Dissertations / Theses on the topic 'Brain on a chip'

Many decades ago, Carver Mead established the foundations of neuromorphic systems. Neuromorphic systems are analog circuits that emulate biology. These circuits utilize subthreshold dynamics of CMOS transistors to mimic the behavior of neurons. The objective is to not only simulate the human brain, but also to build useful applications using these bio-inspired circuits for ultra low power speech processing, image processing, and robotics. This can be achieved using reconfigurable hardware, like field programmable analog arrays (FPAAs), which enable configuring different applications on a cross platform system. As digital systems saturate in terms of power efficiency, this alternate approach has the potential to improve computational efficiency by approximately eight orders of magnitude. These systems, which include analog, digital, and neuromorphic elements combine to result in a very powerful reconfigurable processing machine.

The brain is highly efficient in how it processes information and tolerates faults. Significant research is therefore focused on harnessing this efficiency and to build artificial neural systems that can emulate the key information processing principles of the brain. However, existing software approaches are too slow and cannot provide the dense interconnect for the billions of neurons and synapses that are required. Therefore, it is necessary to look to new custom hardware architectures to address this scalability issue and to enable the deployment of brain-like embedded systems processors. This thesis presents a novel Hierarchical Networks-on-Chip (H-NoC) architecture for SNN hardware, which aims to address the scalability issue by creating a modular array of clusters of neurons using a hierarchical structure of low and high-level routers. The proposed H-NoC architecture can be viewed as a flat 3D structure, which mimics to a degree the hierarchical organisation found in biological neural systems. Furthermore, this H-NoC architecture also incorporates a novel spike traffic compression technique to exploit SNN traffic patterns and locality between neurons, thus reducing traffic overhead and improving throughput on the network. In addition, novel adaptive routing capabilities between clusters, balance local and global traffic loads to sustain throughput under bursting activity. The thesis also reports on analytical results based on five large-scale scenarios, which demonstrate the scalability of the proposed H-NoC approach under varied traffic load intensities. Simulation and synthesis analysis using 65-nm CMOS technology demonstrate a good trade-off between high throughput and low cost area/power footprints per cluster. The thesis concludes with results on the mapping of the IRIS and Wisconsin Breast Cancer data sets using the proposed H-NoC architecture, and validates in FPGA hardware, the analytical performance. Most importantly, the FPGA implementation of both benchmarks demonstrates that the H-NoC architecture can provide up to 100x speedup when compared with biological real-time system equivalents.

Glioblastoma multiforme (GBM) is a malignant brain tumour for which there is currently no effective treatment regime. It is thought to develop due to the overexpression of a number of genes, including the epidermal growth factor receptor (EGFR), which is found in over 40% of GBM. Novel forms of treatment such as antisense therapy may allow for the specific inhibition of aberrant genes and thus they are optimistic therapies for future treatment of GBM. Oligodeoxynucleotides (ODNs) are small pieces of DNA that are often modified to increase their stability to nucleases and can be targeted to the aberrant gene in order to inhibit it and thus prevent its transcription into protein. By specifically binding to mRNA in an antisense manner, they can bring about its degradation by a variety of mechanisms including the activation of RNase H and thus have great potential as therapeutic agents. One of the main drawbacks to the utilisation of this therapy so far is the lack of techniques that can successfully predict accessible regions on the target mRNA that the ODNs can bind to. DNA chip technology has been utilised here to predict target sequences on the EGFR mRNA and these ODNs (AS 1 and AS2) have been tested in vitro for their stability, uptake into cells and their efficacy on cellular growth, EGFR protein and mRNA. Studies showed that phosphorothioate and 2'O-methyl ODNs were significantly more stable than phosphodiester ODNs both in serum and serum-free conditions and that the mechanism of uptake into A431 cells was temperature dependent and more efficient with the use of optimised lipofectin. Efficacy results show that AS 1 and AS2 phosphorothioate antisense ODNs were capable of inhibiting cell proliferation by 69% ±4% and 65% ±4.5% respectively at 500nM in conjunction with a non-toxic dose of lipofectinTM used to enhance cellular delivery. Furthermore, control ODN sequences, 2' O-methyl derivatives and a third ODN sequence, that was found not to be capable of binding efficiently to the EGFR mRNA by DNA chip technology, showed no significant effect on cell proliferation. AS 1 almost completely inhibited EGFR protein levels within 48 hours with two doses of 500nM AS 1 with no effect on other EGFR family member proteins or by control sequences. RNA analysis showed a decrease in mRNA levels of 32.4% ±0.8% but techniques require further optimisation to confirm this. As there are variations found between human glioblastoma in situ and those developed as xenografts, analysis of effect of AS 1 and AS2 was performed on primary tumour cell lines derived from glioma patients. ODN treatment showed a specific knockdown of cell growth compared to any of the controls used. Furthermore, combination therapies were tested on A431 cell growth to determine the advantage of combining different antisense approaches and that of conventional drugs. Results varied between the combination treatments but indicated that with optimisation of treatment regimes and delivery techniques that combination therapies utilising antisense therapies would be plausible.

