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Pireddu, Roberta. "New anticancer drugs : targeting tubulin and signal transduction pathways." Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/54618/.
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The main aim of the study described in this thesis is the development of new anticancer agents. The first chapter is a general introduction to cancer, and the development of chemotherapy anticancer agents during the course of the years. The following four chapters briefly introduce the biological targets in the authors study. Chapter Two describes a general introduction to tubulin and microtubules as anticancer targets. A discussion of those compounds most relevant to this thesis is provided. Chapter Three describes Signal Transducers and Activator of Transcription 3 (STAT3) proteins, their role in cancer and the advances in the search of anticancer agent inhibitors of the STAT3 signalling pathway. Chapter Four focuses on the src homology 2 (SH2) domain containing tyrosine phosphatases SHP-2, a protein-tyrosine phosphatase implicated in pathogenesis of cancer and other human diseases. A brief discussion of the SHP-2 inhibitors is provided. Chapter Five describes the role of proteins Aurora kinases in cancer, promising targets for anticancer drug development, and the advances in the search of their inhibitors targeting the kinase activity at the ATP binding site. The following chapters (6-11) describe the authors own findings. Chapter six focuses on the design and synthesis and biological evaluation of novel styrylchromones, styrylquinazolones, and quinazolones as inhibitors of tubulin polymerization. Styrylchromones Styrylquinazolones Quinazolones Two series of isomeric styrylchromones were initially synthesized in order to establish the methoxy substitution pattern on the A ring favorable for optimal activity. The structure activity relationship on the B ring is also reported. Next, our strategy focused on identifying a chromone core replacement with improved potency. We directed our chemical efforts toward the synthesis of novel styrylquinazoline analogs. The quinazoline core would also provide easy access to the preparation of diverse sets of N-substituted derivatives (methyl and ethyl derivatives). Finally, a novel series of quinazolines were synthesized as conformationally-restricted analogs of chalcones. SAR was conducted around the quinazoline spacer between the aryl rings and systematically investigating the substituent effect in the B ring. Among the synthesized compounds we selected those analogues showing significant cytotoxicity (generally defined as IC50 value < 1.5 uM), and evaluated for activity in vitro tubulin polymerization inhibition assay. Chapter Seven focused on the identification of novel inhibitors of STAT3 dimerization. Computational analyses led us to the development of a T-shape model of molecules that can >2 occupy the pTyr-binding pocket of STAT3 SH2 domain. The rN"s' conjugate addition of nitromethane to a series of amides and the l/ J reduction of the nitro group were combined to give an easy route to onh j. the target T-shape molecules in a combinatorial fashion. The ksJJ methodology was also extended to amides activated by a nitro group. co2r observed a dramatic change in the course of the reaction, which Scaffold of T-shape molecules afforded a mixture of unexpected and unknown products, that each possessed an additional methylene group. A brief study into the mechanism was also conducted. Chapter Eight, Nine and Ten focuss on the development of Aurora kinases and SHP-2 inhibitors. Oxindole derivatives HL10581 and NSC117199 emerged as lead compounds from a high throughput screen for Aurora-A and SHP-2, respectively. f f Chapter Eight describes the synthesis of n-nfh Cl nnhN 2 several derivatives of HL10581 and H03SYYLo H03SYr>o NSC117199, directed to exploration of h h SAR around the oxindole moiety to HLiow.AAKfci-SMM nscii7,99,shp-2,,c5047 mM determine the structural features that are responsible for the activity. Chapters nine and ten report the biological evaluation of oxindole derivatives as inhibitors SHP-2 and Aurora kinases, respectively.
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Ramamoorthy, Divya. "Synthesis of small molecule inhibitors targeting signal transduction pathways." Scholar Commons, 2009. http://scholarcommons.usf.edu/etd/2160.
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The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transduction pathways, thereby treating cancer. We attempted to synthesize compounds based on the hits obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, history of chemotherapeutic drugs and an introduction to signal transduction pathways. The following two chapters briefly introduce the biological targets in the authors study. Chapter Two describes the role of B-cell lymphoma type xL (Bcl-xL), in apoptosis and the development of drugs targeting Bcl-xL. Examples of Bcl-xL drugs relevant to this study have been provided.
Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathways and the recent developments in drug development towards the inhibition of this oncogene. Chapter Four gives a general introduction to microwave-assisted organic synthesis and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author's own findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibitors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL. Our strategy focused on identifying an isoquinolinol lead with increased potency.
Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency.
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Bull, Fiona A. "Targeting opioid receptor signal transduction to produce sustained analgesia." Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/3e42e620-7115-4f45-9100-91c422fce812.
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Mu opioid receptors (MOPs) in the pain pathway contribute to morphine analgesia. Morphine also stimulates reward/reinforcement through disinhibition of dopaminergic (DA) neurones in the ventral tegmental area (VTA), an effect implicated in its abuse and dependence. We hope to develop approaches to achieve sustained analgesia without affecting reward by exploiting differential MOP signalling mechanisms in the pain and reward pathways. MOPs, delta opioid receptors (DOPs) and β-arrestin2 (BAR2) are all necessary components of the signalling complex in nociceptive neurones for morphine analgesic tolerance; c-Src (a tyrosine kinase), thought to couple to MOP receptors through BAR2 has also been implicated. To investigate opioid receptor signalling in response to morphine we used a variety of different techniques that included behavioural measures of nociception, reinforcement and locomotion and electrophysiological methods to study DRG neurones from the pain pathway and brain slices containing VTA neurones. This study in mice confirms that morphine administered subcutaneously (SC) causes analgesia, analgesic tolerance, and has psychomotor effects leading to enhanced locomotion and reinforcement. In VTA neurones morphine and the selective MOP receptor agonist DAMGO caused concentration-dependent inhibition of the frequency of IPSCs. All these actions of morphine were absent from MOP-/- mice. Morphine exhibited reduced potency as 1) an analgesic, 2) stimulator of locomotion, 3) a reinforcer in CPP and 4) an inhibitor of sIPSC frequency, when applied to MOP+/- mice or their VTA neurones. Morphine analgesic tolerance developed faster and to a greater extent in MOP+/- mice than in WT mice. DOP-/- mice exhibited morphine analgesia with less tolerance, as did BAR2-/- mice. BAR2-/- mice also exhibited reduced morphine locomotion and an increased sensitivity to morphine reinforcement. Morphine tolerance was absent from BAR2-/-//DOP-/- mice. The inhibition of sIPSC frequency by morphine was reduced in BAR2+/- and BAR2-/- VTA neurones. Dasatinib and PP2 (c-Src tyrosine kinase inhibitors) prevented the development of morphine tolerance in WT and MOP+/- mice and dasatinib caused its reversal in the latter. The drugs had no significant analgesic effect alone. Dasatinib did not affect morphine preference or locomotor activation. PP2 reduced morphine’s inhibition of sIPSC frequency. As c-Src inhibition does not appear to alter the psychomotor effects produced by morphine and it acts to reduce morphine analgesic tolerance. We believe that cSrc is an attractive target to prevent the development of morphine analgesic tolerance without affecting hedonic homeostasis.
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Faoro, Camilla. "The Signal Recognition Particle: noncanonical functions and drug discovery." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/20168.
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The Signal Recognition Particle (SRP) is an essential ribonucleoprotein complex responsible for co-translational delivery of membrane and secretory proteins to the plasma membrane in prokaryotes and to the endoplasmic reticulum in eukaryotes. In Eubacteria, SRP consists of the GTPase Ffh and the small 4.5S RNA; in eukaryotes, the system is more complex and SRP comprises six proteins (the heterodimer SRP 9/14, SRP 19, SRP 54, and the heterodimer SRP 68/72) along with a large RNA moiety, the 7SL RNA. SRP has been reported to be involved in many cellular processes outside the SRP-targeting cycle. Two layers of the SRP interactome, the SRP proteome and SRP transcriptome, were analysed by LC-MS/MS and RIP-seq, respectively. The majority of identified RNA and protein targets of the SRP subunits have nucleic-acid, chromatin and protein-binding functions and are involved in ribonucleoprotein particles (RNPs) formation, RNA processing and protein transport. Confocal microscopy studies showed that SRP subunits localizes in a dynamic manner during the cell cycle, indicating a spatial-temporal regulation of SRP-binding partners. Truncations or mutations on any of the bacterial components of the SRP system have proven to be either lethal or severely impact cell viability. Here, we propose the bacterial SRP and its interactions with the cognate SRP receptor, FtsY, as an ideal target for the development of novel antibiotics. Using a Fragment-Based Drug Design approach we have identified three fragments from a commercial library that bind to FtsY. We have crystallized FtsY in an Apo form and soaked GTP analogues as well as the three fragments and analogues and determined their X-ray crystal structures at resolution ranging from 1.221.9 Å. Despite the low affinity of the compounds, we were able to identify unambiguously their binding site and show that soaking of fragments, even at low affinity, is possible to aid in our drug discovery project.
