Exploring mechanisms of metastasis suppression in metastatic melaoma

Melanoma is responsible for 76% of deaths from skin cancer, making it the deadliest form of commonly diagnosed skin cancer. The deadly nature of melanoma is due to its tendency towards rapid, early metastasis. Metastasis, the process of cells exiting the primary tumor and forming secondary tumors in other parts of the body, accounts for the majority of morbidity and mortality associated with cancer. Therapeutically targeting and treating melanoma metastases is a challenging clinical goal, as metastatic cells are heterogeneous and can be morphologically and genetically distinct from the primary tumor. This dissertation examines two approaches for prevention or treatment of disseminated melanoma metastases: 1) Re-introduction of metastasis suppressor protein fragments to prevent metastatic colonization, and 2) Treating disseminated metastases with a targeted small molecule treatment. By examining two discrete approaches of treating metastatic melanoma, this work sheds light on the clinical viability of using metastasis suppressors or metastasis-targeting drugs in patients with metastatic melanoma. To examine strategies for metastasis suppression through metastasis suppressor proteins, we examined cleavage products of the metastasis suppressor KISS1, a metastasis suppressor protein. Expression of KISS1 inhibits metastatic colonization at secondary sites, rendering disseminated cells dormant. KISS1 must be secreted outside of the cell to suppress metastasis, where furin cleaves KISS1 into kisspeptins at three dibasic sites. This cleavage liberates an internal kisspeptin, Kisspeptin-54 (KP54, amino acid K67 to F121), which is amidated and can bind a Gq/11-coupled protein receptor KISS1R. The mechanism of action for KISS1 metastasis suppression has long been assumed to be KP54 interacting with KISS1R. However, expression of KISS1R is not necessary for KISS1 metastasis suppression, and the extracellular processing of KISS1 hints at an alternative hypothesis: a different kisspeptin may be responsible for suppressing metastasis. To test this hypothesis, all possible kisspeptins (KISS1 Manufactured Peptides, or KMP) were generated based on known dibasic cleavage sites (M1–Q145; M1–R56; M1–R67; M1–R124; R56–R66; R67-F121; R56–F121; R56–Q145; R67–Q145; R124–Q145) and were used in an experimental metastasis assay to characterize their abilities to suppress metastasis. We found that while KP54 suppressed metastasis, additional KMP lacking the KISS1R binding site (LRF-NH2) were able to completely suppress metastasis (p<0.05). In particular, one kisspeptin (KMP2, M1 – R56) suppressed metastatic traits in vitro as well as completely suppressing metastasis in vivo. To identify the signaling pathways used by KMP2 to suppress metastasis, a genome wide CRISPR/Cas9 screen was performed in KMP2-expressing B16-F10 melanoma cells. As a whole, these data suggest that metastasis suppression by KISS1 is not necessarily contingent on KISS1R activation, and also supports investigation into additional receptors. To investigate the efficacy of targeting metastases with small molecules, we also investigated the impact of ML246 (AKA metarrestin). Metarrestin was discovered by a high throughput assay for molecules which disassemble the perinucleolar compartment (PNC). Perinucleolar compartments are structures composed of RNA and RNA binding proteins near the nucleolus. These structures are enriched in metastatic cells and are druggable targets which target metastases and not normal epithelium. We examined the impact of metarrestin treatment on orthotopic tumor growth, microscopic metastasis formation, and macroscopic metastasis formation. We found that metarrestin treatment had no significant impact on metastatic outgrowth, but suppressed intradermal tumor growth. Based on these data, we can infer that PNC-positive metastases may be too small a population to effectively target in this model. This treatment paradigm may be more effective in conjunction with a more potent approach to metastasis suppression. Overall, the work in this dissertation identified a potent metastasis suppressing fragment of KISS1, KMP2, and described the efficacy of metarrestin treatment of disseminated metastases. The metastasis suppression induced by KMP2 expression was far more potent than the effects of metarrestin treatment on suppressing metastatic colonization and outgrowth, suggesting that treatment deliveries and targets are critical considerations in the development of anti-metastatic therapeutics

Does this Feel Empowering? Using Métissage to Explore the Effects of Critical Pedagogy

The extent to which critical pedagogy disrupts the relations of dominance inside postsecondary classrooms, or empowers students to take socially just action beyond the classroom has been debated and challenged for decades. Through the use of métissage, an interpretive inquiry method that affords collaborative interrogation of individual narrative writings, we five participants in the same critical pedagogy course conducted a post-course inquiry project in order to explore what we had learned through the course. Through this inquiry project, we have come to a deeper understanding of critical pedagogy praxis. Ultimately, what we learned through the use of this inquiry method maintains important implications for postsecondary educators

