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Cyclin-dependent kinase inhibitor drugs drive neutrophil granulocyte apoptosis by transcriptional inhibition of the key survival protein MCL-1
Get PDFThe normal physiological response to bacterial infection or wounding with threat
of infection, termed inflammation, has been shown to be dysregulated in certain
human diseases including (but not limited to): idiopathic pulmonary fibrosis, acute
lung injury, arthritis and glomerulonephritis. The earliest arriving and most
abundant cell responding to an inflammatory stimulus is the neutrophil
granulocyte. It has been shown that under inflammatory conditions neutrophil
granulocytes have extended longevity, enhanced responsiveness and upregulated
activation parameters. In the setting of non-infective, or prolonged, ineffectuallycleared
infective disease where resolution of inflammation does not occur then
neutrophil granulocytes may cause tissue damage which is mediated by excessive,
misdirected exocytosis of toxic granule contents or by spillage of the same
products from necrotic or netotic cell carcasses that have lost membrane integrity.
A key process in the resolution of inflammation is the induction of apoptosis in
recruited neutrophils following a successful response to an inflammatory stimulus.
Cellular signalling from apoptotic cells and from professional phagocytes that have
ingested apoptotic cells has been shown to favour resolution of inflammation and
restoration of tissue homeostasis. Additionally, the removal of key inflammatory
cells in a highly regulated, non-phlogistic fashion robustly assists the resolution
process.
Cyclin-dependent kinase (CDK) inhibitor drugs are being developed as anti-cancer
agents as it is hypothesized that they should interfere with the enhanced cellcycling
ability (increased proliferative capacity and extended longevity) which is
such a key feature of cancer cell biology. The CDKs that drive the cell cycle are
CDKs 1, 2, 4 and 6 and consequently agents were designed to have enhanced
specificity for these targets. CDK inhibitor drugs target the ATP-binding domain
of CDKs and as a result usually have activity against more than one CDK. The
CDK inhibitor drug, R-roscovitine which targets CDKs 2, 5, 7 and 9 was shown to promote neutrophil apoptosis and consequently resolution of inflammation. This
thesis aims to investigate the mechanism by which apoptosis is induced in
neutrophil granulocytes by CDK inhibitor drugs.
The first experimental chapter of this thesis explores in detail the time-course and
active concentration range of CDK inhibitor drugs in comparison to known
promoters and inhibitors of neutrophil apoptosis. It then dissects the apoptotic
machinery which is responsible for the effects of CDK inhibitor drugs before
investigating their capacity to promote apoptosis even in the presence of survival
mediators relevant to the context of inflammatory disease. Flow-cytometry, light
and confocal microscopy as well as western blotting for caspases, mitochondrial
dissipation assay, fluorometric caspase assay and the detection of DNA laddering
demonstrate that CDK inhibitor drugs promote classical neutrophil apoptosis by
the intrinsic pathway and show similar kinetics of apoptosis induction to drugs
that inhibit transcription.
The second experimental chapter investigates the key neutrophil survival protein
and bcl-2 homologue Mcl-1. By flow cytometry, western blotting and RT-PCR it is
demonstrated that Mcl-1 is down-regulated at the level of transcription and that
this occurs even in the presence of inflammatory mediators that would normally
promote neutrophil survival. Additionally, it is shown that pro-apoptotic bcl-2
homologues are affected to a lesser degree suggesting an imbalance of bcl-2
proteins is caused by effects at a transcriptional level mediated by CDK inhibitor
drugs.
The third experimental chapter identifies CDKs and their binding partner cyclins in
neutrophil granulocytes and investigates the impact of CDK inhibitor drugs on
CDK protein levels and cellular distribution by differential lysis and western
blotting as well as by confocal microscopy. The key transcriptional enzyme RNA
polymerase II is also identified and the effect of CDK inhibitor drugs on phosphorylation of this enzyme is documented. Western blotting and confocal
microscopy demonstrate the presence of key CDKs 2, 5, 7, 9 and cyclin binding
partners of CDKs 7 and 9. It is shown that the phosphorylation of RNA
polymerase II mediated by CDKs 7 and 9 is inhibited by CDK inhibitor drugs.