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Lombardi, Zoe. "Optimization of Extracellular signal-regulated kinase 5 (ERK5) targeting in melanoma." Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1227476.
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Melanoma is the deadliest skin cancer with a very poor prognosis in advanced stages. Although targeted and immune therapies have improved survival, not all patients benefit from these treatments. It has been reported that the mitogen-activated protein kinase ERK5 is consistently express in human melanoma and supports melanoma cell proliferation in vitro and in vivo. However, ERK5 inhibition results in cell-cycle arrest rather than appreciable apoptosis. To clarify the role of ERK5 in melanoma growth, we performed transcriptomic analyses following ERK5 knockdown in melanoma cells expressing BRAFV600E and found that cellular senescence was among the most affected processes. In melanoma cells expressing either wild-type or mutant (V600E) BRAF, both genetic and pharmacologic inhibition of ERK5 elicited cellular senescence, as observed by a marked increase in senescence-associated b-galactosidase activity and p21 expression. In addition, depletion of ERK5 from melanoma cells resulted in increased levels of CXCL1, CXCL8, and CCL20, proteins typically involved in the senescence-associated secretory phenotype. Knockdown of p21 suppressed the induction of cellular senescence by ERK5 blockade, pointing to p21 as a key mediator of this process. In vivo, ERK5 knockdown or inhibition with XMD8-92 in melanoma xenografts promoted cellular senescence. Based on these results, small-molecule compounds targeting ERK5 constitute a rational series of prosenescence drugs that may be exploited for melanoma treatment. ERK5 pro-proliferative activities are linked to its presence in the nucleus, but the mechanisms involved in ERK5 nuclear translocation are poorly characterized. We focused on the elucidation of this process to found novel targets and compounds able to prevent ERK5 nuclear shuttling, in order to design new strategies for cancer treatment. To achieve single ERK5 tracking in living cells, we used a Super-Resolution microscope. HeLa cells have been transfected with an expression vector for ERK5, linked to HaloTag, alone or with a vector for a constitutively active form of the ERK5 activator MEK5 (MEK5DD). The cell-permeable photoactivatable chromophore JaneliaFluor646, able to recognise Halo Tag, has been used as detection technique for super resolution imaging. As a complementary approach, HeLa cells, transfected with ERK5 and MEK5DD, have been treated with the alpha/beta importin-mediated transport inhibitor Ivermectin (IVM). MTT and 2D-colony forming assays were performed in A375 cells treated with IVM in combination with the ERK5-i AX-15836. The HaloTag technology provides the JaneliaFluor646 selective binding to ERK5 and Highly Inclined and Laminated Optical sheet (HILO) microscopy allow us to collect the signal of individual chromophores. Data showed that in ERK5-transfected cells, the protein is mainly localized in the cytoplasm, whereas it moves in the nucleus with the activator MEK5DD and this effect is partially reverted in cells treated with IVM. Moreover, ERK5 amount in the nuclear fraction of lysates from IVM treated-cells is lower compared to control, suggesting a role of alpha/beta importins in ERK5 nuclear transport. Finally, we found that ERK5i AX-15836, which has been reported to induce ERK5 nuclear translocation in a paradoxical way, reduced melanoma cell proliferation only in combination with IVM. The HaloTag-JaneliaFluor646 method has proven effective for the localization and tracking of single ERK5 molecules with nanometre accuracy, thus providing a novel approach to evaluate how ERK5 moves to the nucleus. The described technique will also help future studies to investigate the mechanism of action of ERK5 in the nucleus. The actors involved in these processes could be identify as novel targets for ERK5 inhibition, and therefore for a possible anticancer therapy.
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Mitsopoulos, Konstantinos. "The assembly of type III membrane proteins in Escherichia coli." Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310262.
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Skoulding, Nicola Stephanie. "Determining the specificity of peroxisomal targeting signal 1 variants in Arabidopsis thaliana." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549763.
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Peroxisomes are ubiquitous organelles found in most eukaryotic organisms and are involved in ~-oxidation of fatty acids and the degradation of hydrogen peroxide. They contain no DNA thus all proteins required fo: their functions are identified and imported via a peroxisomal targeting signal (PTS). PTS1, a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO·, is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the thermodynamics of PTS1 binding specificity, a fluorescence- based binding assay that enables the quantification of inhibition constants for PTS1- containing peptide complexes with the full length and TPR region of Arabidopsis thaliana PEX5 was used. A library of PTS1 penta-peptides based on the reference sequence YQSKL and deca-peptides based on the reference sequence, VAKTIRPSRV, were synthesised, tested and found to have a Kj between 160 nM to> 100000 nM with only a small difference observed in affinity between the truncated and full length A. thaliana proteins. Circular dichroism studies showed that the TPR domain of AtPEX5 is 60% alpha helical and that this is the only region of the full length AtPEX5 protein that contains secondary structure, independent of the presence of a PTS1. Binding of the PTS1 by the TPR domain was diminished at pH < 6 however secondary structure was still maintained at pH 6 but lost at pH 5. Finally surface plasmon resonance assays using the immobilised peptides YQSKL, VAKTIRPSRL, VAKTIRQSRL and VAKTIRPRSV showed that the binding response of truncated and full length AtPEX5 to the PTS1 is doubly sigmodial in nature and that full length AtPEX5 has a significantly slower kOffthan the TPR domain alone. Overall the results show that PTS1 sequences which target in vivo may not have affinity for AtPEX5 in vitro, showing a difference between the two techniques. Additionally these results show that the mechanism of binding between AtPEX5 and the PTS1 may not simply be 1:1 but involve the formation of higher order multimeric of complexes of AtPEX5-PTS1.
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Gatto, Gregory Joseph. "Peroxisomal targeting signal-1 recognition by the TPR domains of human Pex5p." Available to US Hopkins community, 2003. http://wwwlib.umi.com/dissertations/dlnow/3080661.
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Almarzouki, Amina. "Approaches to incorporate a nuclear targeting signal into non-viral gene delivery systems." Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421483.
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Jiang, Xiaohua, and 蔣曉華. "Targeting cell signaling pathway in treatment of gastric cancer by chemotherapeutic agents." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31244282.
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The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2001-2003<br>published_or_final_version<br>Medicine<br>Doctoral<br>Doctor of Philosophy
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Halic, Mario. "Structures of protein targeting complexes." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2006. http://dx.doi.org/10.18452/15462.
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Sowohl die kotranslationale Translokation von sekretorischen Proteinen durch die Membran als auch die Insertion von Membranproteinen sind essentielle Prozesse in allen lebenden Zellen. Sie erfordern die Sortierung des translatierenden Ribosoms zur Membran mittels des Signalerkenungspartikels (SRP), eines im Verlauf der Evolution konservierten Ribonukleoprotein-Partikels. SRP erkennt die Signalsequenz einer wachsenden Proteinkette, sobald diese aus dem Ribosom hervortritt. Die Bindung von SRP führt zum Anhalten der Peptidelongation (Elongationsarrest) und zum Andocken an den membrangebundenen SRP-Rezeptor (SR). In dieser Arbeit wird die 12 Å Kryo-Elektronenmikroskopie-Struktur eines Sortierungs-Komplexes dargestellt, der aus dem Säugetier-SRP gebunden an ein aktives Ribosom mit Signalsequenz besteht. Ein erstes molekulares Modell von SRP in dieser Konformation wurde erzeugt. Es zeigt wie die S-Domäne von SRP die große ribosomale Untereinheit nahe dem Peptidtunnel-Ausgang kontaktiert, um dort die Signalsequenz zu binden. Außerdem wird deutlich wie die Alu-Domäne von SRP in die Bindungsstelle für Elongationsfaktoren hineinreicht, wodurch die Elongationsarrest-Aktivität der Alu-Domäne erklärt wird. Auf dieser Basis konnte ein erstes Struktur-basiertes Modell der ersten Schritte der kotranslationalen Proteinsortierung entworfen werden. Darüberhinaus wurde auch der Schritt des Andockens an die Membran visualisiert, indem die Struktur des Ribosom-SRP-SR-Komplexes durch Kryo-EM gelöst wurde. Erste Schlüsse hinsichtlich des Mechanismus, der das Ribosom vom SRP zum Translokon transferiert, können hier gezogen werden. Als Nebenergebnis konnte durch die erreichte hohe Auflösung die Position des wichtigen ribosomalen Proteins L30e in der Kryo-EM-Struktur des Weizenkeim-Ribosoms idenifiziert werden.<br>Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires targeting of the translating ribosome to the membrane by the signal recognition particle (SRP), an evolutionary conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to pausing of peptide elongation and docking to the membrane-bound SRP receptor. Here, the 12 Å cryo-electron microscopy structure of a targeting complex is presented consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. A molecular model of SRP in this functional conformation was generated. The model reveals how the S-domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu-domain reaches into the elongation factor binding site of the ribosome explaining its elongation arrest activity. A molecular model of the first steps of protein targeting is presented. Moreover, also the docking step has been visualized by solving a cryo-EM structure of the ribosome-SRP complex bound to the SRP receptor. This structure provides first hints regarding the mechanism of ribosome transfer to the translocon. As a side result the position of the functionally significant ribosomal protein L30e has been identified in the high resolution maps of the wheat germ ribosome.
