Computational biology helps understand how polyploid giant cancer cells drive tumor success

Precision and organization govern the cell cycle, ensuring normal proliferation. However, some cells may undergo abnormal cell divisions (neosis) or variations of mitotic cycles (endopolyploidy). Consequently, the formation of polyploid giant cancer cells (PGCCs), critical for tumor survival, resistance, and immortalization, can occur. Newly formed cells end up accessing numerous multicellular and unicellular programs that enable metastasis, drug resistance, tumor recurrence, and self-renewal or diverse clone formation. An integrative literature review was carried out, searching articles in several sites, including: PUBMED, NCBI-PMC, and Google Academic, published in English, indexed in referenced databases and without a publication time filter, but prioritizing articles from the last 3 years, to answer the following questions: (i) “What is the current knowledge about polyploidy in tumors?”; (ii) “What are the applications of computational studies for the understanding of cancer polyploidy?”; and (iii) “How do PGCCs contribute to tumorigenesis?

Exploiting Self-Organization in Bioengineered Systems: A Computational Approach

The productivity of bioengineered cell factories is limited by inefficiencies in nutrient delivery and waste and product removal. Current solution approaches explore changes in the physical configurations of the bioreactors. This work studies the possibilities of exploiting self-organizing vascular networks to support producer cells within the factory. A computational model simulates de novo vascular development of endothelial-like cells and the resultant network functioning to deliver nutrients and extract product from the cell culture. Microbial factories with vascular networks are evaluated for their scalability, robustness, and productivity compared to the cell factories without a vascular network. Initial studies demonstrate at least an order of magnitude increase in production is possible; the system can be scaled up, and that the self-organization of the efficient vascular network is robust. The work suggests that bioengineered multicellularity may offer efficiency improvements difficult to achieve with physical engineering approaches

Transcriptome analysis of the hypothalamic-pituitary-adrenal axis in the experimentally domesticated fox

Variation in activity of the hormonal stress response, or hypothalamic-pituitary-adrenal (HPA) axis, has been associated with different personality traits and coping styles in humans and animals, while its dysregulation has been implicated in psychological disorders. The molecular basis of HPA axis regulation, however, is not yet well understood. Here, foxes selectively bred for tameness or aggression are used as a model to investigate differences in regulation of the HPA axis. Activity of this axis is markedly reduced in tame compared to aggressive foxes, with reduced levels of HPA axis hormones such as adrenocorticotrophic hormone (ACTH) and cortisol both basally and in response to a stressor. Gene expression differences were analyzed using RNA sequencing in the anterior pituitary and adrenal glands of foxes from the tame and aggressive lines, and variant analysis was performed on RNA reads from hypothalamus, anterior pituitary, and adrenal tissues from the same foxes. Pituitary analysis revealed expression differences in genes related to exocytosis and cellular signaling; adrenals analysis identified differences in similar pathways, in addition to genes related to fatty acid and cholesterol synthesis. Variant analysis also implicated cell signaling and exocytosis, as well as ion transport and DNA damage repair. These findings suggest the importance of regulation of hormone release in the control of ACTH and cortisol levels. They also suggest that metabolism of precursors to cortisol, such as fatty acids and cholesterol, may be of greater importance in HPA axis regulation than synthesis of cortisol itself. Finally, in conjunction with previous genomic findings, they suggest an association between DNA repair mechanisms and selection for tameness. These findings provide possible new lines of investigation into biological underpinnings of the phenotypic differences between the tame and aggressive lines of foxes. More broadly, as the tame foxes are considered experimentally domesticated, the findings from this project may prove applicable to HPA axis regulation differences associated with domestication in other species. Additionally, a deeper understanding of HPA axis regulation and dysregulation may be applicable both to variation in the normal population, particularly as related to behavioral traits such as coping styles, and to a number of psychiatric disorders in humans, as well as to behavioral disorders in other species, such as dogs

On the genetics of intracranial aneurysm and on growth factor induced angiogenesis in the murine brain

