </p><p> Chapter three details the kinetic characterization of Ramo16, a NAD-dependent β-ketoacyl reductase, critical for the formation of the N-acylated fatty acid chain attached to Ramoplanin. Sequence analysis and characterization of the product of the Ramo16 reaction also implies that the biosynthesis of the N-acylated tail of ramoplanin bears significant similarity to type II bacterial fatty acid biosynthesis. These observations have led to a new proposal for the biosynthesis of the N-acylated fatty acid attached to ramoplanin. Further supporting these conclusions, chapter four details the examination of the stereochemistry of the hydrogen removed from the nicotinamide adenine dinucleotide cofactor and the stereochemistry of the product of the enzymatic reaction.</p><p> Chapter five describes the 1.4 Å crystal structure of ramoplanin in the presence of CTAB, an amphipathic membrane phospholipid mimetic. Observation of the location of the N-acylated fatty acid allows interpretation of its critical role in the activity of ramoplanin. The formation of the dimer and the interactions between the two molecules of ramoplanin and ramoplanin and the surroundings were examined in detail and supported earlier structure activity relationships. These observations have led to a new model for ramoplanin and Lipid II recognition in the context of the bacterial membrane. Enduracidin, ramoplanin's sister antibiotic, is produced by S. fungicidicus. Development of fermentation conditions for this strain allowed the isolation of appreciable amounts of enduracidin and 15N-labeled enduracidin, which was used to examine enduracidin's binding to Park's nucleotide, a cell wall precursor to Lipid II, by 2D 1H- 15N HSQC NMR. Although chemical shifts in the HSQC spectrum indicated binding, insoluble fibril formation complicated interpretation and necessitated the use of crystallography to determine the interaction with the substrate. Initial crystal conditions have been determined and diffraction images have been collected at 3.0 Å. Higher resolution images are pending to determine this structure.</p><p> Finally, chapter six discusses the development of a technique to transform S. fungicidicus and introduce plasmids to obtain deletion and insertional disruption mutants. A high throughput PCR screen was determined to enable large batch screening of potential mutants. This technique may be applied to study the function of genes in the pathway by fermenting production of enduracidin and examining the results.</p> / Dissertation
Investigating Alternative Splicing and Polyadenylation of the Interleukin 7 Receptor Exon 6: Implications for Multiple SclerosisEvsyukova, Irina January 2012 (has links)
<p>Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage and its expression is crucial for development and maintenance of T cells. While transcriptional regulation of IL7R expression has been widely studied, its posttranscriptional regulation has only recently been uncovered. Alternative splicing of IL7R exon 6, the only exon that encodes the transmembrane domain of the receptor, results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms, respectively. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis, a prototypic demyelinating disease of the central nervous system. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing.</p><p>We utilized mutagenesis of exon 6 and surrounding introns to identify multiple exonic and intronic cis-acting regulatory elements that impact inclusion of exon 6. At least two of these elements, one exonic splicing silencer and one exonic splicing enhancer, are located in the direct vicinity of the MS-associated SNP. We also uncovered a consensus polyadenylation signal, AAUAAA in intron 6 of IL7R, 16 nucleotides downstream from exon 6 5' splice site, and showed that mutations to this site resulted in an increase in exon 6 inclusion. Additionally, we determined that the 5' splice site of exon 6 is weak. We propose that this site may be responsible for exon 6 splicing regulation.</p><p>Using tobramycin RNA affinity chromatography followed by mass spectrometry, we identified trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein binding candidates. siRNA-mediated knockdown of CPSF1 resulted in an increase in exon 6 inclusion, consistent with the results of mutations to the CPSF1 binding site. Correspondingly, CPSF1 depletion had no effect on a minigene with a mutation in the intronic polyadenylation site. Finally, 3'RACE and RT-PCR experiments on RNA from Jurkat cells suggested that the intronic AAUAAA site is utilized at low frequency by the polyadenylation machinery to produce a novel polyadenylated mRNA isoform. Together, our results suggest that competing pre-mRNA splicing and polyadenylation may regulate exon 6 inclusion and resultant levels of functional IL7R produced. Since the intronic polyadenylated isoform of IL7R is predicted to be translated into a membrane-bound protein product with a shortened, signal transduction-incompetent cytoplasmic tail, this may be relevant for both T cell biology and development of multiple sclerosis.</p> / Dissertation
Diaz Vazquez, Arnaldo Joel
15 May 2009