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Cross, Laura Louise. "Re-design of a receptor-targeting signal interaction to create a new peroxisomal trafficking pathway." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15198/.
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Peroxisomes are crucial for cell survival but do not possess a genome, so protein import is essential for correct functioning of the organelle. The PTS1 (peroxisomal targeting signal 1) pathway is the major, and in some organisms the only, transport pathway for proteins to enter the peroxisome. PTS1 is a recognition sequence at the C-terminus of peroxisomal cargo proteins, which allows their binding to the receptor protein PEX5 (peroxin 5). PEX5 then acts as the vehicle for transporting PTS1-cargo proteins into peroxisomes. This work concerned the disruption of the natural PEX5:PTS1 interaction and the generation of an interaction between a mutated form of PEX5 (PEX5*) and a peptide representing a non-natural PTS1 (PTS1*). An in vitro protein-peptide binding screen was developed to test Arabidopsis thaliana PEX5-C (AtPEX5-C), and variants, with a library of peptides to identify corresponding binding peptides. For wild-type AtPEX5-C, peptides identified were consistent with bioinformatics predictions for plant PTS1 sequences, confirming the validity of the screen. Fluorescence anisotropy was then used to validate binding peptides, revealing that two variants of AtPEX5-C, D505H and N601A, exhibited reduced affinity to a representative native PTS1 (YQSKL-CO2H), yet increased affinity to a non-PTS1 sequence (YQSYY-CO2H). When these two mutations were combined, the affinity for YQSKL was further reduced and the affinity for YQSYY was further enhanced. This effect was amplified when an additional mutation, D507T, was incorporated into the AtPEX5-C double variant. The resulting triple variant was termed PEX5*. Various 9-amino acid sequences were added upstream of YQSYY, and the optimal resulting 14-amino acid sequence was termed PTS1*. The in vitro-validated binding of PEX5* to PTS1* was tested in vivo (in the moss Physcomitrella patens; by R. Paudyal) to investigate whether expression of PEX5* would allow the import of a fluorescent protein with a C-terminal PTS1* tag into peroxisomes.
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Stanley, W. A. "Interaction of Pex5p, a peroxisomal import receptor protein, with the peroxisomal targeting signal type 1." Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413415.
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Schroder, Paul. "Targeting Signal 1 of T cell Activation to Restore Self Tolerance in Type 1 Diabetes." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1381086555.
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Konstantinou, Maria. "Characterising In-111-anti-γH2AX-TAT in targeting the DNA damage signal associated with Wnt activated colorectal cancer". Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/111847/.
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Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the UK and has a poor 60% 5-year survival rate. The Wnt signalling pathway is fundamental for homeostasis of the intestinal epithelium and its deregulation drives development of CRC and induces DNA damage. Histone-2AX (H2AX) is a component of the nucleosome whose phosphorylated form, γH2AX, is a marker of DNA damage. Objectives: Using a well-characterised inducible CRC mouse model of early Wnt deregulation, and established Apc-deficient driven tumour and ex vivo organoid models, we have assessed whether the spontaneous DNA damage generated in these models can be targeted using 111In-anti-γH2AX-TAT (RH2AX), a radio-labelled antibody targeting γH2AX. Methods: Deletion of the Apc gene was effected in the intestine of VilCreERApcfl/fl and Lgr5CreERApcfl/fl models by intraperitoneal or oral induction with tamoxifen. γH2AX immunohistochemical (IHC) characterisation of intestines were performed as well as γH2AX whole mount immunofluorescent analysis on organoids derived from them. RH2AX, an anti-γH2AX antibody conjugated to the cell-penetrating peptide TAT to allow cellular internalisation and nuclear localisation, was used in these models as an imaging agent SPECT/CT imaging and biodistribution studies were conducted after oral induction of VilCreERApcfl/fl and intravenous injection of RH2AX. γH2AX and Lgr5 FACS analysis were carried out on intestinal crypt cells of VilCreERApcfl/fl mice expressing Lgr5-EGFP reporter. Results: Intestinal Apc deficiency increased DNA damage levels in the small intestine of both dysplastic (VilCreERApcfl/fl) and tumour CRC mouse (Lgr5CreERApcfl/fl) models. Apc-deficiency-associated DNA damage is most likely generated through WNT signalling pathway activation and, more specifically, by c-Myc transcription. For the first time, we demonstrated that intestinal dysplasia can be identified through in vivo SPECT imaging, using low SA RH2AX treatment. Low SA RIC treatment in intestinal dysplasia increased the DNA damage levels in healthy and Apc-deficient small and large intestines, increased proliferation in the Apc-deficient tissue and resulted in variable levels of apoptosis depending on the tissue. Conclusion: These findings together indicate that DNA damage is induced by Apc-deficiency, and that there is the possibility to exploit the endogenously-increased DNA damage signal, γH2AX, to attract the RH2AX for in vivo imaging of intestinal dysplasia. This could help diagnose early stages of CRC to provide patients with the appropriate treatment sooner and increase their survival.
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Sathe, Anuja Verfasser], Jürgen E. [Akademischer Betreuer] [Gschwend, Dieter [Akademischer Betreuer] Saur, and Margitta [Akademischer Betreuer] Retz. "Therapeutic targeting of signal transduction pathways in bladder cancer / Anuja Sathe. Gutachter: Dieter Saur ; Margitta Retz. Betreuer: Jürgen E. Gschwend." München : Universitätsbibliothek der TU München, 2015. http://d-nb.info/1077063601/34.
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Deb, Arpan, William A. Johnson, Alexander P. Kline, et al. "Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide." PUBLIC LIBRARY SCIENCE, 2017. http://hdl.handle.net/10150/623017.
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Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV-1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/ function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
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Zu, Yi. "SIRT1 promotes cell proliferation and prevents cellular senescence through targeting LKB1 in primary porcine aortic endothelial cells." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085805.
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Zu, Yi, and 祖毅. "SIRT1 promotes cell proliferation and prevents cellular senescence through targeting LKB1 in primary porcine aortic endothelial cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085805.
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Roulston, Claire. "Occurrence & function of cellular 2A sequences." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7062.
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This thesis describes experiments investigating the translational recoding activities and the novel dual signalling properties of eukaryotic ribosome skipping 2A sequences. Over twenty years ago, the 19 amino acid 2A region of a Picornavirus; namely, Foot-and-Mouth Disease Virus (FMDV) polypeptide was shown to possess apparent “self-cleaving” abilities, cutting at its own C-terminus during translation (Ryan et al., 1991). Active FMDV 2A-like sequences were subsequently found in a number of related viruses (Luke et al., 2008), with several now utilised as essential biotechnology multi-gene transfer tools (Luke et al., 2010b). Then, in 2006, eukaryotic 2A-like sequences were identified from trypanosome non-LTR sequences. These were found to be functional in vitro (Heras et al., 2006). I have been able to identify over 400 putative eukaryotic 2A-like sequences through searching the freely available online proteomic and genomic databases. Data is presented to show that these 2As were encoded in frame with non-LTRs, or metabolic, or immune function genes, from a wide range of eukaryotic organisms; but I could not discern any obvious phylogenetic distribution for 2A. I have discovered that the majority of eukaryotic 2A sequences tested can mediate ribosome skipping in vitro. Modelling in silico indicated that active 2A-like sequences possessed the propensity to form a central alpha-helical region, whereas the models suggested that inactive 2A-like sequences would be essentially unstructured. I also report that some of these eukaryotic 2A peptides constitute a novel form of dual protein targeting as they play a dual role as exocytic pathway signal peptides mediating extracellular protein trafficking. I have shown that this protein trafficking ability is evolutionarily conserved, with an echinoderm sequence able to direct protein targeting in both plant and mammalian cells. I therefore propose that these novel eukaryotic 2A sequences could potentially become extremely valuable in biotechnological engineering.
