Research

A CHANGE IN THE PARADIGM

The natural world consists of hierarchical levels of complexity that range from subatomic particles and molecules to ecosystems and beyond. This implies that, in order to explain the features and behavior of a whole system, a theory might be required that would operate at the corresponding hierarchical level, i.e. where self-organization processes take place.
In the past, biological research has focused on questions that could be addressed by a reductionist program of genetics. The organism (and its development) is currently considered an epiphenomenon of its genes. However, a profound rethinking of the biological paradigm is now underway and it is likely that such a process will lead to a conceptual revolution emerging from the ashes of reductionism. This revolution implies the search for general principles on which a cogent theory of biology might rely. Because much of the logic of living systems is located at higher levels, it is imperative to focus on them. Indeed, both evolution and physiology work on these levels. Therefore, by no means Systems Biology could be viewed a ‘simple’ ‘gradual’ extension of Molecular Biology.

RESEARCH ACTIVITIES – Phase transitions processes

The pathogenesis of several degenerative diseases, including cancer, mostly relies on a cell/tissue differentiation process ‘gone awry’. A complex system is highly sensitive even to subtle changes in the surrounding milieu (the so-called morphogenetic field) when it enters near some critical points (the bifurcation points leading to metastable states, according to the Waddington’s landscape framework). At these points, the system can experience a phase-transition, leading to different cell fate specification, upon the influence of different physical and molecular factors (namely toxicant, like nicotine). Thereby, it is of priceless value understanding the non-linear dynamics of such processes, to capture their architecture by means a proper mathematical modelling, and how to modify them through a pharmacological approach. Namely, a number of natural compounds – including the natural polyol Inositol (and its phosphate derivatives) – are under scrutiny as they showed to modulate the epithelial-mesenchymal transition (EMT vs MET) by targeting the expression of several genes, the activation of the PI3K pathway, as well as the apoptotic process. By modulating EMT vs MET, we can antagonize the first steps of carcinogenesis as well as the metastatic development. In addition, several compounds may act synergistically with chemotherapy in order to improve the overall survival while decreasing the burden of treatment-related side effects. Modulation of EMT is at the core of the Tumor Reversion process, altogether with the inhibition of the Translationally Controlled Tumor Protein (TCTP), which has been demonstrated to play a critical role in reverting cancerous cells toward dormancy or «normalization». We are investigating such processes by means of a model based on the therapeutic potential represented by protein extracts from embryo fish (Zebra and Trout). Embryo fish extracts promote apoptosis and/or foster differentiation/reversion in different cancer types, both in vitro as well as in vivo, mostly through the activation of specific micro-RNA (miRNAs), including mir-218-5p and mir-125a-5p.
Understanding how both inositol and embryo fish extract modulate phase transitions in cancer cells is therefore of critical relevance for translational medicine.
Critical hubs.
A few pathways represent critical hubs in the process that may lead to different phenotypic commitment, including cancer reversion. A relevant role is played by molecular structure participating in shaping the biodynamic interface that regulate the cell-microenvironment cross talk. The balance between phosphoinositides (PI2 versus PIP3), mostly regulated by PI3K, support the transition from a dormant to an aggressive/invasive phenotype. Altogether with the MAPK-ERK pathway, PI3K modulates the activity of several receptors (HER2, EGFR, IGF1, etc.) and ion channels that collectively transduce external biophysical signals into intracellular stimuli. It does without saying that the two principal pathways (PI3K and ERK) play also a central role in chemotherapy resistance. Identification of molecules that target those pathways – especially by inhibiting their genome transcription – is at the core of the laboratory research activity.
Biophysical constraints.
Phenotypic differentiation is underpinned by a complex molecular program, which involves both complex gene-regulatory circuits (behaving according to a non-linear dynamics) and molecular signaling. However, these factors exert only a permissive role, while a more prominent effect is exercised by internal/external constraints (namely of physical origin), acting as instructive cues. A paradigmatic example is provided by gravity, which showed to be instrumental in shaping cell differentiation in many experimental models. We are investigating how microgravity and other physical cues may efficiently influence cell fate transitions in both normal and pathological conditions. Moreover, as the effects of physical cues are mostly conveyed through the cytoskeleton (CSK) and the specialized cell-to-cell and cell-matrix adhesion structures, we are especially committed in investigating cytoskeleton and nucleoskeleton quantitative structure and dynamics. To achieve a reliable systems biology representation of such complex processes, tailored, mathematical modelling approaches and high performance computing studies are usually developed.
Developmental processes and the theory of the organisms.
Organisms are not machines; thus, they require an investigation based on specific theoretical principles. This approach could solve the vexata quaestio engendered by the debate between the opposite stances of vitalism and mechanicism or reductionism. An impressive body of evidence gathered in the last 40 years conclusively demonstrates that the general system’s configuration (architecture), and the dynamic relationships established with the surrounding milieu dictate the function of each molecular factor and not the other way around. Therefore, the central actor of biological processes is the organism. Embracing the organism-based approach permits addressing the issue of biological complexity in very different terms from those usually debated in physics and mathematics. This framework can recognize the relevance of «emergence» i.e., the appearance of novel properties and structures observed at a higher level that cannot be explained entirely in terms of lower-level phenomena. Instead, biological phenomena are strongly influenced by constraints and cues, which arise from higher levels and whose dynamics can be understood as a regimen ruled by «closure of constraints». These considerations explain why a systems biology approach could help establish a new promising methodology for solving issues that a reductionist-based investigation cannot address.
Studies of the developmental dynamics points primarily on early oocyte/embryo differentiating processes occurring during the early life stages. We are investigating pathways involved in oocyte maturation (namely those involving steroidogenesis) by pointing out how changes in the overall cytoskeleton organization and in the oocyte/microenvironment cross talk can efficiently modulate oocyte maturation and further the implantation process. To address these issues we are using a 2D-3D-models – involving both the oocyte and an experimental reconstituted endometrium-like scaffold – as well as rodents. The modulatory role of natural pharmacological compounds (like inositol, melatonin and α-lipoic acid) is under scrutiny to evaluate is such nutraceutical manipulation can improve and regulate the first steps of the morphogenetic process. A central field of investigation is represented by studies focusing upon the endocrine control of ovarian and breast cells. Furthermore, the potential utility of such compounds in treatment of infertility disorders (like Polycystic Ovary Syndrome) is the subject of intensive research.