This dissertation focuses on the development of biological platforms on which the function and characterization of transmembrane proteins can be performed simultaneously utilizing a biomembrane mimic consisting of a solid supported phospholipid bilayer (SLB). The study centered on the platform development, biophysical measurements of transmembrane proteins and membrane species chromatography. Membrane proteins play an essential role in various cellular and physiological processes. Their normal functions are essential to our health, and many impaired proteins have been related to serious diseases. Gaining a better understanding of membrane proteins is an essential step towards the development of more specific and competent drugs. This research study is divided into two main parts. The first part centered on the creation of a new platform for allowing transmembrane proteins to freely move inside supported lipid bilayers with the same mobility that can be found in vesicle systems. SLBs have been extensively used as model systems to study cell membrane processes because they maintain the same two-dimensional fluidity of lipids within the membrane found in live cells. However, one of the most significant limitations of this platform is its inability to incorporate mobile transmembrane species. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the protein, but also passivate the underlying substrate to reduce non-physiological protein-substrate interactions. High diffusion constants and high mobile fractions were obtained for a transmembrane protein reconstituted within this double cushion system. The second area of this study focuses on the creation of a new method to rapidly separate membrane components using electrophoresis in SLBs. This work showed that even subtly different chemical isomers can be well-separated by a simple electrophoretic technique when cholesterol is present in the separation matrix. As a first step towards the purification of proteins, this work showed that streptavidin proteins doubly bound to a bilayer by a biotinylated lipid can be separated from streptavidin proteins which are singly bounded.
Winsor, Tiffany Sabin
<p>Since RNA Polymerase II (RNAPII) transcribes much of the genome, it is well situated to encounter and initiate a response to various types of DNA damage. However, to date very little is known about any response of RNAPII to DNA damage outside of Transcription Coupled Nucleotide Excision Repair (TC-NER). A link between DNA damage response mechanisms and the C-terminal domain of RNAPII (CTD) is suggested by an overlap between proteins that bind the CTD and genes required for resistance to DNA damaging agents. In this thesis, I show that proper deployment of CTD associated proteins is required to respond to DNA damaging agents. Furthermore, I show that a CTD associated protein (Set2) is required for response to DNA damage, but its catalytic activity is not. Finally, I show that the recombinational ability of strains lacking the CTD kinase, Ctk1, is deficient. Based on these lines of evidence, I propose a novel CTD Associated DNA Damage Response (CAR) system of proteins that is required for proper response to DNA damaging agents.</p> / Dissertation
Structural and Cholesterol Binding Properties of the Amyloid Precursor Protein C-Terminal Fragment C99 and the Etiology of Alzheimer's DiseaseBarrett, Paul John 23 September 2013 (has links)
Biochemistry Structural and Cholesterol Binding Properties of the Amyloid Precursor Protein C-Terminal Fragment C99 and the Etiology of Alzheimers Disease Paul John Barrett Dissertation under the direction of Professor Charles Sanders Alzheimers Disease (AD) is a severe form of dementia that currently affects nearly 40 million people worldwide, a number that is estimated to increase by the year 2050 to nearly 120 million. The production and oligomerization of the amyloid beta polypeptides (Aβ) is widely thought to play a central role in AD onset and progression. Aβ oligomers are neurotoxic, such that factors that increase Aβ production and propensity to oligomerize or that reduce its degradation and transport out of the brain are viewed as pro-AD risk factors. Recently, it has been established that cholesterol may be a pro-AD risk factor, but this mechanism is still highly controversial. Aβ polypeptides are derived from two step proteolytic processing of the amyloid precursor protein (APP) by the enzymes β- and γ-secretase. This thesis will investigate both how the structure of C99 and the ability of C99 to bind cholesterol regulate and promote these cleavage events. First, this thesis will focus on the three dimensional structure determination of C99 using NMR spectroscopy. Prior to this work, much had been inferred about how the structure of C99 regulated the cleavage events of the amyloidogenic pathway, but without an actual structure, this work was incomplete. I demonstrate that C99 contains a membrane embedded helical turn N-terminal of the transmembrane helix, and that the transmembrane helix is both highly curved and flexible in nature. We hypothesize from this work that the curved structure of the transmembrane helix allows for proper insertion in the γ-secretase complex, and may be a potential target for modulating Aβ production. Secondly, this thesis will focus on the mechanism that makes cholesterol a pro-AD risk factor. We have shown that not only can C99 specifically bind cholesterol, but that this binding event partitions C99 to cholesterol rich membrane domains. It is known that both β- and γ-secretase reside in these cholesterol rich membranes. These findings show that cholesterol binding by APP may be the first step in promoting Aβ formation during AD.
