Insulin-Like Growth Factor-I (IGF-1), IGF-Binding Protein-3 (IGFBP-3) and mammographic features

Introduction. The IGF system has recently been shown to play an important role in the regulation of breast tumor cell proliferation. However, also breast density is currently considered as the strongest breast cancer risk factor. It is not yet clear whether these factors are interrelated and if and how they are influenced by menopausal status. The purpose of this study was to examine the possible effects of IGF-1 and IGFBP-3 and IGF-1/IGFBP-3 molar ratio on mammographic density stratified by menopausal status. Patients and methods. A group of 341 Italian women were interviewed to collect the following data: family history of breast cancer, reproductive and menstrual factors, breast biopsies, previous administration of hormonal contraceptive therapy, hormone replacement therapy (HRT) in menopause and lifestyle information. A blood sample was drawn for determination of IGF-1, IGFBP-3 levels. IGF-1/ IGFBP-3 molar ratio was then calculated. On the basis of recent mammograms the women were divided into two groups: dense breast (DB) and non-dense breast (NDB). Student’s t-test was employed to assess the association between breast density and plasma level of IGF-1, IGFBP-3 and molar ratio. To assess if this relationship was similar in subgroups of pre- and postmenopausal women, the study population was stratified by menopausal status and Student’s t-test was performed. Finally, multivariate analysis was employed to evaluate if there were confounding factors that might influence the relationship between growth factors and breast density. Results. The analysis of the relationship between mammographic density and plasma level of IGF-1, IGFBP-3 and IGF-1/ IGFBP-3 molar ratio showed that IGF-1 levels and molar ratio varied in the two groups resulting in higher mean values in the DB group (IGF-1: 109.6 versus 96.6 ng/ml; p= 0.001 and molar ratio 29.4 versus 25.5 ng/ml; p= 0.001) whereas IGFBP-3 showed similar values in both groups (DB and NDB). Analysis of plasma level of IGF-1, IGFBP-3 and IGF-1/IGFBP-3 molar ratio compared to breast density after stratification of the study population by menopausal status (premenopausal and postmenopausal) showed that there was no association between the plasma of growth factors and breast density, neither in premenopausal nor in postmenopausal patients. Multivariate analysis showed that only nulliparity, premenopausal status and body mass index (BMI) are determinants of breast density. Conclusions. Our study provides a strong evidence of a crude association between breast density and plasma levels of IGF-1 and molar ratio. On the basis of our results, it is reasonable to assume that the role of IGF-1 and molar ratio in the pathogenesis of breast cancer might be mediated through mammographic density. IGF-1 and molar ratio might thus increase the risk of cancer by increasing mammographic density.

Growth and characterization of III-V compound semiconductor nanostructures by metalorganic chemical vapor deposition

Planar <110> GaAs nanowires and quantum dots grown by atmospheric MOCVD have been introduced to non-standard growth conditions such as incorporating Zn and growing them on free-standing suspended films and on 10° off-cut substrates. Zn doped nanowires exhibited periodic notching along the axis of the wire that is dependent on Zn/Ga gas phase molar ratios. Planar nanowires grown on suspended thin films give insight into the mobility of the seed particle and change in growth direction. Nanowires that were grown on the off-cut sample exhibit anti-parallel growth direction changes. Quantum dots are grown on suspended thin films and show preferential growth at certain temperatures. Envisioned nanowire applications include twin-plane superlattices, axial pn-junctions, nanowire lasers, and the modulation of nanowire growth direction against an impeding barrier and varying substrate conditions.

Growth and application of planar III-V semiconductor nanowires grown with MOCVD

The semiconductor nanowire has been widely studied over the past decade and identified as a promising nanotechnology building block with application in photonics and electronics. The flexible bottom-up approach to nanowire growth allows for straightforward fabrication of complex 1D nanostructures with interesting optical, electrical, and mechanical properties. III-V nanowires in particular are useful because of their direct bandgap, high carrier mobility, and ability to form heterojunctions and have been used to make devices such as light-emitting diodes, lasers, and field-effect transistors. However, crystal defects are widely reported for III-V nanowires when grown in the common out-of-plane <111>B direction. Furthermore, commercialization of nanowires has been limited by the difficulty of assembling nanowires with predetermined position and alignment on a wafer-scale. In this thesis, planar III-V nanowires are introduced as a low-defect and integratable nanotechnology building block grown with metalorganic chemical vapor deposition. Planar GaAs nanowires grown with gold seed particles self-align along the <110> direction on the (001) GaAs substrate. Transmission electron microscopy reveals that planar GaAs nanowires are nearly free of crystal defects and grow laterally and epitaxially on the substrate surface. The nanowire morphology is shown to be primarily controlled through growth temperature and an ideal growth window of 470 +\- 10 °C is identified for planar GaAs nanowires. Extension of the planar growth mode to other materials is demonstrated through growth of planar InAs nanowires. Using a sacrificial layer, the transfer of planar GaAs nanowires onto silicon substrates with control over the alignment and position is presented. A metal-semiconductor field-effect transistor fabricated with a planar GaAs nanowire shows bulk-like low-field electron transport characteristics with high mobility. The aligned planar geometry and excellent material quality of planar III-V nanowires may lead to highly integrated III-V nanophotonics and nanoelectronics.