How can science possibly understand the organ through which the Universe knows itself? The scientific method can be used to study how electro-chemical signals represent information in the brain. However, modelling it by simulating its structures and functions is a computation- and communication-intensive task. Whilst supercomputers offer great computational power, brain-scale models are challenging in terms of communication overheads and power consumption. Dedicated neural hardware can be used to enhance simulation performance, but it is often optimised for specific models. While performance and flexibility are desirable simulation features, there is no perfect modelling platform, and the choice is subordinate to the specific research question being investigated. In this context SpiNNaker constitutes a novel parallel architecture, with communication and memory accesses optimised for spike-based computation, permitting simulation of large spiking neural networks in real time. To exploit SpiNNaker's performance and reconfigurability fully, a neural network model must be translated from its conceptual form into data structures for a parallel system. This thesis presents a flexible approach to distributing and mapping neural models onto SpiNNaker, within the constraints introduced by its specialised architecture. The conceptual map underlying this approach characterizes the interaction between the model and the system: during the build phase the model is placed on SpiNNaker; at runtime, placement information mediates communication with devices and instrumentation for data analysis. Integration within the computational neuroscience community is achieved by interfaces to two domain-specific languages: PyNN and Nengo. The real-time, event-driven nature of the SpiNNaker platform is explored using address-event representation sensors and robots, performing visual processing using a silicon retina, and navigation on a robotic platform based on a cortical, basal ganglia and hippocampal place cells model. The approach has been successfully exploited to run models on all iterations of SpiNNaker chips and development boards to date, and demonstrated live in workshops and conferences.

The study of the central nervous system represents a great challenge in the field of neuroscience. For years, various techniques have been developed to study neuronal cells in-vitro as it is difficult to conduct in-vivo experiments due to ethical problems deriving from its anatomical location. Consequently, both in-vivo and in-vitro animal models have been used extensively to gain new insights into basic functioning principles of neuronal tissue and therapeutic approaches for brain diseases. Over time, we have seen that there is a poor correlation between the clinical diagnosis and the underlying pathological mechanisms. In fact, some symptoms that may occur in the patient are not replicated in the animal, making many promising approaches in animal studies not translatable in the clinic. With the advent of human-induced pluripotent stem cells (h-iPSC) several protocols for the generation of human-neuronal cells are becoming available for all laboratories. The importance of this technique lies in the opportunity to develop a human model derived directly from the patient: the patient's in-vitro cells will exhibit the same genetic and epigenetic modifications as the in-vivo cells. This has raised hopes for the generation of engineered brain models that can be coupled to sensors / actuators in order to better investigate their functional properties in-vitro (i.e. brain-on-a-chip). A reliable method for evaluating the functionality of neuronal cultures is the study of the spontaneous electrophysiological activity using microelectrode arrays (MEA). There are numerous studies in the literature that used h-iPSC on MEAs, showing the characterization of neuronal patterns of patient-derived cultures, demonstrating how this platform is valid for disease phenotyping, drug discovery and translational medicine. Although these models helped to shed light on fundamental biological mechanisms, the majority is based on two-dimensional neuronal cultures, which lack some key features to mimic in-vivo behavior. Three-dimensional h-iPSC-derived models possess a microenvironment, tissue architecture and potential to model network activity with greater complexity than two-dimensional models. Depending on the purpose of the study, we can choose different approaches to recreate 3D in-vitro brain, from those that aim to reproduce the trajectories of neurodevelopment (i.e. brain-organoids) to the use of synthetic materials that reproduce the functionalities of the extracellular matrix (ECM) (i.e. scaffold-based) (Chiaradia and Lancaster, 2020, Tang et al., 2006). Although h-iPSC-derived brain models summarize many aspects of network function in the human brain, they are subject to variability and still do not perfectly mimic behavior in-vivo. Therefore, to reach the full potential of this model we need improvements in differentiation methods and bioengineering, making these models engineered and reproducible. The aim of this PhD thesis was to implement different 3D neuronal culture generation methodologies that can be integrated on MEA devices to offer robust engineered platforms for functional studies.

The aim of my project is to investigate a non-invasive alternative to classical electrical stimulation in the field of neuromodulation techniques, which employ ultrasound (US). Even though ultrasound are collecting enough interest in the scientific community for their several advantages (high spatial resolution, low cost, and non-invasiveness), the mechanisms through which sound waves interact with cells and their activity are still unclear. Under this perspective, I consider a few possible strategies to induce an in vitro electrophysiological response of neuronal assemblies of different sizes to short and low-intensity US pulses; first of all, I had been applied US on neuronal cells treated with piezoelectric barium titanate nanoparticles (BTNPs), in order to exploit their piezoelectric effect to transduce the mechanical stimulus into an electrical one. To make the experimental model closer to the in vivo scenario, I also designed a more complex experimental set-up to investigate the above strategy on heterogeneous (i.e., neurons coming from different brain areas) and three-dimensional (3D) neuronal networks. As it is known, cells in the brain are characterized by a 3D structure and multi-cellular links, so 3D structures are a more powerful model than 2D ones1 in order to emulate the in vivo effects. Moreover, I wanted to merge the two aforementioned strategies to establish an experimental protocol to release a model drug, Doxorubicin, stored in polyelectrolyte microcapsules, fabricated with the layer-by-layer technique, using an ultrasound.