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Phyu, Su Myat. "Targeting of the PI3K/AKT/mTOR signalling pathway and associated kinases in breast and colon cancer cells and response evaluation by molecular imaging techniques." Thesis, University of Aberdeen, 2018. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=238576.
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The phosphatidylinositol-3-kinase/AKT (Protein Kinase B)/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway, downstream of tyrosine kinase receptors, is upregulated in human cancers including breast and colon cancers. Glycogen synthase kinase 3 (GSK 3) is a serine/threonine protein kinase plays important role in various cellular processes including glycogen synthesis mediated by insulin signalling pathway. Moreover, 5' adenosine monophosphate activated protein kinase (AMPK), a crucial cellular energy sensor, has regulatory role in cell growth and proliferation through mTOR pathway. Phosphatidylcholine (PtdCho) is the major phospholipid in the mammalian cell membranes and is mainly synthesized by the CDP-choline pathway. Malignant transformation has been reported to be associated with altered choline metabolism. Hyperactivation of the PI3K/AKT signalling pathway upregulates the key enzymes of phospholipid metabolism. The first line antidiabetic drug, metformin, modulates glucose and concomitant lipid metabolism through AMPK activation. Studies suggest phosphatidylcholine biosynthesis and breakdown through CDP-choline pathway are modulated by glucose metabolism and de novo fatty acid synthesis. Cancer cell growth inhibitory effect of PI3K/AKT/mTOR/GSK3 pathway inhibitors and metformin were investigated by cytotoxic assay, western blot and cell cycle analysis in breast and colon cancer cells. IC50 values of anticancer drugs and combination indices between drug combinations were determined. 31P-NMR was carried out on cell extracts after drug treatments. [14C (U)] glucose and [3H] choline incorporation into lipids were also determined. All inhibitors targeting PI3K/AKT/mTOR signaling pathway, GSK3 and metformin have cancer cell growth inhibition. By 31P-NMR, PI3K/AKT/mTOR pathway inhibition induced agent-specific changes in PCho intensity. Increased UDP-sugars observed in breast and colon cancer cell extracts treated with LY294002 and AZD8055, an effect abrogated by inclusion of a GSK3 inhibitor. A link between glycolytic intermediates and phosphatidylcholine biosynthesis was investigated by metformin and GSK3 inhibitor in breast and colon cancer cells.
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Rondelet, Arnaud. "Caractérisation d'une nouvelle voie d'adressage des protéines à la membrane externe des bactéries à Gram négatif." Phd thesis, INSA de Lyon, 2012. http://tel.archives-ouvertes.fr/tel-00833222.
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Le système Tat (pour Twin Arginine Translocation) exporte des protéines repliées depuis le cytoplasme vers le périplasme des bactéries. L'adressage des protéines à exporter au système Tat repose sur une séquence signal spécifique amino terminale clivée après exportation. Chez le phytopathogène Dickeya dadantii, l'homologue de pectine lyase PnlH possède une séquence signal Tat qui assure son adressage au système Tat mais qui n'est pas clivée après exportation et ancre la protéine dans la membrane externe. Chez les protéobactéries, la majorité des protéines de membrane externe sont soit des lipoprotéines soit des protéines intégrales de membrane en tonneau β. L'adressage de ces protéines à la membrane externe repose sur des voies spécifiques du type de protéine : la voie Lol pour les lipoprotéines et la combinaison des chaperons périplasmiques SurA, Skp et DegP et du complexe de membrane externe Bam (β barrel assembly machinery) pour les protéines en tonneau β. Au cours de ce travail, l'étude de l'adressage de PnlH à la membrane externe a montré que SurA se liait à la séquence signal hydrophobe de PnlH pour la protéger de l'environnement hydrophile au cours de son transit dans le périplasme. La séquence signal de PnlH (41 acides aminés) porte l'intégralité de l'information nécessaire à son adressage à la membrane externe. La nature de l'information adressant les protéines au système Tat est bien connue et dans ce travail nous nous sommes efforcés d'identifier les informations requises pour les deux dernières étapes de l'adressage de PnlH à la membrane externe : la traversée du périplasme et l'insertion dans la membrane externe. La délétion d'une région conservée comprise entre les résidus 28 et 41 de la séquence signal de PnlH affecte l'adressage de cette dernière à la membrane externe. Des substitutions des acides aminés conservés de cette région ne semblent pas affecter l'adressage de PnlH, indiquant que l'information nécessaire à l'adressage de PnlH à la membrane externe après exportation ne réside pas dans la séquence en acides aminés de la séquence signal de PnlH. En revanche, nos données suggèrent que la présence d'une hélice α hydrophobe dans la séquence signal de PnlH est importante pour son adressage à la membrane externe. Cette observation est particulièrement intéressante puisqu'une telle structure est généralement considérée comme une caractéristique des protéines de membrane interne.
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Walker, Michelle Elisabeth. "Mitochondrial protein targeting signals." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/14627.
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Raine, Amanda. "Ribosome Associated Factors Recruited for Protein Export and Folding." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4839.
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Reyes, Resina Irene. "Heterómeros de receptores CB1, CB2, GPR55 y GPR18: Señalización celular, farmacología y análisis de su potencial como dianas terapéuticas de enfermedades neurodegenerativas." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/586258.
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Los receptores cannabinoides CB1R y CB2R son GPCRs que forman parte del sistema endocannabinoide y son considerados dianas potenciales para numerosas enfermedades. Su compleja farmacología junto con la naturaleza lipofílica de sus ligandos perjudica la traslacionalidad de la investigación centrada en el sistema endocannabinoide. Para comprender mejor la farmacología de CB2R se puso a punto una técnica homogénea y no radioactiva para estudiar la unión de ligando en célula viva, gracias a la cual se identificó un segundo centro de afinidad en CB2R, y cambios conformacionales en el receptor cuando éste se coexpresa con GPR55, un GPCR huérfano que une cannabinoides.
Los GPCRs pueden formar heterooligómeros, con propiedades farmacológicas diferentes a las de los receptores individuales. Dado que existen complejos CB1R-CB2R y CB2R-GPR55, se investigó si CB1R y GPR55 podían formar complejos heteroméricos. Se confirmó esta posibilidad en un sistema de expresión heterólogo y en muestras de sistema nervioso central, donde se detectó la huella del heterómero.
Se postula que en microglía la activación de los receptores de cannabinoides produce un efecto neuroprotector. El GPCR huérfano GPR18, que une cannabinoides y comparte con CB2R un papel regulador de la respuesta inmune, se expresa en microglía. Dado que se ha sugerido un posible cross-talk entre CB2R y GPR18, se estudió si GPR18 era capaz de interaccionar con CB1 y CB2. Se observó que GPR18 no es capaz de formar complejos heteroméricos con CB1R, pero si con CB2, tanto en un sistema heterólogo como en microglía, y en ambos casos se detectó la huella del heterómero. En microglía activada y en cultivos primarios de ratones modelo de la enfermedad de Alzheimer la expresión de CB2R-GPR18 se encuentra aumentada.
Dado el papel de CB2R en microglía, se estudió la presencia y la función de los complejos CB1R-CB2R en microglía en reposo y activada. La expresión aumentada de complejos CB1R-CB2R y su señalización en microglía activada indican que la función de los cannabinoides es la de mantener la microglía en reposo, pero desencadenar una señalización robusta cuando la microglía se activa. En cerebro de animales modelo de enfermedades neurodegenerativas (Parkinson y Alzheimer), donde se encontraron marcadores de microglía activada, se observaron resultados similares a los encontrados en una línea de microglía tratada con lipopolisácarido e interferón gamma. Ello sugiere que CB1R, CB2R y CB1R-CB2R en microglía activada tienen potencial como dianas en el tratamiento de enfermedades neurodegenerativas que cursan con neuroinflamación.
Los receptores CB2R y GPR55 tienen un relevante papel en mecanismos de respuesta a estrés, ansiedad y depresión. Considerando la estrecha relación entre las enfermedades neuropsiquiátricas y el suicidio, se evaluó si la expresión de los complejos CB2R-GPR55 está alterada en suicidas. Se detectó la presencia de heterómeros CB2-GPR55 en corteza prefrontal, y un aumento en la expresión de estos heterómeros en corteza prefrontal de muestras de suicidas.