Probing the physics of narrow-line regions of Seyfert galaxies I: The case of NGC 5427

We have used the Wide Field Spectrograph (WiFeS) on the ANU 2.3m telescope at Siding Spring to observe the nearby, nearly face-on, Seyfert 2 galaxy, NGC 5427. We have obtained integral field spectroscopy of both the nuclear regions and the HII regions in the spiral arms. We have constrained the chemical abundance in the interstellar medium of the extended narrow line region (ENLR) by measuring the abundance gradient in the circum-nuclear \ion{H}{ii} regions to determine the nuclear chemical abundances, and to use these to in turn determine the EUV spectral energy distribution for comparison with theoretical models. We find a very high nuclear abundance, $\sim 3.0$ times solar, with clear evidence of a nuclear enhancement of N and He, possibly caused by massive star formation in the extended ($\sim 100$pc) central disk structure. The circum-nuclear narrow-line region spectrum is fit by a radiation pressure dominated photoionisation model model with an input EUV spectrum from a Black Hole with mass $5\times10^7 M_{\odot}$ radiating at $\sim 0.1$ of its Eddington luminosity. The bolometric luminosity is closely constrained to be $\log L_{\mathrm bol.} = 44.3\pm 0.1$ erg s$^{-1}$. The EUV spectrum characterised by a soft accretion disk and a harder component extending to above 15keV. The ENLR region is extended in the NW-SE direction. The line ratio variation in circum-nuclear spaxels can be understood as the result of mixing \ion{H}{ii} regions with an ENLR having a radius-invariant spectrum.Comment: Accepted for publication in Astronomy and Astrophysics, 14 pages, 13 figure

Skills and Knowledge for Data-Intensive Environmental Research.

The scale and magnitude of complex and pressing environmental issues lend urgency to the need for integrative and reproducible analysis and synthesis, facilitated by data-intensive research approaches. However, the recent pace of technological change has been such that appropriate skills to accomplish data-intensive research are lacking among environmental scientists, who more than ever need greater access to training and mentorship in computational skills. Here, we provide a roadmap for raising data competencies of current and next-generation environmental researchers by describing the concepts and skills needed for effectively engaging with the heterogeneous, distributed, and rapidly growing volumes of available data. We articulate five key skills: (1) data management and processing, (2) analysis, (3) software skills for science, (4) visualization, and (5) communication methods for collaboration and dissemination. We provide an overview of the current suite of training initiatives available to environmental scientists and models for closing the skill-transfer gap

Probing the Physics of Narrow Line Regions in Active Galaxies II: The Siding Spring Southern Seyfert Spectroscopic Snapshot Survey (S7)

Here we describe the \emph{Siding Spring Southern Seyfert Spectroscopic Snapshot Survey} (S7) and present results on 64 galaxies drawn from the first data release. The S7 uses the Wide Field Spectrograph (WiFeS) mounted on the ANU 2.3m telescope located at the Siding Spring Observatory to deliver an integral field of $38\times25$~ arcsec at a spectral resolution of $R=7000$ in the red ($530-710$nm), and $R=3000$ in the blue ($340-560$nm). {From these data cubes we have extracted the Narrow Line Region (NLR) spectra from a 4 arc sec aperture centred on the nucleus. We also determine the H$\beta$ and [OIII]~$\lambda$5007 fluxes in the narrow lines, the nuclear reddening, the reddening-corrected relative intensities of the observed emission lines, and the H$\beta$ and \lOIII\ luminosities {determined from spectra for which the stellar continuum has been removed.} We present a set of images of the galaxies in [OIII]~$\lambda$5007, [NII]~$\lambda$6584 and H$\alpha$ which serve to delineate the spatial extent of the extended narrow line region (ENLR) and {\bf also to} reveal the structure and morphology of the surrounding \HII\ regions. Finally, we provide a preliminary discussion of those Seyfert~1 and Seyfert~2 galaxies which display coronal emission lines in order to explore the origin of these lines.Comment: Accepted for publication 9 Jan 2015, Astrophysical Journal Supplements. 49pages, 8 figure