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 50-58).
With up to 40% of cancer patients showing metastatic lesions to the brain and a 30% five-year survival rate post-diagnosis, secondary tumors to the brain are a leading cause of cancer-related deaths. Understanding the mechanisms of tumor cell extravasation at the brain is therefore crucial to the development of therapeutic agents targeting this step in cancer metastasis, and to the overall improvement of cancer survival rates . Investigating the interactions between tumor cells and brain stroma is of particular interest due to the site's unique microenvironment. In fact, the interface between brain and blood, known as the blood-brain barrier (BBB), is the tightest endothelial barrier in humans. The presence of tight junctions between brain endothelial cells, coupled with the spatial organization of pericytes and astrocytes around the vasculature, restrict the entry of most solutes and cells into the brain. Yet, the brain constitutes a common metastatic site to many primary cancers originating from the lung, breast and skin. This suggests that tumor cells must employ specific mechanisms to cross the blood-brain barrier. While in vitro models aimed at replicating the human blood-brain barrier exist, most are limited in their physiological relevance. In fact, the majority of these platforms rely on a monolayer of human brain endothelial cells in contact with pencytes, astrocytes and neurons. While this approach focuses on incorporating the relevant cell types of the brain microenvironment, it fails to accurately replicate the geometry of brain capillaries, the barrier tightness of the BBB, and the juxtacrine and paracrine signaling events occurring between brain endothelial cells and stromal cells during vasculogenesis. To integrate these features into a physiologically relevant blood-brain barrier model, we designed an in vitro microvascular network platform formed via vasculogenesis, using endothelial cells derived from human induced pluripotent stem cells, primary human brain pericytes, and primary human brain astrocytes. The vasculatures formed with brain pericytes and astrocytes exhibit decreased cross-section areas, increased endothelial cell-cell tight junction expression and basement membrane deposition, as well as reduced and more physiologically relevant values of vessel permeability, compared to the vasculatures formed with endothelial cells alone. The addition of pericytes and astrocytes in the vascular system was also coupled with increased extravasation efficiencies of different tumor cell subpopulations, despite the lower permeability values measured in this BBB model. Moreover, an increase in the extravasation potential of metastasized breast tumor cells collected from the brain was recorded with the addition of pericytes and astrocytes, with respect to the parental breast tumor cell line. These results were not observed in metastasized breast tumor cells collected from the lung, thus validating our BBB model and providing useful insight into the role of pericytes and astrocytes in extravasation. Our microfluidic platform certainly provides advantages over the current state-of-the-art in vitro blood-brain barrier models. While being more physiologically relevant than most in vitro platforms when it comes to geometry, barrier function and juxtacrine/paracrine signaling between the relevant cell types, our model provides a robust platform to understand tumor cell-brain stromal cell interactions during extravasation.
by Cynthia Hajal.
S.M.

The blood-brain barrier (BBB) is crucial to maintain brainhomeostasis and prevent toxic substances from entering the brain.Endothelial cells (EC) are essential for the BBB and in this thesistwo different BBB-on-chips were designed for electricalcharacterization of immortalized mouse EC (bEnd3).Indium-Tin-Oxide (ITO) coated glass slides were etched, creating ITOelectrodes with increasing distance between them. The glass slideswere attached to a 3D-printed plastic well with UV-glue. The second prototype was an extension of the first prototype with acopper printed circuit board (PCB) attached to the ITO glass slidesusing silver epoxy to connect the ITO electrodes to the copperelectrodes. The aim with these two chips was to create chips withtransparent electrodes for live imaging of the cells with an opticalmicroscope. The chips were characterized with scanning electron microscopy (SEM) and a profilometer beforeseeding the cells inside the well. The absolute impedance wasmeasured across two parallel electrodes at a time. The impedance wasplotted against the distance between the electrodes. The method usedis called transmission line measurements (TLM) and is used to extractthe sheet impedance between the electrodes to evaluate the barriertightness of the cells. Only one chip from each prototype remained intact after thefabrication and sterilization, making it difficult to drawconclusions from the impedance measurements. However, based on thetwo chips, the TLM for the first prototype followed a linear trendwith a high R-square value whereas, the second prototype showed largevariations, causing the R-square value to decrease

Les organes sur puce (OoC) sont des systèmes miniaturisés basés sur la microfluidique qui permettent de reproduire la dynamique, les fonctions et les réponses physiologiques et pathologiques de mini-organes dans un micro-environnement contrôlé. Le cerveau est un organe majeur pour l'étude des maladies neurodégénératives, et le schéma de propagation des NDD dans le cerveau reste peu clair. Ainsi, la reconstruction de réseaux neuronaux sur une puce pourrait fournir une plateforme pour comprendre les mécanismes de propagation de ces maladies. Pour construire des réseaux neuronaux dans de tels systèmes miniatures, il faut tenir compte de l'unidirectionnalité du réseau neuronal et de l'efficacité des connexions entre les nœuds. Dans ma thèse de doctorat, j'ai d'abord montré la construction in vitro d'un réseau neuronal cortico-striatal unidirectionnel en utilisant des techniques de patterning dans le moule. J'ai ensuite vérifié la connectivité et la fonctionnalité du réseau neuronal par imagerie calcique et coloration par immunofluorescence. Afin de modéliser les mécanismes de propagation des maladies neurodégénératives. Afin de modéliser les mécanismes de propagation des maladies neurodégénératives, j'ai utilisé de l'a-synucléine pour infecter les réseaux neuronaux et j'ai observé de la synucléine phosphorylée dans les réseaux neuronaux. En outre, j'ai montré deux nouvelles méthodes de fabrication des puces pour améliorer la survie des neurones dans les puces. Globalement, les réseaux neuronaux sur puce pourraient offrir davantage de possibilités pour l'étude des maladies neurodégénératives