Para investigar el potencial terapéutico de los fitocannabinoides es necesario conocer mejor su relación con CB1R y CB2R, ya que el mecanismo de acción del cannabidiol y el cannabigerol (dos importantes componentes no psicotrópicos de la planta del cannabis) sobre estos receptores es aún confuso. Se observó que a concentraciones nanomolares el cannabidiol es capaz de modular de manera negativa la tanto la afinidad de la unión de los agonistas a CB2R como la señalización mediada por este receptor, por lo que se postula que el cannabidiol es un modulador alostérico de CB2R. En cambio, cannabigerol parece unirse al centro ortostérico de CB1R y CB2R, sobre los que actúa como un agonista con selectividad funcional, y modula la señalización de los cannabinoides sobre los heterómeros CB1R-CB2R.<br>Cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors are GPCRs of the mammalian endocannabinoid system.Their complex pharmacology is delaying the translational success of medications targeting the endocannabinoid system. To better understand CB2R pharmacology, a novel homogeneous technique was developed to study ligand binding to CB2R in living cells, which disclosed a second affinity state of CB2R and conformational changes when CB2R interacts with another GPCR.
To know how cannabinoid signaling is affected by receptor-receptor interactions, we studied whether CB1R and CB2R interact with two orphan GPCRs, namely GPR55 and GPR18. CB1R-GPR55 and CB2R-GPR18 heteromers were detected both in transfected cells and in the central nervous system, and their fingerprint was also revealed. Given the neuroprotective role of CB2R activation to prevent neuroinflammation, the presence of CB1R-CB2R and CB2R-GPR18 heteromers was studied in both resting and activated microglia. The higher number of complexes and the differential signalling found both in activated microglia and in the brain of animal models of neurodegenerative diseases, indicate that CB1R-CB2R and CB2R-GPR18 heteromers have a role in neuroprotection. Other pathologies where cannabinoid receptors have shown an important role are anxiety and depression disorders, which are related to suicide. The presence of the previously described CB2R-GPR55 heteromer was detected in human prefrontal cortex, and a higher amount of heteroreceptor complexes was found in samples from suicide victims, indicating that CB2R-GPR55 heteromers could have a role in depression.
To further exploit the therapeutic potential of the cannabinoids, it is also important to understand how phytocannabinoids, such as cannabidiol and cannabigerol, two compounds found in the cannabis plant and which lack psychoactive effects, interact with CB1R and CB2R. Nanomolar concentrations of cannabidiol modulated in a negative way the affinity of the binding of agonists to CB2R as well as the signaling mediated by this receptor. Thus, it is postulated that cannabidiol is an allosteric modulator of CB2R. In contrast, cannabigerol appears to bind to the orthosteric center of CB1R and CB2R, where it acts as an agonist with functional selectivity, and modulates the signaling of the cannabinoids on CB1R-CB2R heteromers.
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Landgraf, Pablo. "Identifizierung des peroxisomalen Targeting-Signals des Adrenoleukodystrophie-Proteins." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-28682.
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Olivier, Lisa Marie. "Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9981970.
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Deng, Qingwei 1968. "Identification of dendritic targeting signals of voltage-gated potassium channel 3." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82219.
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Members of voltage-gated potassium channel subfamily 3 (Kv3) have been extensively demonstrated to play a significant role in facilitating function of "fast-firing" neurons in the central nervous system. Kv3.1 and Kv3.3 channels, members of Kv3 channel subfamily, have different distribution profiles on the regional level of brain and on the subcellular level of neurons in mammals and in weakly electric fish, according to mRNA hybridizations in situ and immunohistochemical analysis. In mammals, Kv3.1 channels are expressed in soma, axon and proximal dendrites as well as presynaptic membrane of "fast-firing" neurons. In weakly electric fish (Apteronotus), Kv3.1 channels are distributed in the soma, in the basilar dendrites and in the proximal apical dendrites of pyramidal neurons; on the other hand, Kv3.3 channels are expressed in a larger region: soma, basilar dendrites and entire apical dendrites of these cells. Mechanisms underlying differential subcellular distribution of Kv3.1 and Kv3.3 channels in the apical dendritic compartment of pyramidal neurons are unknown. In order to identify peptide sequences responsible for the differential subcellular localization, I have used Semliki Forest virus as a modified viral expression system (PDE) in vivo to study dendritic targeting mechanisms in the pyramidal neurons of electrosensory lateral line lobe (ELL), where the primary processing for afferent input occurs in the apteronotid electrosensory system.
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Groot, Marcel. "Die Rolle des Tyrosinkinase-Rezeptors VEGFR-2 im neuronalen Kontext." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1166623362738-03904.
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Im Rahmen dieser Arbeit wurde die Rolle des Rezeptors VEGFR-2, Flk-1, im neuronalen Kontext untersucht. In einem ersten Schritt wurde in embryonalen Stammzellen der Maus das fluoreszierende Protein eGFP unter der Kontrolle regulatorischer Sequenzen des flk-1-Promotors, -Enhancers exprimiert. Nach der Differenzierung zu Sphäroiden wurden Endothelzellen nachgewiesen, die sowohl eGFP als auch das zelltypspezifische Oberflächenantigen CD31 ausprägen. Ebenso wurden nach der neuronalen Differenzierung in Gegenwart von Stromazellen eGFP-exprimierende Zellen identifiziert. Diese standen mit Zellen, die das für neuronale Vorläuferzellen charakteristische Protein Nestin ausprägten, in einem räumlichen Zusammenhang. Die Vorgehensweise, die Inaktivierung des flk-1-Gens mit der Differenzierung embryonaler Stammzellen in vitro zu kombinieren, sollte hier die Interpretation des Phänotyps des flk-1-defizienten Mausmodells ermöglichen. Der Rezeptor war während der neuronalen Differenzierung der Stammzellen auf Stromazellen in vitro für die Regulation der Anzahl der Vorläuferzellen essentiell. Ferner spielte der Rezeptor im Rahmen eines weiteren Differenzierungsmodells, das auf der Zugabe relevanter Wachstumsfaktoren beruht, eine instruktive Rolle im Hinblick auf die Identität der Neuronen. Kriterium war hier die differentielle Expression Homeobox-enthaltender Transkriptionsfaktoren. In einem zweiten Schritt wurden mit Hilfe dieses Modells differentiell-exprimierte Gene von Stammzellen des Wildtyps sowie Zellen mit einer Inaktivierung des flk-1-Gens nach der neuronalen Differenzierung durch subtraktive Hybridisierung in Verbindung mit der PCR identifiziert. Tatsächlich wurde das Protein PEA-15 nicht nur differentiell exprimiert sondern auch als Bestandteil des VEGFR-2-vermittelten Signalwegs identifiziert. Die biologischen Funktionen des Proteins PEA-15 wurden durch VEGF-vermittelte Phosphorylierung reguliert. Die Stimulation durch VEGF führte zunächst zu einer Aktivierung des Proteinkinase B-, Akt-Signalwegs. Für die Stimulation des Akt-Signalwegs war die Phosphorylierung der intrazellulären Tyrosinreste Y1052 und Y1057 des Rezeptors essentiell. Damit einhergehend wurde PEA-15 gegenüber der proteasomalen Degradation stabilisiert. Es wurde gezeigt, daß das Protein PEA-15 die Teilungsaktivität von Zellen beeinflusst. Die VEGF- vermittelte Stimulation führte zur Phosphorylierung der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2. Die weitere Phosphorylierung der Substrate dieser Kinasen im Zellkern wurde durch Interaktion mit PEA-15 unterdrückt. Die Regulation des c-fos-Promotors war zugleich Indikator der Inhibition der Phosphorylierung betreffender Substrate sowie der proliferativen Aktivität. Auf diese Weise ist die Phosphorylierung von PEA-15 nach Stimulation durch VEGF für die Selektivität des Flk-1-vermittelten Signalwegs von unmittelbarer Bedeutung. Die Regulation der biologischen Funktion von PEA-15 erklärt die differentielle Ausprägung im Rahmen der neuronalen Differenzierung embryonaler Stammzellen in vitro. So war die Anzahl GFAP- beziehungsweise PEA-15-exprimierender Zellen nach Differenzierung muriner Stammzellen mit einer Inaktivierung des flk-1-Gens deutlich geringer. Die differentielle Expression identifizierter Gene wurde im Mausmodell nach konditionaler Inaktivierung des flk-1-Gens überprüft. Tatsächlich wurde Vimentin in verschiedenen Arealen des Gehirns differentiell ausgeprägt. Ein Zusammenhang zwischen der differentiellen Expression des Proteins PEA-15, der Anzahl GFAP-exprimierender Zellen und der Ausprägung des Rezeptors Flk-1 ergab sich aus der Identifikation einer Zellpopulation in der subgranulären Zone des Gyrus Dentatus. Dort wurde in flk-1-defizienten, adulten Mäusen eine geringere Anzahl GFAP-exprimierender Zellen nachgewiesen. Schließlich wurden sowohl im Cerebellum als auch im Cortex histologische Unterschiede deutlich, die sich im adulten Organismus aus der Inaktivierung des Rezeptors Flk-1 ergeben. Die vorliegende Arbeit zeigt, daß der Rezeptor VEGFR-2, Flk-1, im neuronalen Kontext eine Rolle spielt, die sich nicht ausschließlich auf die Vermittlung eines Schutzmechanismus gegenüber der neuronalen Apoptose beschränkt, sondern auch auf eine Beteiligung an der Neurogenese hinweist. Die Vorgehensweise, mit Hilfe der subtraktiven Hybridisierung Bestandteile Rezeptor-vermittelter Signalwege vor dem Hintergrund der Differenzierung embryonaler Stammzellen zu identifizieren, verdeutlicht die Eignung der Methode auch bei komplexen Zellpopulationen.