Cerebrovascular diseases continue to challenge us by robbing lives and leaving many disabled still in their prime working age. Some cerebrovascular diseases are more acute in nature, and some erode the quality of life over a long period of time. A life-threatening form of acute cerebrovascular disease is brought on by the rupture of an intracranial aneurysm (IA). Most IAs are berry-shaped pouches at the forking site of cerebral arteries. Since according to autopsy results, 2-5% of the population harbours IA, it is a common disease. Most IA go unnoticed during one s lifetime, however, often the first symptom they give is their deadly rupture. Likely, both environmental factors and a compound genetic susceptibility, contribute to the risk of IA, making it a complex disease. The aim of studies I-III was to test whether in humans common genetic variants contribute to the susceptibility to IA (I,II), and to seek genetic evidence for their pathomechanism (III). In multinational genome-wide association studies (I,II) we identified 5 loci with strong statistical evidence of association with IA, and a further 14 loci with suggestive evidence. Further, we found that suggestive IA risk locus at 5q26 is strongly associated with high systolic blood pressure in over 210 000 individuals of European descent, highlighting the connection between hypertension and IA (III). To gain further insight into cerebral vasculopathies and to facilitate the development of novel therapies, in study (IV) we turned our attention to vascular growth factor induced angiogenesis in a model organism. We tested by viral gene transfer the known vascular growth factors in the murine central nervous system and characterised extensively the angiogenesis upon treatment. The aim of the study was to identify the best candidate vascular growth factor(s) for therapeutic brain angiogenesis. We identified placenta growth factor as the most safe and efficient candidate for therapeutic revascularisation of the central nervous system. We envision a placenta growth factor enhanced multiple bur hole indirect extracranial-intracranial bypass as a novel therapeutic approach in the brain, possibly aiding the treatment of diseases such as chronic cerebral hypoperfusion, complex IAs and stroke

Development of novel anticancer agents targeting G protein coupled receptor: GPR120

The G-protein coupled receptor, GPR120, has ubiquitous expression and multifaceted roles in modulating metabolic and anti-inflammatory processes. GPR120 - also known as Free Fatty Acid Receptor 4 (FFAR4) is classified as a free fatty acid receptor of the Class A GPCR family. GPR120 has recently been implicated as a novel target for cancer management. GPR120 gene knockdown in breast cancer studies revealed a role of GPR120-induced chemoresistance in epirubicin and cisplatin-induced DNA damage in tumour cells. Higher expression and activation levels of GPR120 is also reported to promote tumour angiogenesis and cell migration in colorectal cancer. A number of agonists targeting GPR120 have been reported, such as TUG891 and Compound39, but to date development of small-molecule inhibitors of GPR120 is limited. This research applied a rational drug discovery approach to discover and design novel anticancer agents targeting the GPR120 receptor. A homology model of GPR120 (short isoform) was generated to identify potential anticancer compounds using a combined in silico docking-based virtual screening (DBVS), molecular dynamics (MD) assisted pharmacophore screenings, structure–activity relationships (SAR) and in vitro screening approach. A pharmacophore hypothesis was derived from analysis of 300 ns all-atomic MD simulations on apo, TUG891-bound and Compound39-bound GPR120 (short isoform) receptor models and was used to screen for ligands interacting with Trp277 and Asn313 of GPR120. Comparative analysis of 100 ns all-atomic MD simulations of 9 selected compounds predicted the effects of ligand binding on the stability of the “ionic lock” – a characteristic of Class A GPCRs activation and inactivation. The “ionic lock” between TM3(Arg136) and TM6(Asp) is known to prevent G-protein recruitment while GPCR agonist binding is coupled to outward movement of TM6 breaking the “ionic lock” which facilitates G-protein recruitment. The MD-assisted pharmacophore hypothesis predicted Cpd 9, (2-hydroxy-N-{4-[(6-hydroxy-2-methylpyrimidin-4-yl) amino] phenyl} benzamide) to act as a GPR120S antagonist which can be evaluated and characterised in future studies. Additionally, DBVS of a small molecule database (~350,000 synthetic chemical compounds) against the developed GPR120 (short isoform) model led to selection of the 13 hit molecules which were then tested in vitro to evaluate their cytotoxic, colony forming and cell migration activities against SW480 – human CRC cell line expressing GPR120. Two of the DBVS hit molecules showed significant (\u3e 90%) inhibitory effects on cell growth with micromolar affinities (at 100 μM) - AK-968/12713190 (dihydrospiro(benzo[h]quinazoline-5,1′-cyclopentane)-4(3H)-one) and AG-690/40104520 (fluoren-9-one). SAR analysis of these two test compounds led to the identification of more active compounds in cell-based cytotoxicity assays – AL-281/36997031 (IC50 = 5.89–6.715 μM), AL-281/36997034 (IC50 = 6.789 to 7.502 μM) and AP-845/40876799 (IC50 = 14.16-18.02 μM). In addition, AL-281/36997031 and AP-845/40876799 were found to be significantly target-specific during comparative cytotoxicity profiling in GPR120-silenced and GPR120-expressing SW480 cells. In wound healing assays, AL-281/36997031 was found to be the most active at 3 μM (IC25) and prevented cell migration. As well as in the assessment of the proliferation ability of a single cell to survive and form colonies through clonogenic assays, AL-281/36997031 was found to be the most potent of all three test compounds with the survival rate of ~ 30% at 3 μM. The inter-disciplinary approach applied in this work identified potential chemical scaffolds –spiral benzo-quinazoline and fluorenone, targeting GPR120 which can be further explored for designing anti-cancer drug development studies