Organ-on-a-chip (OoC) is a microfluidic-based miniaturized system that enables to mimic dynamics, functions, physiological and pathological responses of mini-organs in a controlled microenvironment. The brain is a major organ for studying neurodegenerative diseases, and the pattern of NDD propagation in the brain remains unclear. Thus, reconstructing neural networks on a chip could provide a platform for understanding the spreading mechanisms of these diseases. Building neural networks in such miniature systems require addressing the neural network's unidirectionality and the efficiency of the connections between the nodes. In my PhD thesis, I first showed in vitro construction of a unidirectional cortico-striatal neural network using in-mold patterning technics. Then I verified the neural network's connectivity and functionality by calcium imaging immunofluorescence staining. Further, multi-node neural networks were constructed as well. In order to model the propagation mechanisms of neurodegenerative diseases. I used a-synuclein to infect neural networks and observed phosphorylated synuclein in the neural networks. In addition to this, I showed two new methods of fabricating chips to improve the survival of neurons in the chips. Overall, neural networks on a chip could offer more possibilities for studying neurodegenerative diseases

The present dissertation presents my doctoral work developed during the last three years at the Italian Institute of Technology (IIT) and the University of Genoa. The work was focused on the development of different ferrite nanoparticles and their magnetic characterization. Another objective of this work was the use of these magnetic nanoparticles for magnetic hyperthermia as a suitable mean to enhance the blood brain barrier passage. The first chapter deals with the synthesis and characterization of different divalent ions substituted ferrite nanocubes (NCs). In particular, trough non-hydrolytic synthesis, cobalt ferrite, zinc ferrite and mixed ferrite NCs, i.e. cobalt-manganese and cobalt-zinc, were obtained. The size and the composition were controlled by modifying the synthesis parameters, obtaining cubic-shaped nanoparticles with a cube edge ranging from 5 nm to 65 nm at different ions stoichiometry. The full characterization of these NCs was carried out to find the combination of composition and size that better suits their application in magnetic hyperthermia treatment (MHT), magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). Additionally, their use to prepare magnetic clusters by controlling the aggregation of these nanocubes into polymeric beads, here named magnetic nanobeads, was also studied. In chapter 1 is shown that these nanocubes, especially cobalt ferrite and zinc ferrite, revealed outstanding heating properties in magnetic hyperthermia. The same nanocubes were showing good performances as MRI contrast agent and generates MPI signals that were better than commercially available Resovist magnetic nanoparticles. Thanks to the large portfolio of NCs here prepared, it was possible to correlate their structural and chemical properties to the hysteresis loops measured under alternating magnetic field (AMF), probing heat losses as a function of media viscosity, concentration and aggregation status. The results obtained revealed that among all the different compositions, the zinc ferrite NCs are the most promising material for MHT, MPI and MRI applications, thanks also to his biocompatibility. In the second chapter, the functionalization and the exploitation of magnetic nanoparticles for enhancing central nervous system delivery is reported. In particular, the main goal of this study was to increase the NCs transportation through the blood-brain barrier (BBB) for the treatment of neurodegenerative diseases and brain tumors by using magnetic hyperthermia and molecular targeting. To reach this scope two strategies were followed. The first approach consists on the temporary and local damage of the BBB driven by the heat properties of the iron oxide and cobalt ferrite NCs thus increasing the para-cellular transportation through the BBB. The second approach consists on the functionalization of the same NCs with the trans-activating transcriptional activator peptide (TAT) to enhance the trans-cellular transportation through the BBB. The experiments were carried on a functional in vitro model of BBB using bEnd3 cells. First a suitable coating for the nanoparticles was developed. The results showed the importance of coating the NPs with polyethylene glycol (PEG) to increase the stability in biological media, enhancing the passive passage through the BBB. Then, the heating performances of both iron oxide and cobalt ferrite NCs were compared to induce thermal damage to the BBB. Due to their ability to heat up using lower NPs dose, cobalt ferrite NCs were chosen over iron oxide ones for further studies. The experiments of BBB transportation of these nanoparticles in presence of magnetic hyperthermia revealed a double fold dose increase in the passage when the barrier was thermally damaged. Nevertheless, the complete recovery from the temporarily induced damage was demonstrated. Concerning the second BBB transportation approach, the TAT coated NPs were successfully prepared. Further experiments will be done to test them on the BBB model. Finally, being most of the neurodegenerative disorders characterized by peptide fibrils accumulation in to the brain, a preliminary study focused on the use of Ferulic acid (FA) as a potential compound for disassembling aggregated insulin fibrils in a protein plaque model was followed. The effect of the FA on the fibrils was found to be concentration dependent, increasing with the increase of compound concentration. Further studies should be done to delivery this compound to the brain.