GENETIC AND BIOCHEMICAL ANALYSIS OF THE INTERACTION BETWEEN THE YEAST FATTY ACID SYNTHESIS ENZYME YBR159W AND THE TRANSLATION INITIATION COMPLEX eIF2BBrowne, Christopher Michael 04 June 2013 (has links)
This dissertation focuses on the biochemical and genetic characterization of the protein-protein interaction in the budding yeast Saccharomyces cerevisiae between the cytosolic translation initiation guanine nucleotide exchange factor eIF2B and the endoplasmic reticulum (ER) membrane-embedded very-long-chain fatty acid (VLCFA) synthesis beta-keto-reductase enzyme YBR159W (IFA38). The dissertation is divided between the physical characterization of the interaction and examination of the functional consequences the ybr159wΔ deletion has on the yeast cells physiology. I first look at how the interaction is occurring in yeast. I utilize yeast 2-hybrid analysis to show that eIF2B subunits GCD6 and GCD7 interact with YBR159W. My experiments show that eIF2B does not interact with other VLCFA synthesis enzymes and that YBR159W does not interact directly with the other canonical components of the eIF2B complex. Compared to a wild type strain, a ybr159wΔ null yeast strain has a reduced growth rate and the hallmarks of a reduced translation activity including reduced 35S-methionine incorporation and low levels of polyribosomes. It is unknown if the reduced translation rate is a direct or indirect consequence of the ybr159wΔ mutation. The total cellular abundance of eIF2B complex is reduced in a ybr159wΔ null strain but the stoichiometry of the eIF2B complex and its enzymatic activity appears equivalent to wild-type. Deletion of YBR159W or other VLCFA synthesis enzymes significantly alters sphingolipid production in yeast. Deletion of the eIF2B subunit GCN3 does not cause a significant change in sphingolipid production in yeast. In the second section, I examine what effect YBR159W has on the localization of the cytoplasmic eIF2B complex. In yeast, eIF2B forms one or two large foci known as eIF2B bodies. I discover that YBR159W is important for either the formation or maintenance of the eIF2B body. In ybr159wΔ null yeast, eIF2B forms many smaller foci throughout the cell. Other VLCFA synthesis enzyme mutants display this same phenotype. I also find that a fraction of the eIF2B complex associates with lipid membranes. This lipid association is not dependent on the presence of YBR159W and is not mediated by rough ER bound ribosomes. Further experiments are required to determine the mechanistic and functional role of YBR159W interacting with eIF2B.
Pappius, Hanna Maria (Kwiecinska).
An extensive investigation of blood preservation, plasma 'substitutes' and the survival of blood cells after transfusion has been carried out in the Department of Biochemistry, McGill University, since the first months of World War II, under the direction of Dr. O.F. Denstedt and with the financial support of the National Research Council of Canada and, more recently, of the Defence Research Board of Canada.
Kochen, Joseph A.
Cancer has developed into one of the most complex of biological problems and research into its causes, development, manifestations and control is the focal point of a large number of scientific disciplines. It is unlikely that any investigator can completely confine his work or interest within the boundaries of only one of its component disciplines. Cowdry, in a discussion on modern trends in cancer research, has remarked "it is said that in cancer the most productive research is between the sciences, as in the cell itself very fundamental processes take place at the surfaces between different materials". [...]
Factors affecting the anaerobic glycolysis of brain tissue and the effects of sodium and potassium on brain metabolism.Rosenfeld, Michael. W. January 1956 (has links)
The experiments to be presented consist of an attempt to extend the observations of Dickens and Greville on the effects of anoxia and lack of substrate on the subsequent carbohydrate metabolism of brain tissue. Dickens and Greville (1933) working with brain slices, and Elliott and Henry (1946) with suspensions, showed that deprivation of both oxygen and substrate for relatively short periods caused a marked inhibition of the subsequent anaerobic glycolysis, while respiration was inhibited to a lesser degree.
Townsend, Edith. E.
The work to be presented in this thesis concerns the divergent pathways of tryptophan metabolism to the 5-hydroxyindoles and to kynurenine. More specifically an attempt has been made to elucidate the role of dietary tryptophan in the operation of these two pathways. Previous investigators have implied that thiamine is involved in the kynurenine-forming pathway. Both nutritional studies and experiments in vitro were carried out to determine just where thiamine (or thiamine pyrophosphate, its coenzyme form) exerts its action.
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