S7 : Probing the physics of Seyfert Galaxies through their ENLR & HII Regions

Here we present the first results from the Siding Spring Southern Seyfert Spectroscopic Snapshot Survey (S7) which aims to investigate the physics of ~140 radio-detected southern active Galaxies with z<0.02 through Integral Field Spectroscopy using the Wide Field Spectrograph (WiFeS). This instrument provides data cubes of the central 38 x 25 arc sec. of the target galaxies in the waveband 340-710nm with the unusually high resolution of R=7000 in the red (530-710nm), and R=3000 in the blue (340-560nm). These data provide the morphology, kinematics and the excitation structure of the extended narrow-line region, probe relationships with the black hole characteristics and the host galaxy, measures host galaxy abundance gradients and the determination of nuclear abundances from the HII regions. From photoionisation modelling, we may determine the shape of the ionising spectrum of the AGN, discover whether AGN metallicities differ from nuclear abundances determined from HII regions, and probe grain destruction in the vicinity of the AGN. Here we present some preliminary results and modelling of both Seyfert galaxies observed as part of the survey.Comment: 6 pages, 2 figures, Invited Talk at the IAU symposium 30

Probing the Physics of Narrow Line Regions in Active Galaxies III: Accretion and Cocoon Shocks in the LINER NGC1052

We present Wide Field Spectrograph (WiFeS) integral field spectroscopy and HST FOS spectroscopy for the LINER galaxy NGC 1052. We infer the presence of a turbulent accretion flow forming a small-scale accretion disk. We find a large-scale outflow and ionisation cone along the minor axis of the galaxy. Part of this outflow region is photoionised by the AGN, and shares properties with the ENLR of Seyfert galaxies, but the inner ($R \lesssim 1.0$~arcsec) accretion disk and the region around the radio jet appear shock excited. The emission line properties can be modelled by a "double shock" model in which the accretion flow first passes through an accretion shock in the presence of a hard X-ray radiation, and the accretion disk is then processed through a cocoon shock driven by the overpressure of the radio jets. This model explains the observation of two distinct densities ($\sim10^4$ and $\sim10^6$ cm$^{-3}$), and provides a good fit to the observed emission line spectrum. We derive estimates for the velocities of the two shock components and their mixing fractions, the black hole mass, the accretion rate needed to sustain the LINER emission and derive an estimate for the jet power. Our emission line model is remarkably robust against variation of input parameters, and so offers a generic explanation for the excitation of LINER galaxies, including those of spiral type such as NGC 3031 (M81).Comment: Accepted for publication in Astrophysical Journal. 16 pages, 12 figure

Dissecting Galaxies: Separating Star Formation, Shock Excitation and AGN Activity in the Central Region of NGC 613

The most rapidly evolving regions of galaxies often display complex optical spectra with emission lines excited by massive stars, shocks and accretion onto supermassive black holes. Standard calibrations (such as for the star formation rate) cannot be applied to such mixed spectra. In this paper we isolate the contributions of star formation, shock excitation and active galactic nucleus (AGN) activity to the emission line luminosities of individual spatially resolved regions across the central 3 $\times$ 3 kpc$^2$ region of the active barred spiral galaxy NGC$\sim$613. The star formation rate and AGN luminosity calculated from the decomposed emission line maps are in close agreement with independent estimates from data at other wavelengths. The star formation component traces the B-band stellar continuum emission, and the AGN component forms an ionization cone which is aligned with the nuclear radio jet. The optical line emission associated with shock excitation is cospatial with strong $H_2$ and [Fe II] emission and with regions of high ionized gas velocity dispersion ($\sigma > 100$ km s$^{-1}$). The shock component also traces the outer boundary of the AGN ionization cone and may therefore be produced by outflowing material interacting with the surrounding interstellar medium. Our decomposition method makes it possible to determine the properties of star formation, shock excitation and AGN activity from optical spectra, without contamination from other ionization mechanisms.Comment: 16 pages, 12 figures. Accepted for publication in MNRA

Probing the physics of narrow-line regions of Seyfert galaxies I. The case of NGC 5427

The spectra of the extended narrow-line regions (ENLRs) of Seyfert 2 galaxies probe the physics of the central active galaxy nucleus (AGN), since they encode the energy distribution of the ionising photons, the radiative flux and radiation pressure, nuclear chemical abundances and the mechanical energy input of the (unseen) central AGN

Discovery of α-Amidobenzylboronates as Highly Potent Covalent Inhibitors of Plasma Kallikrein

Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin swelling in subcutaneous and submucosal membranes in various locations of the body. Herein, we describe a series of potent α-amidobenzylboronates as potential covalent inhibitors of PKa. These compounds exhibited time-dependent inhibition of PKa (compound 20 IC50 66 nM at 1 min, 70 pM at 24 h). Further compound dissociation studies demonstrated that 20 showed no apparent reversibility comparable to d-Phe-Pro-Arg-chloromethylketone (PPACK) (23), a known nonselective covalent PKa inhibitor