Seed and vegetative propagation methods for the rare Florida native species Chionanthus pygmaeus (pygmy fringetree)

Chionanthus pygmaeus Small (pygmy fringetree) (Oleaceae) is an endemic and rare Florida species, which has an attractive, small habit giving it great potential for use in managed landscapes. Members of the genus Chionanthus are difficult to propagate via cuttings and possess complex seed dormancies that are not well understood. Conservation of pygmy fringetree and its potential for commercial propagation for use in managed landscapes is contingent on a better understanding of its complex seed dormancy and enhancement of its propagation. I conducted two experiments to assess sexual and asexual propagation methods for pygmy fringetree. The first experiment was conducted to determine what factors are involved in overcoming seed dormancy. Various scarification treatments, which mimicked conditions seeds are exposed to in the wild, were investigated to determine their effects on germination of 20-year-old seeds originally collected from the species’ native range. Treatments included endocarp removal, sulfuric acid, boiling-water, and smoke-water treatments. Prior to treatment initiation, seed viability was estimated to be 12%. Treated seeds went through two cold- and two warm-stratification periods of 4°C and 25°C, respectively, in a dark growth chamber. After 180 days, none of the treatments induced early germination. Seeds were then tested for viability, which was 11%. Seed dormancy of the species is apparently complex, allowing some of the seeds to retain some degree of viability, but without dormancy requirements satisfied. The second experiment was conducted to assess if pygmy fringetree could be successfully propagated via hardwood or root cuttings if the appropriate combination of environmental conditions and hormones were applied. Hardwood and root cuttings were treated with either 1000 ppm IBA talc, 8000 ppm IBA talc, or inert talc. All cuttings were placed on a mist bench in a greenhouse for 9 weeks. Hardwood cuttings were supplemented with bottom heat at 24 °C. No treatments were successful in inducing adventitious root formation. I conclude that pygmy fringetree seeds possess complex dormancy that was not able to be overcome by the treatments utilized. However, this result is confounded by the age of the seeds used in the experiment. I also conclude that vegetative propagation of pygmy fringetree is highly dependent on the time of year cuttings are harvested. Further research of both seed and asexual propagation methods need to be explored before pygmy fringetree can be propagated on a commercial scale.

Estimates of genetic parameters for growth and wood properties in Eucalyptus pellita F. Muell. to support tree breeding in Vietnam

Eucalyptus pellita demonstrated good growth and wood quality traits in this study, with young plantation grown timber being suitable for both solid and pulp wood products. All traits examined were under moderate levels of genetic control with little genotype by environment interaction when grown on two contrasting sites in Vietnam. Eucalyptus pellita currently has a significant role in reforestation in the tropics. Research to support expanded of use of this species is needed: particularly, research to better understand the genetic control of key traits will facilitate the development of genetically improved planting stock. This study aimed to provide estimates of the heritability of diameter at breast height over bark, wood basic density, Kraft pulp yield, modulus of elasticity and microfibril angle, and the genetic correlations among these traits, and understand the importance of genotype by environment interactions in Vietnam. Data for diameter and wood properties were collected from two 10-year-old, open-pollinated progeny trials of E. pellita in Vietnam that evaluated 104 families from six native range and three orchard sources. Wood properties were estimated from wood samples using near-infrared (NIR) spectroscopy. Data were analysed using mixed linear models to estimate genetic parameters (heritability, proportion of variance between seed sources and genetic correlations). Variation among the nine sources was small compared to additive variance. Narrow-sense heritability and genetic correlation estimates indicated that simultaneous improvements in most traits could be achieved from selection among and within families as the genetic correlations among traits were either favourable or close to zero. Type B genetic correlations approached one for all traits suggesting that genotype by environment interactions were of little importance. These results support a breeding strategy utilizing a single breeding population advanced by selecting the best individuals across all seed sources. Both growth and wood properties have been evaluated. Multi-trait selection for growth and wood property traits will lead to more productive populations of E. pellita both with improved productivity and improved timber and pulp properties.