The human brain is the most complex organ of our body. in which neurons are the interacting elements. These cells are coupled through physical connections and complex biochemical processes. They are able to self-organization and exhibit a rich repertoire of spatiotemporal patterns and dynamics states. However, because of such high complexity, understanding human physiology as well as pathogenesis is not straightforward. Set-ups for in vivo studies are often very complicated, time consuming, and with low reproducibility. For this reason, there is the need to develop new in vitro systems capable of mimicking as much as possible the human brain. In addition, a successful model would minimize animal use for drug screening applications, deliver a highly reproducible system, and significantly lower costs in light of the current demand for pharmacological development. Primary dissociated neuronal cultures are an elegant yet powerful experimental tool to investigate and describe both electrophysiological and morphological properties of neuronal networks, which guarantee a good trade-off between controllability/observability and similarity to the in vivo nervous system. The electrophysiological activity of such neuronal assemblies can be extracellularly recorded by means of Micro-Electrode Arrays (MEAs). Up to now, most of the works make use of homogeneous networks, which do not fully mimic the complex organization as well as the functional and structural complexity of the human brain in vivo. The goal of this research project is to recreate in vitro 2D and 3D engineered neuronal networks made up of interacting sub-populations to recreate interconnected brain regions on a chip.

Il gene Sox2 codifica per un fattore di trascrizione attivo nelle cellule staminali durante lo sviluppo del SNC nei vertebrati. Mutazioni eterozigoti di Sox2 nell'uomo causano uno spettro caratteristico di anomalie del SNC, che coinvolgono l'ippocampo e l'occhio, e che causano epilessia, disabilità di apprendimento e difettivo controllo motorio. Per comprendere il ruolo di Sox2 nello sviluppo neuronale, il nostro laboratorio ha generato KO condizionali di Sox2 nel topo. Le conseguenze della delezione di Sox2 in diversi momenti dello sviluppo producono importanti difetti cerebrali. Il KO condizionale permette di osservare una funzione importante di Sox2 anche nel mantenimento del self-renewal e delle colture a lungo termine di NSC in vitro. Sox2-mut NSC, coltivate come neurosfere, derivate dal prosencefalo di topi P0, si auto-rinnovano per diversi passaggi in coltura, ma poi vanno incontro a esaurimento della coltura. La formazione delle sfera viene recuperata da lentivirus Sox2. Questo rivela un ruolo essenziale per Sox2 nel mantenimento delle NSC. Per comprendere i meccanismi delle funzioni di Sox2, una questione centrale è quali geni Sox2 regola come un fattore di trascrizione, con quali meccanismi, e quali geni Sox2-regolati sono mediatori critici della sua funzione. Un nuovo modo in cui Sox2 regola i suoi targets è stato recentemente osservato nel nostro laboratorio: Sox2 mantiene un elevato numero di interazioni a lungo raggio tra geni ed enhancer distali, che regolano l'espressione genica. Abbiamo determinato mediante Chia-PET l’intero pattern di interazioni a lungo raggio in NSC wt e Sox2-mut. La delezione di Sox2 causa una vasta perdita di interazioni a lungo raggio e ridotta espressione di un sottogruppo di geni associati. L'espressione di uno di questi geni, SOCS3, recupera il difetto di self-renewal delle cellule mut. Il nostro lavoro identifica Sox2 come un importante regolatore della connettività funzionale cromatinica nelle NSC e dimostra il ruolo di geni associati con interazioni Sox2-dipendenti nel mantenimento delle NSC e, potenzialmente, in disturbi dello sviluppo neurologico. Abbiamo studiato il differenziamento delle cellule Sox2-mut in neuroni e glia, rispetto ai controlli: in stadio avanzato, poche cellule β-tub-positive sono state osservate nei mut differenziati, con scarsa morfologia differenziata. Questo risultato ha mostrato l'importanza di Sox2 nello sviluppo in neuroni maturi. Abbiamo anche analizzato i cambiamenti nell'espressione genica derivati dalla delezione di Sox2 mediante analisi RNA-seq di tre campioni per entrambe le cellule wt e Sox2-mut indifferenziate, e in due condizioni di differenziamento (giorno 4 e il giorno 11). Centinaia di geni sono deregolati in cellule mutanti. Il gene più down-regolato è SOCS3, quindi abbiamo trasdotto le cellule Sox2-mut con un lentivirus SOCS3. Le cellule mut trasdotte inizialmente crescono come le cellule non trasdotte (solo una parte delle cellule era stata trasdotta), ma continuano a crescere anche dopo che le cellule mut non trasdotte si sono completamente esaurite.Questi risultati suggeriscono che SOCS3 recupera parzialmente il difetto di proliferazione delle cellule mut. Ho anche provato se la reintroduzione di SOCS3 potrebbe recuperare il difetto nel differenziamento neuronale delle cellule mut e i miei esperimenti iniziali suggeriscono che potrebbe essere così: le cellule SOCS3-trasdotte erano tutte GFAP-negative e sembravano β-tub-positive, anche se sembravano avere una morfologia sofferente. Altro scopo è verificare il ruolo di alcuni degli altri geni più deregolati come mediatori della funzione di Sox2 nel self-renewal e nel differenziamento, con esperimenti di rescuing. Infine mi propongo di verificare se la reintroduzione di Sox2 nelle cellule mut potrebbe ripristinare le interazioni a lungo raggio, perse nei mutanti, di un piccolo numero di geni bersaglio identificati, con esperimenti di 3C.