MAIN TOPICS

Microenvironment, cytoskeleton, epithelial-mesenchymal reversion, tumor reversion, PI3K-pAKT, ERK, miRNAs, steroidogenesis, receptors (ER, HER2, IGF1, FSHr, EGFR), ion channels, TCTP, MDM2, critical transitions, complexity, constraints, Embryo Fish Extracts (FEEs), Grape Seed Extracts (GSEs), Inositol, melatonin.

Current research programs:

– ONCOTROUT and ONCOTROUT-MICE: these projects aim at investigating how miRNAs extracts from trout embryos induce tumor reversion in a model of liver cancer, as well as in vivo, with rodents bearing hepatic tumors.
– PI3K-ERK. The project focuses on the joint inhibition of the PI3K-pAKT and MAPK-ERK pathways induced by inositol. A central is the identification of those miRNAs (namely miR-125a-5p) through which inositol exerts its inhibitory effects.
– PI3K-ER+: this program specifically strives to understand the differential regulation of the PI3K/ERK pathway in ER+ breast cancer cells through the complementary and synergistic activity of different drugs.
– PI3K and CT resistance. The project aims at verifying how addition of some natural factors (including inositol, grape seed extracts, FEEs) can prevent chemotherapy (CT) resistance or rescue cancer cells from acquired drug resistance.
– CHIROSTEROIDO. The research program aims at assessing the anti-aromatase activity of D-Chiro-Inositol in ER+ cancer cells, and if this new molecule could overcome the acquired resistance to conventional anti-estrogenic treatments.
– OVODCI. Similarly, this program aims at assessing the anti-aromatase activity of D-Chiro-Inositol in ovarian cells, from both normal and Polycystic Ovary Syndrome (PCOS)
– CALCIFIBRO. The project is currently ongoing to assess how inositol (and other natural compounds) can modify collagen and fibronectin release from the tumor microenvironment.
– OVOINO II. This research program is the continuation of a previous one (Ovoino I), in which we demonstrated that myo-Inositol and D-Chiro-Inositol exert opposite function upon steroidogenesis in ovarian cells. This program will investigate the steroidogenesis modulation in human cells.