Growth and development of three key summer grasses in Australian cotton systems

Awnless barnyard grass, feathertop Rhodes grass, and windmill grass are important weeds in Australian cotton systems. In October 2014, an experiment was established to investigate the phenological plasticity of these species. Seed of these species were planted in a glasshouse every four weeks and each cohort grown for 6 months. A developmental response to day length was observed in barnyard grass but not in the other species. Days to maturity increased with each planting for feathertop Rhodes and windmill grass for the first six cohorts. Barnyard grass showed a similar pattern in growth for seeds planted from October to December with an increase in the onset of maturity from 51 to 58 days. However, the onset of maturity for cohorts planted between January and March decreased to between 50 and 52 days. All species had a decrease in the total number of panicles produced from the first four plantings. Feathertop Rhodes grass planted in October produced 41 panicles compared to those planted at the end of December producing 30 panicles, barnyard grass had a decrease from 99 to 47 panicles and windmill grass 37 to 15 panicles on average. By comparing the development of these key weed species over 12 months, detailed information on the phenological plasticity of these species will be obtained. This information will contribute to more informed management decisions by improving our understanding of appropriate weed control timings or herbicide rates depending on weed emergence and development.

Seed layer technique for high quality epitaxial manganite films

We introduce an innovative approach to the simultaneous control of growth mode and magnetotransport properties of manganite thin films, based on an easy-to-implement film/substrate interface engineering. The deposition of a manganite seed layer and the optimization of the substrate temperature allows a persistent bi-dimensional epitaxy and robust ferromagnetic properties at the same time. Structural measurements confirm that in such interface-engineered films, the optimal properties are related to improved epitaxy. A new growth scenario is envisaged, compatible with a shift from heteroepitaxy towards pseudo-homoepitaxy. Relevant growth parameters such as formation energy, roughening temperature, strain profile and chemical states are derived.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Examining the growth and evolution of magnetic fields in clusters of galaxies: a numerical perspective

Magnetic fields are ubiquitous in galaxy cluster atmospheres and have a variety of astrophysical and cosmological consequences. Magnetic fields can contribute to the pressure support of clusters, affect thermal conduction, and modify the evolution of bubbles driven by active galactic nuclei. However, we currently do not fully understand the origin and evolution of these fields throughout cosmic time. Furthermore, we do not have a general understanding of the relationship between magnetic field strength and topology and other cluster properties, such as mass and X-ray luminosity. We can now begin to answer some of these questions using large-scale cosmological magnetohydrodynamic (MHD) simulations of the formation of galaxy clusters including the seeding and growth of magnetic fields. Using large-scale cosmological simulations with the FLASH code combined with a simplified model of the acceleration of cosmic rays responsible for the generation of radio halos, we find that the galaxy cluster frequency distribution and expected number counts of radio halos from upcoming low-frequency sur- veys are strongly dependent on the strength of magnetic fields. Thus, a more complete understanding of the origin and evolution of magnetic fields is necessary to understand and constrain models of diffuse synchrotron emission from clusters. One favored model for generating magnetic fields is through the amplification of weak seed fields in active galactic nuclei (AGN) accretion disks and their subsequent injection into cluster atmospheres via AGN-driven jets and bubbles. However, current large-scale cosmological simulations cannot directly include the physical processes associated with the accretion and feedback processes of AGN or the seeding and merging of the associated SMBHs. Thus, we must include these effects as subgrid models. In order to carefully study the growth of magnetic fields in clusters via AGN-driven outflows, we present a systematic study of SMBH and AGN subgrid models. Using dark-matter only cosmological simulations, we find that many important quantities, such as the relationship between SMBH mass and galactic bulge velocity dispersion and the merger rate of black holes, are highly sensitive to the subgrid model assumptions of SMBHs. In addition, using MHD calculations of an isolated cluster, we find that magnetic field strengths, extent, topology, and relationship to other gas quantities such as temperature and density are also highly dependent on the chosen model of accretion and feedback. We use these systematic studies of SMBHs and AGN inform and constrain our choice of subgrid models, and we use those results to outline a fully cosmological MHD simulation to study the injection and growth of magnetic fields in clusters of galaxies. This simulation will be the first to study the birth and evolution of magnetic fields using a fully closed accretion-feedback cycle, with as few assumptions as possible and a clearer understanding of the effects of the various parameter choices.