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Wolff, Samuel Carey Nicholas Robert Allen. "Identification of targeting signals in human P2Y receptors in polarized MDCK(II) epithelial cells." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1269.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.<br>Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Neurobiology." Discipline: Neurobiology; Department/School: Medicine.
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Khazaipoul, Siavash. "Studies of recombinant protein expression : targeting signals, 3' untranslated regions and trans-acting factors." Thesis, University of Newcastle upon Tyne, 2011. http://hdl.handle.net/10443/2181.
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At present mammalian cell factories are being employed for recombinant protein production. However, the yields of proteins produced from such systems are often poor. This thesis describes experiments to study the effects of altering targeting signals (signal peptide) and 3’ untranslated regions (3’UTR) in an expression vector on protein expression. A variety of gene constructs containing Gaussia princeps luciferase as reporter were created using a seamless cloning method. In these constructs a variety of signal peptides, some with altered hydrophobicity, were combined with the Gaussia luciferase coding region and either the native Gaussia luciferase or human albumin 3’UTR. These were then transfected into CHO AA8 Tet-Off cells to measure how modification of the signal peptide/3’UTR affects protein expression. The results indicate that the Albumin 3’UTR, in conjunction with an appropriate signal peptide, boosts protein production by approximately 3 fold compared to the native Gaussia luciferase 3’UTR. Deletion analysis of the Albumin 3’UTR showed that deletion of regions 1-50, 1-100, 1-150, 101-150 significantly reduces protein production compared with deletion of regions 51-100, 51-150 and 1-50&101-150. Interestingly, mRNA abundance levels were significantly decreased for constructs containing deletions in regions 1-50, 1-150 and 1-50&101-150. UV Cross linking and electrophoretic mobility gel shift competition assays showed strong competition by RNA transcripts from the deletion construct 1-50, which was then used as bait for isolating bound protein/s from a CHO cell extract. Three proteins, including CUG-BP1 an RNA-binding protein involved in mRNA stability and translation were identified by mass spectrophotometry analysis. Knock down of CUG-BP1 expression using siRNA, led to impairment of complex formation between CHO cell protein extract and Albumin 3’UTR RNA transcripts, and in addition it led to an increase in the reporter activity and mRNA expression level in cells expressing the reporter gene with the full length Albumin 3’UTR and deletion variant 51-100. It is hypothesised that the differences in mRNA expression levels and secreted luciferase activity were due to CUG-BP1 binding to the Albumin 3’UTR. Further work is needed to explore the effects of CUG-BP1 on mRNA translation and stability.
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Pröhl, Michael [Verfasser], Ulrich Sigmar [Gutachter] Schubert, and Wolfgang [Gutachter] Weigand. "Synthesis of D-fructose conjugated metal complexes and polymers for breast cancer targeting / Michael Pröhl ; Gutachter: Ulrich Sigmar Schubert, Wolfgang Weigand." Jena : Friedrich-Schiller-Universität Jena, 2018. http://d-nb.info/1170396542/34.
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Milbank, Edward. "Extracellular vesicles as a therapeutic strategy to prevent or reverse obesity and its metabolic complications in the field of nanomedicine Extracellular vesicles: Pharmacological modulators of the peripheral and central signals governing obesity Microparticles from apoptotic RAW 264.7 macrophage cells carry tumour necrosis factor-a functionally active on cardiomyocytes from adult mice." Thesis, Angers, 2016. http://www.theses.fr/2016ANGE0074.
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A ce jour, les thérapies anti-obésité restent limitées. De récente études ont fourni des résultats prometteurs en démontrant une diminution du poids de la souris via une injection stéréotaxique d’une forme dominante négative de l’AMPK (AMPK DN) directement dans le noyau ventromédial hypothalamique (VMH). Cependant, le potentiel thérapeutique de cette thérapie génique se voit entravé par une libération non spécifique de l’AMPK suite à une injection intraveineuse, plus adaptée à une approche clinique. Nous avons donc développé une approche de « nanobiomédecine » en utilisant des exosomes - nanovésicules contenant des lipides, des protéines et des acides nucléiques - pour délivrer l’AMPK DN spécifiquement au niveau du VMH. Des cellules dendritiques immatures ont été utilisées pour produire des exosomes non-inflammatoires. Pour permettre le ciblage spécifique du VMH par les exosomes, les cellules dendritiques ont été transfectées pour exprimer Lamp2b, une protéine exosomale, fusionnée au peptide de ciblage neuronal RVG. De façon intéressante, les exosomes Lamp2b-RVG ont été localisés au niveau du cerveau suite à une injection intraveineuse. Les exosomes Lamp2b-RVG ont ensuite été chargés par l’AMPK DN sous le contrôle d’un promoteur spécifique du VMH, apportant une double spéficité tissulaire aux exosomes. Les exosomes Lamp2b-RVG chargés avec l’AMPK DN induisaient une diminution de la phosphorylation de l’acetyl-CoA carboxylase dans des cellules Neu2A in vitro. De plus, l’injection intraveineuse d’exosomes Lamp2b-RVG chargés avec l’AMPK DN induisait une perte de poids de l’animal après 6 jours de traitement, démontrant le potentiel de cette approche de « nanobiomédecine »<br>Actual pharmacological therapies for treating obesity are limited. Promising results on decreasing mice body weight were obtained using a ventromedial nucleus hypothalamic (VMH) stereotaxic injection of a dominant negative isoform of AMPK (AMPK DN). However, DNA-mediated therapeutic potential is hampered by inadequate tissue specific delivery following a systemic injection - more adapted to a bedside approach -. Herein, we developed a nanobiomedicine approach using exosomes - nano-scaled endogenous vesicles containing lipids, proteins and nucleic acids - to deliver DNA in a hypothalamic specific way. Immature dendritic cells were used to generate non inflammatory exosomes. Exosome neuronal targeting aptitudes were achieved by constraining the dendritic cells to express Lamp2b, an exosomal protein, fused to the neuron-specific RVG peptide. Interestingly, DID-labelled Lamp2b-RVG exosomes were found into the mice brain following an intravenous injection. Isolated Lamp2b-RVG exosomes were then loaded by transfection-mediated techniques with AMPK DN under the control of a VMH specific promoter conferring double tissue expression specificity to the exosomes. AMPK DN-loaded exosomes induced a decrease of acetyl-CoA carboxylase phosphorylation in Neu2a neuronal cells in vitro. Furthermore, intravenously injected AMPK DN loaded exosomes induced a decrease of mice body weight following 6 days of treatment, demonstrating the potential of this nanobiomedicine approach
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Mellick, Albert S. "The characterisation and In vivo targeting of Gzα". Master's thesis, 1997. http://hdl.handle.net/1885/145963.
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RAHMAN, Md Bytul Mokaddesur. "NMR analyses on recognition of the mitochondrial targeting signal by Tim50." Thesis, 2014. http://hdl.handle.net/2237/20002.
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Chen, Nien-tzu, and 陳念慈. "Identification of the mitochondrial targeting signal of yeast glutamyl-tRNA synthetase." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/72264724593825241374.
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碩士<br>國立中央大學<br>生命科學研究所<br>98<br>In Saccharomyces cerevisiae, the cytoplasmic glutamyl-tRNA synthetase (cGluRS) is encoded by GUS1. In cytoplasm, cGluRS attaches Glu to the cognate tRNAGlu to form Glu-tRNAGlu. In addition to the cytoplasmic activity, cGluRS can enter the mitochodria to synthesize Glu-tNRAGln (a misacylated product), which is then converted to correct Gln-tRNAGln by Glu-tNRAGln amidotransferase. A pervious study in our lab indicated that Arc1p, which binds cGluRS in cytoplasm, also has a mitochondrial targeting signal. Our results showed that cGluRS and Arc1p each carry a mitochondrial targeting signal and thus can enter the mitochondria by itself. Next, we used the cytoplasmic form of valyl-tRNA synthetase as the reporter gene to map the mitochondrial targeting signal of cGluRS. As it turned out, this signal was embedded in the N-terminal amino acids 1-29 of cGluRS. In contrast, no mitochondrial targeting signal was found in Schizosaccharomyces pombe or Candida albicans GluRS. Moreover, Arc1p from Pichia guilliermodii, Candida albicans, and Schizosaccharomyces pombe does not possess a mitochondrial targeting signal.