The Sox2 gene encodes a transcription factor active in stem/progenitor cells during the development of central nervous system in vertebrates. Heterozygous Sox2 mutations in humans cause a characteristic spectrum of CNS abnormalities, involving the hippocampus and the eye, and causing epilepsy, learning disabilities and defective motor control. In order to understand the role of Sox2 in neural development, our laboratory generated Sox2 conditional KO mutations in mouse. The consequences of Sox2 ablation at different developmental time points produced important brain defects, more serious when the ablation was early. Sox2 conditional KO allowed to observe an important function for Sox2 also in the maintenance of NSC self-renewal in long-term in vitro NSC cultures. Sox2-mut NSC, cultured as neurospheres from P0 mouse forebrain, self-renewed for several passages in culture, but then underwent a decrease in growth, with progressive culture exhaustion. Sphere formation could be rescued by lentiviral Sox2. This reveled an essential role for Sox2 in the development of multiple CNS regions and in the maintenance of NSC. To understand the mechanisms of Sox2 function, a central question is which genes Sox2 regulates as a transcription factor, by what mechanisms Sox2 acts in regulating them, and which Sox2-regulated genes are critical mediators of its function. A new way in which Sox2 regulates its targets has been recently observed in our laboratory: Sox2 maintains a high number of long-range interactions between genes and distal enhancers, that regulate gene expression. We determined, by genome-wide chromatin interaction analysis (RNApolII ChIA-PET) the global pattern of long-range chromatin interactions in normal and Sox2-mut mouse NSC. Sox2 deletion caused extensive loss of long-range interactions and reduced expression of a subset of genes associated with Sox2-dependent interactions. Expression of one of these genes, Socs3, rescued the self-renewal defect of Sox2-mut NSC. Our work identifies Sox2 as a major regulator of functional chromatin connectivity in NSC, and demonstrates the role of genes associated with Sox2-dependent interactions in NSC maintenance and, potentially, in neurodevelopmental disorders. We studied the differentiation of Sox2-mut cells into neurons and glia, as compared to controls: at advanced stage, very few β-tub-positive cells were observed in Sox2-mut cells differentiated, with poor differentiated morphology. This result showed the importance of Sox2 in the development into mature neurons. We also analyzed the changes in gene expression resulting from Sox2 deletion by RNA-seq analysis of three samples for both wt and Sox2-mut cells in undifferentiated cells, and two differentiation conditions (day 4 and day 11). Hundreds of genes were deregulated in mutant cells. The most down-regulated gene was Socs3, so we transduced Sox2-mut cells with a lentiviral Socs3–vector, coexpressing GFP. Socs-3 transduced mut cells initially grew as the untransduced cells (only a proportion of the cells had been tranduced), but continued to grow even after the untransduced mut cells were completely exhausted, and transduced cells were positively selected. These results suggested that Socs3 partially rescued the proliferation defect of mut cells. I also tested if the reintroduction of Socs3 could rescue the neuronal differentiation defect of mut cells and my initial experiments suggest that this might be the case: Socs3-transduced cells were all GFAP-negative, and they all appeared β-tub-positive, though they seemed to have a suffering morphology. I aimed to test the role of some of the other most deregulated genes as mediators of Sox2 function in self-renewal and differentiation, by rescuing experiments of mut cells. I aim to test if Sox2 reintroduction in mut cells could rescue the long-range interactions of a small number of identified target genes, lost in Sox2-mut cells, by 3C experiments.