SCIENTIFIC COMMITTEE.
Prof. Mariano Bizzarri, Ph.D, MD, from the Experimental Medicine Department of the Sapienza University, heads the Laboratory.
Senior and Junior scientists: Noemi Monti, Valeria Fedeli, Alessandro Querqui, Aurora Piombarolo, Guglielmo Lentini.
Consulting committee: Prof. Antonio Angeloni, Cinzia Marchese Marco Tafani, Francesco Fedele, David Della Morte, Agostino Tafuri, Franco Marinozzi, Fabiano Bini, Andrea Fuso, Alessandro Giuliani, Angela Catizone, Giulia Ricci, Roberto Verna, Andrea Pensotti.
Corresponding scientists: Jan Brabek (Prague University); Ana Soto, Carlos Sonnenschein (TUFT Medical School, Boston), Adolfo Saiardi (University College, London), Andras Paldi (École des Hautes Études, Paris), Giuseppe Longo, Maël Montevil (ENS, Paris), Scott F. Gilbert (Swarthmore College, USA), Masami Murakami (Gunma University, Japan). Halim Harrath (KSU, Riyadh, Saudi Arabia), Masa Tsuchiya (SEIKO Life Science Laboratory, Japan), Oleg Naimark (Polytechnique University, perm, Russia). Zdravko Kamenov (Sofia University, Bulgaria), Josè Nieto-Villar (La Havana University, Cuba).

LIFE IN SPACE: AN UNEXPECTED THOUGHTFUL MODEL

Gravity has constantly influenced both physical and biological phenomena throughout Earth’s history. The gravitational field has played a major role in shaping evolution when life moved from water to land. However, gravity may influence in a more deep and subtle fashion the way the cells behave and build themselves. Cells may indeed ‘sense’ changes in the microgravity field through (1) an indirect mechanism (mainly based on the modification of physical properties of their microenvironment); (b) the development of specialized structures for the mechanical perception and transduction of gravitational forces (like the cytoskeleton); (c) changes in the dynamics of enzymes kinetics or protein network self-assembly. It is worth noting that the latter two processes are dramatically affected by non-equilibrium dynamics. Non-linear dynamical processes far from equilibrium, involve an appropriate combination of reaction and diffusion, and the pattern arising from those interactions are tightly influenced by even minimal changes in reactant concentrations or modification in the strength of the morphogenetic field. Processes of this kind are called dissipative structures, given that a consumption of energy is required to drive and maintain the system far from equilibrium. That prerequisite is needed in order to allow the system to promptly change its configuration, according to the system’s needs. In turn, the dissipative energy provides the thermodynamic driving force for the self-organization processes. Accordingly to some preliminary results, gravity seems to influence non-equilibrium processes (like the cytoskeleton reorganization), acting as an ‘inescapable’ constraint that obliges living beings to adopt only a few configurations among many others. By ‘removing’ the gravitational field, living structures are free to recover more degrees of freedom, thus acquiring new phenotypes and new functions/properties.

These data raise several crucial questions. Some of these entail fundamentals of theoretical biology, as they question the gene-centric paradigm, according to which biological behavior can be explained by solely genetic mechanisms. Indeed, influence of physical cues in biology (and, in particular, on gene expression) is still now largely overlooked. This is why it has been argued that the ultimate reason for human space exploration is precisely to enable us to discover ourselves. Undoubtedly, the microgravity space-field presents an unlimited horizon for investigation and discovery. Controlled studies conducted in microgravity can advance our knowledge, providing amazing insights into the biological mechanism underlying physiology as well as many relevant diseases, like cancer. Thereby, space-based investigations may serve as a novel paradigm for innovation in basic and applied science.