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Jaru-Ampornpan, Peera. "Post-Translational Membrane Protein Targeting by the Chloroplast Signal Recognition Particle." Thesis, 2011. https://thesis.library.caltech.edu/6429/9/Thesis_Peera.pdf.
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Post-translational transport of membrane proteins poses enormous challenges to the cells. The transport factors must accurately select and deliver the cargos to the appropriate target membranes. In addition, they have to provide chaperone for their hydrophobic cargos. To understand capacity and limitation of a post-translational transport factor, we studied one of the most efficient membrane protein transport pathways, the delivery of light-harvesting chlorophyll-binding (LHC) proteins to the thylakoid membrane. This targeting reaction is mediated by the chloroplast Signal Recognition Particle (cpSRP) and its receptor. Although the core SRP GTPases are close homologues of those in cytosolic SRP pathways, the unique features of cpSRP that might reflect its adaptation to the challenges in post-translational targeting include (i) the lack of the otherwise universally conserved SRP RNA, and (ii) the exclusive presence of a novel protein, cpSRP43. In the first part of this thesis, we define the thermodynamic and kinetic framework for the GTPase cycles of cpSRP and its receptor and uncover the molecular bases that enable their intrinsically fast interactions, such that they can bypass an SRP RNA, an essential accelerator for the cytosolic SRP–receptor interaction. The second part of the thesis is devoted to characterization of the chaperone function of cpSRP43. We show that cpSRP43 specifically and effectively prevents and reverses the aggregation of its cargo, LHC proteins. We further investigate the molecular mechanism of this novel disaggregase activity, using a combination of biochemical and structural approaches. In summary, this dissertation aims to understand how cpSRP and its receptor adapt to their unique requirements in efficiently transporting a family of highly abundant membrane proteins.
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Badolato, Mariateresa, Sebastiano Andò, Antonio Garofalo, and Francesca Aiello. "Preclinical and mechanistic studies of small-molecule drugs targeting stat3." Thesis, 2018. http://hdl.handle.net/10955/1838.
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Lee, Sejeong. "Dynamic interactions during ribosome targeting to the membrane." Doctoral thesis, 2014. http://hdl.handle.net/11858/00-1735-0000-0022-5FD8-1.
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Huang, Tzyy-Nan, and 黃子南. "Identification of A Targeting Signal In The C-Terminal of Potassium Channel Subunit Kv4.3S." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/56161553745236317504.
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碩士<br>國立陽明大學<br>神經科學研究所<br>89<br>In the alignment of amino acid sequences, Kv4 family members except Kv4.3S have VXXSXL motif at the end of their C-termini. The motif is similar to the VXXSL, a motif found at the C-terminal of many Kv1 members is crucial for surface expression. Kv4.3S and Kv4.3S△C42 were transfected into MDCK cells and selected by G418 for permanent clones. They were expressed differently. Kv4.3S was expressed in the regions near nucleus, but Kv4.3△C42 was expressed in the cytosol. Using GFP fusion proteins (including GFP-Kv4.2, GFP-Kv4.3S, and GFP-Kv4.3SΔC6) to observe living cell expression, we found that subcellular localization of GFP-Kv4.3S was similar to Kv4.3S. The others were also expressed in the cytosol. The same results were found in HA fusion proteins. The data indicated that Kv4.3S contains targeting signals at the end of C-terminal. Using markers specific for ER and Golgi apparatus respectively, we found that Kv4.3S was localized in the Golgi apparatus.
When we analyzed the last 22 amino acids of Kv4.3S C-terminal, there were three motifs —, RGR, TCK and IITLCI, which might be involved in subcellular targeting. By constructing point mutations in Kv4.3S, we demonstrated that RGR and IITLCI were crucial for Kv4.3S expression in Golgi apparatus, in both MDCK and Hela cells.
Tac chimeras were used to measure the ratio of cell surface expression. Out of our expect, two chimeras related to IITLCI ; Tac-Kv4.3ΔC6 and Tac-Kv4.3S, C610A could decrease surface expression. As is remarked above, IITLCI, the last six amino acids of Kv4.3S, could change both Kv4.3S and Tac chimeras expression region.
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Yuan-yi, Hsu, and 徐元翊. "Identification of the Coiled Body-Specific Targeting Signal in the U1 snRNP C Protein." Thesis, 1998. http://ndltd.ncl.edu.tw/handle/85742030408296842030.
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碩士<br>國立臺灣大學<br>分子醫學研究所<br>87<br>Coiled bodies are special subnuclear structures in most eukaryotic cells. The role of coiled bodies in cellular fuctions is still unclear. However, because of their complexity in protein content, including factors involved in RNA processing, coiled bodies were suggested to be involved in assembling, storage or sorting RNA processing proteins. U1 snRNP specific protein C ( U1C ) had been proposed to target to Xenopus oocyte coiled body homologue, C snurpsome. Utilizing transfection of HeLa cell line as the model system to identify the property of U1C targeting to coiled bodies, we get results that U1C is concentrated in coiled bodies and its targeting peptide locate near its N termianl region. Protein interaction is also characterized in the thesis. Immunofluorescence staining of transfected full length U1C show wide-spread distribution pattern in nuclear plasma and concentrated dots colocalized iwth coiled bodies, but not colocalized with nuclear speckes. Keeping only 30 residues in the N terminal region of U1C, caused the protein distribute in nucleus without concentrated in coiled bodies. However, deletion of the N termianl 30 residues of U1C caused the inability of nuclear targeting. With SV40 viral T antigen, the N terminal deletion form enternuclear but can't concentrated in coiled bodies. The conclusion is that the N terminal 30 residues, which are enough for nuclear targeting , are essential for entering coiled bodies, but until now only N terminal 72 amino acids were shown to be enough for coiled body-specific targeting. To identify protein interactions with different truncate U1C, Sm core protein and coilin were analyzed by Y12 snri-Sm immunoprecipitation. Until now we can't directly link coiled body-specific targeting with specific protein interaction.
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Stoyanova, Tsenka. "RF signal modeling and deployment strategy targeting outdoor RSS-based localization and tracking applications in wireless sensor network." Thesis, 2011. http://hdl.handle.net/10889/5237.
Full textAbstract:
The localization of the sensor nodes is a fundamental issue in the area of wireless
sensor networks (WSNs). An attractive way for estimating the location of mobile or
static wireless objects is by using the received signal strength (RSS) attenuation with the distance, which does not require any additional hardware. This is possible due to the fact that in most sensor nodes radios the received signal strength indicator (RSSI) is a standard feature and can be obtained automatically by the received messages. On the other hand the RSS is known for being noisy, unstable, variable and difficult to use in practice. For achieving a better understanding of the nature of these difficulties and limitations, and for identifying the range of applicability of the RSS in localization and tracking scenarios, a thorough study about the RSS and its dependence on the various factors and environmental conditions is essential.
The present doctoral dissertation investigates the feasibility of sensor node
localization and target tracking with the resources of the WSN technology, when using
only the RSS of the exchanged messages. Moreover, it offers experimental support to
the hypothesis that proper modeling of the RSS behavior and appropriate selection of
the topology parameters are essential for the applicability of WSN in real world
conditions. In brief, the present doctoral dissertation concerns with: (i) identifying the main factors that influence the accuracy, the variability and the reliability of the obtained RSS, (ii) modeling the RF signal propagation in the context of WSNs, and (iii)defining the basic deployment constraints and evaluation of the topology parameters that can guarantee successful localization and tracking.
For assessing the practical value of various RF-models, experiments using Tmote Sky and TelosB sensor nodes in real-field outdoor environment were carried out. The impact of a number of factors, such as the operating frequency of the radio, the transmitter–receiver distance, the variation of transceivers hardware due to manufacturing tolerances, the antenna orientation, and the environmental conditions, on the RSS was investigated. The influence of the various factors that affect the RF signal propagation and some constraints imposed by the WSN nature was accounted in order to design practical models, suitable for outdoor unobstructed and outdoor tree-obstructed environments.
A pre-deployment simulation framework has been introduced and in its context a
RF signal propagation-based connectivity strategy (RFCS) has been developed to fulfill
three deployment provisions: (i) discovering the most appropriate height from the
ground and distances for the sensor nodes, (ii) reducing the transmission power, and
(iii) minimizing the interference from non-neighbor nodes. The RFCS uses a RF signal
propagation model to predict the RSS in order to identify the most appropriate
communication-based deployment parameters, i.e. T-R distance, height from the ground and transmission power.