Drug Repurposing

This book focuses on various aspects and applications of drug repurposing, the understanding of which is important for treating diseases. Due to the high costs and time associated with the new drug discovery process, the inclination toward drug repurposing is increasing for common as well as rare diseases. A major focus of this book is understanding the role of drug repurposing to develop drugs for infectious diseases, including antivirals, antibacterial and anticancer drugs, as well as immunotherapeutics

4,6-Diphenyl-pyridines/pyrimidines and pyrazolo[3,4-d]pyrimidines: promising scaffolds as antiviral, anticancer and theranostic agents.

Nitrogen-based heterocyclic molecules received increasing attention in biological and chemical sciences, becoming a significant moiety in drug design. In this thesis, the research work has been focused on the design and the synthesis of novel derivatives based on the 4,6-diphenyl-pyridine/pyrimidine and pyrazolo[3,4-d]pyrimidine scaffolds as key pharmacophores for their antiviral and anticancer activities, respectively. The first part deals with the design and synthesis of a set of small molecules able to interfere with the Influenza (flu) RNA-dependent RNA Polymerase (RdRp) functions, exploiting the protein-protein interactions (PPIs) approach. An introduction of influenza disease and its pathogens is provided, focusing on the replicative mechanism of the influenza viruses and on the structural and functional information of the flu RdRp. The PPIs of the three polymerase subunits PA, PB1 and PB2, are reported and the inhibitors targeting the heterodimer PA-PB1 are introduced. Finally, the rational design and synthesis of new hybrid compounds bearing the 4,6-diphenyl-pyridine/pyrimidine cores are described togheter with the biological evaluation and molecular modeling studies. The second part of the dissertation focuses on the pyrazolo[3,4-d]pyrimidine compounds, on which both a lead optimization study and a theranostic design have been performed. The pyrazolo[3,4-d]pyrimidines have shown a promising activity both in in vitro and in in vivo as protein kinase inhibitors. The lead optimization study has been performed with the aim of obtaining a new class of derivatives as potent Src kinase inhibitors. The rational design, synthesis and biological analysis of the novel compounds are discussed. A further part of the project is dedicated to the design and development of potential theranostic prodrugs of two promising in-house pyrazolo[3,4-d]pyrimidines, SI306 and SI113. These prodrugs have been synthesized as potential agents for the diagnosis and the treatment of glioblastoma multiforme (GBM). The state of the art on theranostic applications of drugs, whose use is continually increasing, is also described

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Biosimulation of Vocal Fold Inflammation and Healing

Personalized, pre-emptive and predictive medicine is the capstone of contemporary medical care. The central aim of this dissertation is to address clinical challenges in prescribing personalized therapy to patients with acute phonotrauma. Inflammation and healing, which are innate tissue responses to mechanical stress/ trauma, are regulated by a complex dynamic system. A systems biology approach, which combines empirical, mathematical and computational tools, was taken to study the biological complexity of this dynamic system in vocal fold injury.Computational agent-based models (ABMs) were developed to quantitatively characterize multiple cellular and molecular interactions around inflammation and healing. The models allowed for tests of various hypothetical effects of motion-based treatments in individuals with acute phonotrauma. A phonotrauma ABM was calibrated and verified with empirical data of a panel of inflammatory mediators, obtained from laryngeal secretions in individuals following experimentally induced phonotrauma and a randomly assigned motion-based treatment. A supplementary ABM of surgically induced vocal fold trauma was developed and subsequently calibrated and verified with empirical data of inflammatory mediators and extracellular matrix substances from rat studies, for the purpose of gaining insight into the &ldquo net effect &rdquo of cellular and molecular responses at the tissue level.ABM simulations reproduced and predicted trajectories of inflammatory mediators and extracellular matrix as seen in empirical data of phonotrauma and surgical vocal fold trauma. The simulation results illustrated a spectrum of inflammatory responses to phonotrauma, surgical trauma and motion-based treatments. The results suggested that resonant voice exercise may optimize the combination of para- and anti-inflammatory responses to accelerate healing. Moreover, the ABMs suggested that hyaluronan fragments might be an early molecular index of tissue damage that is sensitive to varying stress levels - from relatively low phonatory stress to high surgical stress.We propose that this translational application of biosimulation can be used to quantitatively chart individual healing trajectories, test the effects of different treatment options and most importantly provide new understanding of laryngeal health and healing. By placing biology on a firm mathematical foundation, this line of research has potential to influence the contour of scientific thinking and clinical care of vocal fold injury