Neuronal networks are at the base of information processing in the brain. They are series of interconnected neurons whose activation defines a recognizable linear pathway. The main goal of studying neural ensembles is to characterize the relationship between the stimulus and the individual or general neuronal responses and the relation amongst the electrical activities of neurons within the network, also understanding how topology and connectivity relates to their function. Many techniques have been developed to study these complex systems: single-cell approaches aim to investigate single neurons and their connections with a limited number of other nerve cells; on the opposite side, low-resolution large-scale approaches, such as functional MRI (Magnetic Resonance Imaging) or electroencephalography (EEG), record signal changes in the brain that are generated by large populations of cells. More recently, multisite recording techniques have been developed to overcome the limitations of previous approaches, allowing to record simultaneously from huge neuronal ensembles with high spatial resolution and in different brain regions, i.e. by using implantable semiconductor chips. Local Field Potentials (LFPs), the part of electrophysiological signals that has frequencies below 500 Hz, capture key integrative synaptic processes that cannot be measured by analyzing the spiking activity of few neurons alone. Several studies have used LFPs to investigate cortical network mechanisms involved in sensory processing, motor planning and higher cognitive processes, like memory and perception. LFPs are also promising signals for steering neuroprosthetic devices and for monitoring neural activity even in human beings, since they are more easily and stably recorded in chronic settings than neuronal spikes. In this work, LFP profiles recorded in the rat barrel cortex through high-resolution CMOS-based needle chips are presented and compared to those obtained by means of conventional Ag/AgCl electrodes inserted into glass micropipettes, which are widely used tools in electrophysiology. The rat barrel cortex is a well-known example of topographic mapping where each of the whiskers on the snout of the animal is mapped onto a specific cortical area, called a barrel. The barrel cortex contains the somatosensory representation of the whiskers and forms an early stage of cortical processing for tactile information, along with the trigeminal ganglion and the thalamus. It is an area of great importance for understanding how the cerebral cortex works, since the cortical columns that form the basic building blocks of the neocortex can be actually seen within the barrel. Moreover, the barrel cortex has served as a test-bed system for several new methodologies, partly because of its unique and instantly identifiable form, and partly because the species that have barrels, i.e. rodents, are the most commonly used laboratory mammal. The barrel cortex, the whiskers that activate it and the intervening neural pathways have been increasingly the subject of focus by a growing number of research groups for quite some time. Nowadays, studies (such this one) are directed not only at understanding the barrel cortex itself but also at investigating issues in related fields using the barrel cortex as a base model. In this study, LFP responses were evoked in the target barrel by repeatedly deflecting the corresponding whisker in a controlled fashion, by means of a specifically designed closed-loop piezoelectric bending system triggered by a custom LabView acquisition software. Evoked LFPs generated in the barrel cortex by many consecutive whiskers' stimulations show large variability in shapes and timings. Moreover, anesthetics can deeply affect the profile of evoked responses. This work presents preliminary results on the variability and the effect of commonly used anesthetics on these signals, by comparing the distributions of evoked responses recorded from rats anesthetized with tiletamine-xylazine, which mainly blocks the excitatory NMDA receptors, and urethane, which conversely affects both the excitatory and inhibitory system, in a complex and balanced way yet preserving the synaptic plasticity. Representative signal shape characteristics (e.g., latencies and amplitude of events) extracted from evoked responses acquired from different cortical layers are analyzed and discussed. Statistical distributions of these parameters are estimated for all the different depths, in order to assess the variability of LFPs generated by individual mechanical stimulations of single whiskers along the entire cortical column. Preliminary results showed a great variability in cortical responses, which varied both in latency and amplitude across layers. We found significant difference in the latency of the first principal peak of the responses: under tiletamine-xylazine anesthetic, the responses or events of the evoked LFPs occurred later than the ones recorded while urethane was administered. Furthermore, the distributions of this parameter in all cortical layers were narrower in case of urethane. This behavior should be attributed to the different effects of these two anesthetics on specific synaptic receptors and thus on the encoding and processing of the sensory input information along the cortical pathway. The role of the ongoing basal activity on the modulation of the evoked response was also investigated. To this aim, spontaneous activity was recorded in different cortical layers of the rat barrel cortex under the two types of anesthesia and analyzed in the statistical context of neuronal avalanches. A neuronal avalanche is a cascade of bursts of activity in neural networks, whose size distribution can be approximated by a power law. The event size distribution of neuronal avalanches in cortical networks has been reported to follow a power law of the type P(s)= s^-a, with exponent a close to 1.5, which represent a reflection of long-range spatial correlations in spontaneous neuronal activity. Since negative LFP peaks (nLFPs) originates from the sum of synchronized Action Potentials (AP) from neurons within the vicinity of the recording electrode, we wondered if it were possible to model single nLFPs recorded in the basal activity traces by means of only one electrode as the result of local neuronal avalanches, and thus we analyzed the size (i.e. the amplitude in uV) distribution of these peaks so as to identify a suitable power-law distribution that could describe also these single-electrode records. Finally, the results of the first ever measurements of evoked LFPs within an entire column of the barrel cortex obtained by means of the latest generation of CMOS-based implantable needles, having 256 recording sites arranged into two different array topologies (i.e. 16 x 16 or 4 x 64, pitches in the x- and y-direction of 15 um and 33 um respectively), are presented and discussed. A propagation dynamics of the LFP can be already recognized in these first cortical profiles. In the next future, the use of these semiconductor devices will help, among other things, to understand how degenerating syndromes like Parkinson or Alzheimer evolve, by coupling detected behaviors and symptoms of the disease to neuronal features. Implantable chips could then be used as 'electroceuticals', a newly coined term that describes one of the most promising branch of bioelectronic medicine: they could help in reverting the course of neurodegenerative diseases, by constituting the basis of neural prostheses that physically supports or even functionally trains impaired neuronal ensembles. High-resolution extraction and identification of neural signals will also help to develop complex brain-machine interfaces, which can allow intelligent prostheses to be finely controlled by their wearers and to provide sophisticated feedbacks to those who have lost part of their body or brain functions.