The localization and tracking considerations, by means of localization and tracking techniques, topology parameters and factors influencing the localization and tracking accuracy, are combined in illustrative simulation examples to evaluate their significance concerning the performance of the localization and tracking task.
Furthermore, the propagation model and the topology parameters being identified
were validated in real outdoor sensor node localization and target tracking tests.<br>-
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Hwang-Fu, Yu-Hsien. "Dynamic Regulations of Co-translational Protein Targeting by the Signal Recognition Particle Receptor in E. coli and Human." Thesis, 2019. https://thesis.library.caltech.edu/11309/9/YuHsien_HwangFu_2019_thesis_final.pdf.
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<p>The Signal Recognition Particle (SRP) and its receptor (SR) co-translationally deliver the majority of secretory and membrane proteins to their membrane destinations. SRP recognizes and binds to the cargo, and SR recruits the transit complex to the target membrane. Precise timing and coordination are encoded into the interactions between SRP and SR in response to the cargo and environment to enable efficient and accurate targeting. This dissertation explores the regulation mechanisms of SRP membrane recruitment by SR in both bacteria and eukaryotes.</p>
<p>The bacterial SR is a peripheral membrane protein and recruits SRP by adjusting its membrane-binding modes. A complete kinetic model of SRP membrane recruitment by the SR was established based on direct observations and quantitative analyses of protein-membrane binding events at single-molecule resolution. Biochemical and cell biological examinations validated the physiological significance of the newly discovered dynamic membrane-binding mode of the SR. The two-stage SR membrane binding mechanism ensures both speed and specificity in bacterial co-translational protein targeting.</p>
<p>In eukaryotes, a proper initial recognition of SRP sets the right course for membrane delivery of the transit complex, as eukaryotic SR is anchored on the endoplasmic reticulum. A Molecular Recognition Feature (MoRF) in the disordered linker domain of eukaryotic SR was identified and characterized. The SR MoRF element enables rapid SRP-SR assembly in response to the ribosomal content of the cargo. The stimulation by SR MoRF is only present in eukaryotes and is likely a functional replacement of the tetraloop in bacterial SRP RNA whose stimulatory role was abolished during evolution.</p>
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Liu, Hsin-Yu, and 劉欣瑜. "The functional and mitochondrial targeting signal studies of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) subunit in mitochondrial complexⅠ." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/03608895926813533123.
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碩士<br>國立清華大學<br>分子醫學研究所<br>97<br>Mammalian NADH-ubiquinone oxidoreductase (complex I) is the first, largest and most complicated respiratory complex in mitochondria. Seven subunits of complex I, including ND1-6 and ND4L, are encoded by mitochondrial DNA (mtDNA), and the other thirty-eight subunits are encoded by nuclear DNA (nDNA). NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is one of the core nucleus-encoded subunits existing in human mitochondrial complex I. It contains one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), which may play a role in the prevention of oxidative damage. The defect of NDUFV2 subunit is associated with neurodegenerative diseases, including Parkinson disease, Alzheimer’s disease, Bipolar disorder and Schizophrenia. In this study, we applied the RNA interference (RNAi) technology in human T-REx293 cells to investigate the function of NDUFV2 subunit. We found that suppression of NDUFV2 expression in the cells would cause a slowing growth cell rate in galactose medium, decreasing oxygen consumption rate, reducing mitochondrial membrane potential (MMP) and increasing reactive oxygen species (ROS) generation, but did not affect complex I assembly. These observations provided the evidences that NDUFV2 plays an essential role for energy production in cells. In addition, we designed various truncation constructs to investigate the mitochondrial targeting mechanism of NDUFV2. We identified that the cleavage site of NDUFV2 was located around amino acid residue 32 and the first 22 residues of NDUFV2 was enough to function as a mitochondrial targeting sequence (MTS) to carry the passenger protein, enhanced green fluorescent protein (EGFP), into mitochondria successfully. Furthermore, we used the site-directed mutagenesis to study the basic, hydrophobic and hydroxylated residues in this identified N-terminal MTS. We found that the basic and hydrophobic residues were important for the MTS of NDUFV2, but the hydroxylated residues were not. In a recent study, the patients of the hypertrophic cardiomyopathy and encephalomyopathy were found to contain 4 bp deletion in the second intron of NDUFV2 (IVS2+5_+8delGTAA) to cause the exon 2 losing. To dissect the pathogenetic mechanism caused by this mutation, we established the human disease model and found that lost of this exon 2 cause NDUFV2 to lose its mitochondrial targeting ability. In this report, we proved that the NDUFV2 plays an important role for energy production in mammalian cells and identified the location of mitochondrial targeting sequence in this protein.
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Nyathi, Yvonne, and M. R. Pool. "Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC." 2015. http://hdl.handle.net/10454/17895.
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Yes<br>The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These
factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit
site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site
where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of
NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and
SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation
before translocation.<br>This work was supported by grants from the BBSRC [H007202/1] and Wellcome Trust [097820/Z/11/A].
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Panda, U. "A Low Complexity High Speed Architecture Design Methodology For Reduced 3-Lead to 12-Lead ECG Signal Reconstruction Targeting Remote Health Care." Thesis, 2014. http://raiith.iith.ac.in/967/1/EE12M1038.pdf.
Full textAbstract:
Cardiovascular diseases is one of the prime causes of human corporeality and mobidity in
society . In order to abate this researchers had paid heed in the field of detection and pre-
vention in both hospital-based and remotely accessed environments . Advancements in
wireless technology and tale-monitoring can be used to provide the accessibility of state-of
-the-art(Sot A) facilities to patients in remote and rural areas. However, bandwidth and storage
limitations and data transmission time are major challenges in wireless transmission . Though
cardiologists are habituated to standard 12-lead (S12) system because of its decade old usage
and widespread acceptability, however generally, for such remote healthcare environments a
reduced lead(RL)ECG is suitable for aforementioned reasons , which however , may not be
clinically acceptable for diagnosis . Several efficient algorithms for reconstruction of RL to
SotA 12 lead have been proposed. The overall Cardio Vascular Disease detection system can
be characterized to 6 different sections namely Data Acquisition , Preprocessing , Data
Transmission, Coefficient Generation, Signal Reconstruction and Display on Monitor. The
thesis work includes a low complexity and high speed architecture design ( for the preprocess-
sing section) and its implementation on FPGA and ASIC platform which intern can be used
for the accurate reconstruction of 3 lead to 12 lead ECG signal reconstruction .
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Gopalan, Archana. "Targeting breast cancer with natural forms of vitamin E and simvastatin." Thesis, 2012. http://hdl.handle.net/2152/ETD-UT-2012-05-5520.
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Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models.<br>text
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Mishra, Akaash K. "Developing small molecule inhibitors targeting Replication Protein A for platinum-based combination therapy." Thesis, 2014. http://hdl.handle.net/1805/6466.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)<br>All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.
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Huang, Wei. "Small molecule compounds targeting DNA binding domain of STAT3 for inhibition of tumor growth and metastasis." Thesis, 2014. http://hdl.handle.net/1805/5221.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)<br>Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including proliferation, survival, self-renewal, migration, invasion, angiogenesis and immune response by regulating the expression of diverse downstream target genes. Thus, inhibiting STAT3 promises to be an attractive strategy for treatment of advanced tumors with metastatic potential. We firstly identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding site of STAT3 using an in-silico screening approach; however, inS3-54 was finally found not to be appropriate for further studies because of low specificity on STAT3 and poor absorption in mice. To develop an effective and specific STAT3 inhibitor, we identified 89 analogues for the structure-activity relationship analysis. By using hematopoietic progenitor cells isolated from wild-type and STAT3 conditional knockout mice, further studies showed that three analogues (A18, A26 and A69) only inhibited STAT3-dependent colony formation of hematopoietic progenitor cells, indicating a higher selectivity for STAT3 than their parental compound, inS3-54. These compounds were found to (1) inhibit STAT3-specific DNA binding activity; (2) bind to STAT3 protein; (3) suppress proliferation of cancer cells harboring aberrant STAT3 signaling; (4) inhibit migration and invasion of cancer cells and (5) inhibit STAT3-dependent expression of downstream targets by blocking the binding of STAT3 to the promoter regions of responsive genes in cells. In addition, A18 can reduce tumor growth in a mouse xenograft model of lung cancer with little effect on body weight. Taken together, we conclude that it is feasible to inhibit STAT3 by targeting its DNA-binding domain for discovery of anticancer therapeutics.
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Ma, Changle. "Subcellular and functional analyses of two small heat shock proteins and protein kinases from peroxisomes of Arabidopsis thaliana L." Doctoral thesis, 2006. http://hdl.handle.net/11858/00-1735-0000-0006-AC21-A.
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