Early growth response genes 2 and 3 regulate the expression of Bcl6 and differentiation of T follicular helper cells

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GC) and Tfh differentiation requires the function of B cell lymphoma 6 (Bcl6). We have now discovered that early growth response gene (Egr) 2 and 3 directly regulate the expression of Bcl6 in Tfh cells which is required for their function in regulation of GC formation. In the absence of Egr2 and 3, the expression of Bcl6 in Tfh cells is defective leading to impaired differentiation of Tfh cells resulting in a failure to form GCs following virus infection and defects in production of anti-viral antibodies. Enforced expression of Bcl6 in Egr2/3 deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of Egr2/3 which is important for Tfh cell development and Tfh cell mediated B cell immune responses.

Characterization of TRIB2-mediated resistance to anti-cancer drugs

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Hormone-Mediated Gene-Specific Translation Regulation

The central role of translation regulation in the control of critical cellular processes has long been recognized. Yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to specific stimuli has only recently become technically feasible. Using a genetic approach, we have identified new Arabidopsis weak-ethylene insensitive mutants that also display defects in translation, which suggested the existence of a previously unknown molecular module involved in ethylene-mediated translation regulation of components of this signaling pathway. To explore this link in detail, we implemented for Arabidopsis the ribosome-footprinting technology, which enables the study of translation at a whole-genome level at single codon resolution[1]. Using ribosome-footprinting we examined the effects of short exposure to ethylene on the Arabidopsis translatome looking for ethylene-triggered changes in translation rates that could not be explained by changes in transcript levels. The results of this research, in combination with the characterization of a subset of the aforementioned weak-ethylene insensitive mutants that are defective in the UPF genes (core-components of the nonsense-mediated mRNA decay machinery), uncovered a translation-based branch of the ethylene signaling pathway[2]. In the presence of ethylene, translation of a negative regulator of ethylene signaling EBF2 is repressed, despite induced transcription of this gene. These translational effects of ethylene require the long 3´UTR of EBF2 (3´EBF2), which is recognized by the C-terminal end of the key ethylene-signaling protein EIN2 (EIN2C) in the cytoplasm once EIN2C is released from the ER-membrane by proteolytic cleavage. EIN2C binds the 3´EBF2, recruits the UPF proteins and moves to P-bodies, where the translation of EBF2 in inhibited despite its mRNA accumulation. Once the ethylene signal is withdrawn, the translation of the stored EBF2 mRNAs is resumed, thus rapidly dampening the ethylene response. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator. Translation regulatory elements can be located in both 3′ and 5′ UTRs. We are now focusing on the ead1 and ead2 mutants, another set of ethylene-signaling mutants defective in translational regulation. Ribosome-footprinting on the ead1 mutant revealed an accumulation of translating ribosomes in the 5´UTRs of uORF-containing genes and reduction in the levels of ribosomes in the main ORF. The mutant is also impaired in the translation of GFP when this reporter is fused to WT 5´UTR of potential EAD1 targets but not when GFP is fused to the uORF-less versions of the same 5´UTRs. Our hypothesis is that EAD1/2 work as a complex that is required for the efficient translation of mRNAs that have common structural (complex 5´UTR with uORFs) and functional (regulation of key cellular processes) features. We are working towards the identification of the conditions where the EAD1 regulation of translation is required. [1] Ingolia, N. et al. (2009) Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science, 324; 218-222 [2] Merchante, C. et al. (2015) Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2. Cell, 163(3): 684-697

Mechanisms of force-mediated regulation of transcription, chromatin remodeling and cell fate decisions