RESEARCH ACTIVITIES – Phase transitions processes

Space biomedicine and biotechnology studies are chiefly developed according to the following area of interest:

1. THEORETICAL STUDIES and BASIC BIOLOGY. The theoretical foundations that allow us to understand how gravity can significantly influence numerous processes and functions of living structures are still essentially unknown. The interactions occur according to different mechanisms and principles in relation to the level of observation – organism as a whole, organs, cells – and the development of an integrated project. These themes requires in particular the possibility of studying critical hubs and pathways (like the endocrine control) in 3D-models, properly integrated with microfluidic devices and real-time imaging, allowing for extended periods of investigation. Furthermore, a specific area of investigation is related to the discovery of useful molecules (e.g. antibiotics, anti-inflammatories, MABs, protein crystals) produced by unicellular organisms in weightlessness. The lab will develop systematic access solutions to space laboratories currently available (e.g. ISS) and in the near future (e.g. AXIOM, SpaceRider), and shall dedicate facilities for research, manufacturing and testing in space. A special interest is currently driven to the development of a test strategy (e.g. which facilities to use, which key partners to include) to optimize availability and cost of results based on the scientific requirements generated. Other area of interest include: specification and spatialization of the infrastructure(s) for the generation of innovative molecules, which can be hosted within the platforms available and under development; study of natural molecules for their ability to counteract the effects induced by radiation, insulin resistance, hormonal alterations, muscle damage, oxidative effects. 

2. PERSONALIZED ASTRONAUT MEDICINE

The need to develop wearable sensors and systems capable of detecting a useful set of parameters of physiological interest constitutes a critical need for the purposes of human space exploration and colonization of the Moon in the context of identifying new markers of muscle damage…

Investigate solutions related to the use of sensors (not necessarily wearable) useful for a predictive interpretation of patterns related to the health of astronauts. Definition of environmental, PA and interface requirements for diagnostic sensor systems. Spatialization of sensor systems for diagnostics. Creation of the data collection and processing platform, potentially using AI algorithms. Ergonomics studies in VR related to the use of sensors for diagnostics by the crew. Development of augmented reality applications to support the crew in the correct use of sensors for diagnostics. Ground validation on patient/volunteer cohorts affected by pathologies similar to dysfunctions detected in space.

Identification of the physiological needs to be supported and monitored in long-term missions with specific attention to the human transfer phases (e.g. journey to Mars), preliminary identification of transfer strategies (e.g. torpor, wake/sleep shifts), solution support architectures (e.g. capsules for transfer), identification of the need for physiological support (sensors, muscle tissue stimulation, support for effects on the bone system), identification of variables impacting the genome (e.g. passage within variable magnetic fields), architectures for using the identified solutions (e.g. sensors, stimulators), use of AI for physiological support and diagnosis.

3. FOOD FOR SPACE

Ensuring the correct nutrition of astronauts is an absolute imperative whose relevance has grown over the years. Define the correct metabolic fingerprint of astronauts in a microgravity or reduced gravity environment or in any case different from that of Earth. Support the correct development of astronauts’ metabolism, also through nutraceutical tools. Definition of the microbiota characteristics that the space environment must guarantee to astronauts and therefore identify the aspects deriving from and affecting Life Support systems. Design and implementation of systems for the in situ production of food for astronauts: development of production systems in a microgravity environment, or in any case with gravity different from Earth (Moon 0.16 g, Mars 0.38 g); integration of the water cycle with food production systems for astronauts. Study of plants and water systems for the purification and decontamination of environments.

Understanding of the impact of macro and micronutrients in conditions of reduced microgravity, not only in nutritional but also physiological terms (e.g. impact on tissues, metabolic impact, hormonal impact), analysis of differentiated nutritional models for long-term missions.