This suggests that a key mechanism by which neutrophil apoptosis is induced by
CDK inhibitor drugs is the inhibition of transcription of key proteins and suggests
that neutrophils require survival proteins for functional longevity.
The fourth experimental chapter addresses the production and use of HIV-tat
dominant negative CDK 7 and 9 proteins to knockdown CDKs 7 and 9 in
neutrophil granulocytes in vitro to provide a molecular biology surrogate for the
pharmacological data already presented. The cloning, production, purification and
use of HIV-tat dominant negative CDK proteins are described.
The final chapter describes the use of a more specific pharmacological inhibitor of
CDKs 7 and 9, DRB, in the mouse bleomycin lung injury model. Resolution of
inflammation by a compound specifically targeting CDKs 7 and 9 is described.
This thesis identifies CDKs 7 and 9 as key targets of CDK inhibitor drugs in
neutrophilic inflammation. It shows these drugs acting at the level of transcription
to drive neutrophil apoptosis by exploiting the unique dependency of neutrophils
on the short-lived survival protein Mcl-1. In so doing the presence of functional
and essential transcriptional machinery is identified in neutrophils and the
transcriptional profile of resting, stimulated and inhibited neutrophils is delineated.
These findings suggest novel approaches to the pharmacological promotion of
resolution of inflammation and indicate key new targets for rational drug design. In
future, it will be important to further characterize the effects of CDK inhibitor
drugs on other cell-types including epithelial cells, fibroblasts and mononuclear
cells. This information should prove important to the continued investigation of CDK inhibitor drugs in resolution of inflammation and also to the ongoing
experimental trial of these drugs in idiopathic pulmonary fibrosis
Light activatable nanosystems for drug delivery
Get PDFPolymers are showing great promise in advancing the field of “Smart Medicines”, the area on which this PhD thesis is focused. The contents of this thesis explore the versatility of light in combination with polymeric systems to achieve selective drug delivery in three ways: using light to monitor cell uptake, the release of drugs from a polymeric backbone, and drug release via nanoparticle cleavage.
The first project includes the modification of RAFT agents to enable the controlled synthesis of size-defined monodisperse polymers with defined end-groups. Here, modifications included fluorophores and lysine-based cell-penetrating peptides. The modified RAFT agents then allowed for the synthesis of polymer chains that were fluorescently “tagged” and demonstrated enhanced cell uptake due to the cell penetrating peptide attached. The polymers showed high biocompatibility and were found to be localised inside endosomes within cells. This work demonstrates the first use of RAFT agents modified with both a fluorophore and cell-penetrating peptide, which endows both a tracking ability and high cell uptake. It allowed for polymer uptake to be measured quantitatively using fluorescence to compare the cellular uptake abilities of three different length cell-penetrating peptides.
The second route explored within the PhD thesis was light-mediated drug release. Here two approaches were taken:
(i). Drug attachment along a polymeric backbone with drug release facilitated by
photo-cleavage. Here, the water insoluble chemotherapy agent camptothecin was conjugated to a coumarin methacrylate monomer via a light cleavable bond, rendering the camptothecin 'inactive'. The coumarin camptothecin monomer was then co-polymerised with the water soluble N,N-dimethyl acrylamide (DMA) to yield a water soluble, light cleavable, camptothecin containing polymer. The light sensitivity of the polymer under 365 nm light was validated in solution and in vitro. Biological testing showed high biocompatibility of the polymer under dark conditions, with the polymer showing high levels of toxicity following irradiation. This work demonstrates the versatility of polymers to be used for selective cell-killing, but also their ability to enhance the water solubility of typically ‘difficult to formulate’ chemotherapy drugs.