Tissue mechanics and cellular interactions influence every single cell in our bodies to drive morphogenesis. However, little is known about mechanisms by which cells sense physical forces and transduce them from the cytoskeleton to the nucleus to control gene expression and stem cell fate. We have identified a novel nuclear-mechanosensor complex, consisting of the nuclear membrane protein emerin (Emd), actin and non-muscle myosin IIA (NMIIA), that regulates transcription, chromatin remodeling and lineage commitment. Force-induced enrichment of Emd at the outer nuclear membrane leads to a compensation between H3K9me2,3 and H3K27me3 on constitutive heterochromatin. This strain-induced epigenetic switch is accompanied by the global rearrangement of chromatin. In parallel, forces promote local F-actin polymerization at the outer nuclear membrane, which limits the availability of nuclear G-actin. Subsequently, the reduction of nuclear G-actin results in attenuated global transcription and therefore increased H3K27me3 occupancy to reinforce gene silencing. Restoring nuclear actin levels in the presence of mechanical strain counteracts PRC2-mediated silencing of transcribed genes. This mechanosensory circuit is also observed in vivo. Depletion of NMIIA in mouse epidermis leads to decreased H3K27me3 levels and precocious lineage commitment, thus abrogating organ growth and patterning. Our results reveal how mechanical signals regulate nuclear architecture, chromatin organization and transcription to control cell fate decisions.

The natural plant stress elicitor cis-jasmone causes cultivar-dependent reduction in growth of the stink bug, Euschistus heros and associated changes in flavonoid concentrations in soybean, Glycine max.

To test the hypothesis that the plant stress related elicitor cis-jasmone (cJ) provides protection in soybean pods against the seed-sucking stink bug pest, Euschistus heros, the growth of E. heros on cJ-treated pods was investigated using three soybean cultivars differing in insect susceptibility, i.e. BRS 134 (susceptible), IAC 100 (resistant) and Dowling (resistant). E. heros showed reduced weight gain when fed cJ-treated Dowling, whereas no effect on weight gain was observed when fed other treated cultivars. Using analysis of variance, a three factor (cultivar x treatment x time) interaction was observed with concentrations of the flavonoid glycosides daidzin and genistin, and their corresponding aglycones, daidzein and genistein. There were increases in genistein and genistin concentrations in cJ-treated Dowling at 144 and 120 h post treatment, respectively. Higher concentrations of malonyldaidzin and malonylgenistin in Dowling, compared to BRS 134 and IAC 100, were observed independently of time, the highest concentrations being observed in cJ-treated seeds. Levels of glycitin and malonylglycitin were higher in BRS 134 and IAC 100 compared to Dowling. Canonical variate analysis indicated daidzein (in the first two canonical variates) and genistein (in the first only) as important discriminatory variables. These results suggest that cJ treatment leads to an increase in the levels of potentially defensive isoflavonoids in immature soybean seeds, but the negative effect upon E. heros performance is cultivar-dependent.

Peptidomimetic ligands for tuning integrin-mediated signalling: rational design, synthesis and theranostic applications

Integrins are α/β-heterodimeric transmembrane adhesion receptors that mediate cell-cell and cell-ECM interactions. Integrins are bidirectional signalling receptors that respond to external signals (“outside-in” signalling) and in parallel, transduce internal signals to the matrix (“inside-out” signalling), to regulate vital cellular functions including migration, survival, growth and differentiation. Therefore, dysregulation of these tightly regulated processes often results in uncontrolled integrin activation and abnormal tissue expression that is responsible for many diseases. Because of their important roles in physiological and pathological events, they represent a validated target for therapeutic and diagnostic purposes. The aim of the present Thesis was focused on the development of peptidic ligands for α4β1 and αvβ3 integrin subtypes, involved in inflammatory responses (leukocytes recruitment and extravasation) and cancer progression (angiogenesis, tumor growth, metastasis), respectively. Following the peptidomimetic strategy, we designed and synthesized a small library of linear and cyclic hybrid α/β-peptidomimetics based on the phenylureido-LDV scaffolds for the treatment of chronic inflammatory autoimmune diseases. In order to implement a fast and non-invasive diagnostic method for monitoring the course of the inflammatory processes, a flat glass-surface of dye-loaded Zeolite L-crystal nanoparticles was coated with bioactive α4β1-peptidomimetics to detect specific integrin-expressing cells as biomarkers of inflammatory diseases. Targeted drug delivery has been considered a promising alternative to overcome the pharmacokinetic limitations of conventional anticancer drugs. Thus, a novel Small-Molecule Drug Conjugate was synthesized by connecting the highly cytotoxic Cryptophycin to the tumor-targeting RGDfK-peptide through a protease-cleavable linker. Finally, in view to making the peptide synthesis more sustainable and greener, we developed an alternative method for peptide bonds formation employing solvent-free mechanochemistry and ultra-mild minimal solvent-grinding conditions in common, inexpensive laboratory equipment. To this purpose, standard amino acids, coupling agents and organic-green solvents were used in the presence of nanocrystalline hydroxyapatite as a reusable, bio-compatible inorganic basic catalyst.