OBJECTIVES

The main objective of the initiative is to pool the capabilities and potential of the partners, starting an activity that is capable of self-sustaining, generating scientific and economic results in the context of the New Space Economy.

Technology Transfer Table. The laboratory’s activities will be enhanced thanks to the creation of a Technology Transfer Table, to which stakeholders and research institutions interested in the terrestrial applications of the results achieved in space will be invited, according to a scheduled deadline. A first operational opportunity will be offered by the projects relating to Food for Space, with a roundtable coordinated by the Ministry of Agriculture in which the Space Biomed Laboratory will participate, together with representatives of the ASI and the agri-food companies involved. The objective is obviously aimed at valorizing patents and industrial applications, through the possible establishment of spin-offs and startups.

Educational. The Laboratory intends to launch, in cooperation with La Sapienza University and TAS-Italia, – also involving other actors such as the Italian Space Agency, the Centre for AeroSpace Research (CRAS), and the Pratica di Mare Flight Experimental Center of the Air Force – an educational program with the launch of advanced training courses, masters and specific teachings within the context of the current degree courses in Medicine and Space Engineering. The teaching commitment component, associated with research, is considered preparatory for the training of highly specific skilled personnel who could tomorrow find work placements in public structures and private bodies in the sector. In this capacity the laboratory will stipulate agreements with research institutions and Italian and foreign companies for the creation of advanced training courses. To this end, a specific website and appropriate communication strategies will be set up.

Deliverables. Once fully operational, the collaboration will allow the marketing of a series of services for the implementation of medical and biological tests and experiments on the ground and in orbit, to be directed towards industries, space agencies, research institutions and pharmaceutical companies. For this purpose, a partner may be defined with the specific purpose of representing the team as a Service Provider towards private customers and the possibility of expanding the partnership between the University and Thales Alenia Space by involving other entities and companies.

Current research programs:

Ongoing research programs. The Sapienza team is already committed in a number of research studies, including:
– OVOSPACE: evaluation of endocrine function in microgravity. This program is supported by an investigation performed on board of the International Space Station (ISS).
– ORION: pharmacological modulation of ovarian alterations induced by microgravity. This program is supported by an investigation performed on board of the ISS.
– GRAVI-HEART: evaluation of new markers of muscle damage (heart-muscle) in hypoxia/microgravity and development of sensors for salivary analysis
– MONSTRE: PNRR-PE15 – Spoke 9: analysis of markers and pathways of muscle damage. The program is included in the National PNRR program

THE SCIENTIFIC GROUP.

SAPIENZA UNIVERSITY. The Sapienza team has acquired – since 2005 – a role of national pre-eminence for studies conducted in microgravity. The laboratory owns a Random Positioning Machine – through which it carries out experiments in microgravity – and has participated/is participating in space missions on the International Space Station. The focus is to investigate the functional behavior, morphological characterization and gene/protein expression during microgravity. A patent, related to the discovery of antibiotics produced in microgravity is currently ongoing.

THALES ALENIA SPACE. The TASI team that will support the Laboratory has great systems and specialist know-how in the field of human space missions and is interested in continuing its R&D activity in support of the development of its products (e.g. SPACE HOME, Space Rider), in field of inhabited modules, and participation in the ASI/ESA/NASA and Commercial programs (e.g. AXIOM).

Furthermore, there is great interest in supporting technologies that will support life in long-duration missions, requiring the implementation of new engineering concepts related to life science, such as transfer pods, life support control systems, extremely more complicated and complex than current systems.

The main themes related to the activities planned for the Laboratory are:

• Development of greenhouses and systems for food supply in orbit, in transit and/or on the lunar/Martian surface

• Design and implementation of medical payloads, sensors and human physiology experiments on space stations

• Integrability of wearable systems/sensors in space structures for radiation protection

• Astronaut Digital Twin

• Test facilities and software simulators for the characterization of systems and the space environment

• Space systems for «In-orbit manufacturing» and «pharmaceutics in space» applications

• Ability to design complex systems such as transport modules fully equipped with capsules for the transfer and permanent support of human life on distant planets.