(ii). Integrating a light responsive coumarin
moiety containing two orthogonal
functional groups in between the two blocks of an amphiphilic block copolymer
comprised of polyethylene glycol (PEG) and poly-L-lactide (PLA). Such photocleavable amphiphilic polymers were used to form light-sensitive nanoparticles, with the ability to encapsulate drugs within their core. Here the chemotherapy drug doxorubicin was encapsulated and consequently liberated by illumination under 365 nm light. Nanoparticles were found to release over 60 % of encapsulated drug after only 2 minutes of light irradiation. In vitro results validated the novel strategy, showing high biocompatibility under dark conditions and selective toxicity upon irradiation.
In summary the developed polymer systems showed promise as drug delivery agents by demonstrating good biocompatibility, trackability, high cell uptake, improved drug solubility, controlled drug activation, and selective drug delivery
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Molecular toxicology studies on the quartz hazard.
Get PDFSilicon makes up almost 28% of the Earth's crust and within that crust, quartz (crystalline silica) is one of the most abundant minerals. Exposure to quartz can occurin a number of occupations, including the mining and construction industries in which respirable quartz particles are generated and become airborne. Inhalation of quartz canlead to the fibrosing lung disease silicosis and cancer. Silicosis has been recognised for many decades as one of the most prevalent occupational lung diseases. In 1997, anIARC working Group classified quartz as a class 1 lung carcinogen, but only in some industries, suggesting that the quartz hazard is a variable entity. The reactivity of thequartz surface may underlie its ability to cause inflammation and treatments that ameliorate this reactivity would then reduce the quartz hazard.In the present study the effect of treating quartz with aluminium lactate, a procedure reported to decrease the quartz hazard, on the highly reactive quartz surface and on proinflammatory events in the rat lung were explored. Aluminium lactate-treated quartz showed a reduced surface reactivity as measured by electron spin resonance. Eighteenhours post-instillation of quartz into the rat lung, there was massive inflammation as indicated by the number of neutrophils in the bronchoalveolar lavage (BAL) and anincrease in BAL macrophage inflammatory protein-2 (MIP-2). However, aluminium lactate-treated quartz had no significant effect when compared to control. Epithelial damage as indicated by BAL protein and gamma glutamyl transpeptidasea lso increased with quartz instillation but not with aluminium lactate-treated quartz and furthermore,quartz induced an increase in MIP-2 mRNA content of BAL cells while aluminium lactate-treated quartz had no effect compared to controls. There was an increase innuclear binding of the transcription factor nuclear factor-kappa B (NF-xB) in the quartz exposed BAL cells and again, no effect on nuclear NF-xB binding in BAL cells fromaluminium lactate-treated quartz instilled rats.In addition, the effect of aluminium lactate and PVNO quartz treatment on DNA damage, cell cytotoxicity and particle uptake by A549 cells was assessed. DNA strandbreakage, as produced by quartz at non-toxic concentrations, could be completely prevented by both coating materials. Particle uptake by A549 cells appeared to be significantly inhibited by the PVNO coating, and to a lesser extent by the aluminium lactate coating, demonstrating that respirable quartz particles induce oxidative DNA damage in human lung epithelial cells and indicating that the surface properties of the quartz as well as particle uptake by these target cells are important in the cytotoxic and genotoxic effects of quartz in vitro.Finally, the role played by surface area and specific reactivity in the acute inflammatory response to particles was investigated. Acute inflammatory response followinginstillation of particles has been used to evaluate hazard but has been criticised because of the non-physiological delivery and the problems of local overload. Here, a numberof low toxicity dusts of various particle sizes were instilled and the neutrophil influx into the lung 18-24 hours post-instillation assessed. The extent of inflammation wasshown to be a function of the surface area instilled and ultrafine particles, which present a case of high surface area per unit mass, were inflammogenic pro rata with theirsurface area. There is no evidence that ultrafine particles of carbon black, titanium dioxide or polystyrene have any special reactivity in addition to their large surface area.We further tested whether this approach could be used to model the reactivity of highly toxic dusts. Rats were instilled with either quartz or aluminium lactate-treated quartzand, as anticipated, the high specific surface reactivity of quartz meant that it was much more inflammogenic than was predicted using the relationship described for `low toxicity'dusts. This approach represents the possibility of modelling potential toxicity for nuisance dusts based on the inflammatory response of a given instilled surface area dose
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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