SCIENTIFIC COMMITTEE. Prof. Mariano Bizzarri, Ph.D, MD, from the Experimental Medicine Department of the Sapienza University, heads the Laboratory.

Members of the Board: Antonio Angeloni, Cinzia Marchese (Dept. of Experimental Medicine), Giorgio Boscheri, Ivano Musso (Thales Alenia Space).

Consulting committee: Marco Tafani, Francesco Fedele, Paolo Gaudenzi, David Della Morte, Agostino Tafuri, Franco Marinozzi, Fabiano Bini, Andrea Fuso, Alessandro Giuliani, Angela Catizone, Giulia Ricci.

Senior and Junior scientists: Noemi Monti, Valeria Fedeli, Alessandro Querqui, Aurora Piombarolo, Guglielmo Lentini.

LABORATORY LAYOUT AND FACILITIES

The laboratory makes use of numerous equipment and instruments located in the main center in via Scarpa 16 (building RM039), also distributed in other structures within the University. The main ones of which are:
• Laboratory of simulated weightlessness. The lab owns a Random Positioning Machine through which selective gravity values – from the almost ~0g of the International Space Station to the 0.16 and 0.38 g of the Moon and Mars respectively – can be properly simulated and investigated.
Laboratory of Analytical studies. This section includes HPLC/MS devices for advanced analytical studies.
Laboratory of Molecular Biology. Within this section Western-blot and immunofluorescence analysis can be performed, altogether with investigations based upon PCR. The section can provide cell and organoids cultures, in both 2D and 3D.
Laboratory of Confocal Microscopy. This section deals with morphological and ultra-structural studies carried out on cells, tissues and organoids.
Animal facility. The animal facility support in vivo experiments for animal housing and management.

The Somatic Mutation Theory (SMT) of carcinogenesis encompasses significant inconsistencies. Indeed, the increasingly burden of unexplained paradoxes and shortfalls is driving the current carcinogenesis theory toward a blind alley.

Ignoring these paradoxes is unsustainable. By avoiding these conundrums, the scientific community is depriving itself of the opportunity to achieve real progress in this important biomedical field.

Environment-organism interaction: the epigenetic point of view.

What molecular mechanisms regulate cell senescence and organism aging? Why aging processes show different onset, duration and intensity in different subjects? Why the normal and healthy aging could deviate toward a pathological condition? Why so many diseases are age-related? The complexity of the aging processes makes it difficult to simply answer to these questions. Moreover a bias is added by the possibility that changes leading to pathological aging originate earlier in life and show a more or less “asymptomatic” period. Paradoxically, exactly these two features could suggest one possible answer: epigenetic modifications retain the ability to orchestrate complex processes that result in altered gene expression and can reveal the associated, pathological, phenotype even after many years. Moreover, epigenetic modifications can represent the effectors mediating the environmental insults that a subject encounters during his life.

A section of the Systems Biology Group lab carries on neuro-epigenetic studies with particular interest toward the role of DNA methylation in neurodegenerative processes. This line of the research, aimed at deciphering the complex interaction between environmental factors or natural compounds (such as S-adenosyl-methionine, vitamin K, inositol, alpha-lipoic acid, inositol) and DNA methylation, found its applicative fields not only in neurodegenerative models but virtually in all the experimental models studied in the lab.

Therefore, cancer research should be reframed by embracing new theoretical perspectives, taking the cells-microenvironment interplay as the privileged level of observation. This implies we have to adopt radically different premises as such provided by The Tissue Organization Field Theory (TOFT), according to which cancer arises as a consequence of altered cross-talk among cells and their microenvironment. In turn, modification in the cell-stroma interaction may efficiently modulate key phase-transition, thus promoting true tumor reversion.

However, experimental and clinical evidences suggest that cancers can be induced to become quiescent, differentiate, die or form completely normal tissues, if provided with the correct set of complex signals, as conveyed by embryonic tissues or other microenvironmental cues.

These data suggest that by manipulating cancer microenvironment may help in opening new avenues for therapeutic solutions.