Le reti neuronali sono alla base della codifica dell'informazione cerebrale. L'obiettivo principale dello studio delle popolazioni neuronali è quello di caratterizzare la relazione tra uno stimolo e la risposta individuale o globale dei neuroni e di studiare il rapporto tra le varie attività elettriche dei neuroni appartenenti ad una particolare rete, comprendendo anche come la topologia e la connettività della rete neuronale influiscano sulla loro funzionalità. Fino ad oggi, molte tecniche sono state sviluppate per studiare questi sistemi complessi: studi a singola cellula mirano a studiare singoli neuroni e le loro connessioni con un numero limitato di altre cellule; sul lato opposto, approcci su larga scala e a bassa risoluzione, come la risonanza magnetica funzionale o l'elettroencefalogramma, registrano segnali elettrofisiologici generati nel cervello da vaste popolazioni di cellule. Più recentemente, sono state sviluppate tecniche di registrazione multisito che mirano ad abbattere le limitazioni dei precedenti approcci, rendendo possibile la misurazione ad alta risoluzione di segnali generati da grandi ensamble neuronali e da diverse regioni del cervello simultaneamente, ad esempio mediante l'uso di chip impiantabili a semiconduttore. I potenziali di campo locali (LFP) catturano processi sinaptici chiave che non possono essere estratti dall'attività di spiking di qualche neurone isolato. Numerosi studi hanno utilizzato gli LFP per studiare i meccanismi corticali coinvolti nei processi sensoriali, motori e cognitivi, come la memoria e la percezione. Gli LFP rappresentano anche dei segnali interessanti nell'ambito delle applicazioni neuroprotesiche e per monitorare l'attività cerebrale negli esseri umani, dal momento che possono essere registrati più stabilmente e facilmente in impianti cronici rispetto agli spike neuronali. In questo studio, sono riportati dei profili LFP registrati dalla barrel cortex di ratto tramite chip ad ago ad alta risoluzione basati su tecnologia CMOS e confrontati con quelli ottenuti tramite elettrodi convenzionali in Ag/AgCl inseriti in micropipette di vetro, strumenti comunemente usati in elettrofisiologia. La barrel cortex di ratto è un esempio ben noto di mapping topografico, nel quale ogni baffo sul muso dell'animale è mappato in una specifica area corticale, chiamata barrel. La barrel cortex contiene la rappresentazione sensoriale dei baffi dell'animale e rappresenta uno dei primi stadi di elaborazione dell'informazione tattile, insieme al ganglio del trigemino e al talamo. Essa è un'area di primaria importanza per lo studio del funzionamento della corteccia cerebrale, visto che le colonne corticali che formano i blocchi di base della neocorteccia possono essere visualizzati facilmente all'interno della barrel cortex. La barrel cortex inoltre è utilizzata come sistema di test in numerose metodologie innovative, grazie alla sua struttura unica ed istantaneamente identificabile, e grazie anche al fatto che le specie dotate di barrel, i roditori, sono gli animali da laboratorio più comuni. La barrel cortex e le sue interconnessioni neuronali sono stati oggetto delle ricerche più disparate in questi ultimi decenni. Attualmente, alcuni studi (come questo) non mirano solamente a comprendere meglio la barrel cortex, ma anche ad analizzare problematiche in campi scientifici collegati, utilizzando la barrel cortex come modello base. In questo lavoro, sono stati evocati segnali LFP nella barrel cortex tramite deflessioni ripetute dei baffi dell'animale, realizzate in modo controllato tramite un sistema di deflessione piezoelettrica a closed-loop innescato da un sistema di acquisizione LabView. Le risposte evocate generate nella barrel dalla stimolazione ripetuta dei baffi presentano elevata variabilità nella forma e nelle latenze temporali. Inoltre, il tipo di anestesia utilizzata può influenzare profondamente il profilo della risposta evocata. Questo studio riporta i risultati preliminari sulla variabilità della risposta neuronale e sull'effetto di due anestetici di uso comune su questi segnali, confrontando le distribuzioni delle risposte evocate in ratti anestetizzati con tiletamina-xylazina (il quale agisce prevalentemente sui recettori eccitatori di tipo NMDA) e uretano (che agisce in modo più bilanciato e complesso su entrambi i sistemi eccitatori ed inibitori, preservando la plasticità sinaptica). Sono state analizzate e discusse alcune caratteristiche rappresentative del segnale evocato (ad esempio, le latenze temporali e l'ampiezza degli eventi), registrato a varie profondità corticali. Per tutte le prondità corticali acquisite, sono state stimate le distribuzioni statistiche di tali parametri, in modo da valutare la variabilità degli LFP evocati dalle stimolazioni meccaniche individuali delle vibrisse del ratto lungo l'intera colonna corticale. I primi risultati presentano una grande variabilità nelle risposte corticali, sia in latenza che in ampiezza. Inoltre, è stata riscontrata una differenza significativa nella latenza del primo picco principale delle risposte evocate: gli LFP evocati in animali anestetizzati con tiletamina-xylazina presentavano una latenza più lunga di quelli registrati in ratti anestetizzati con uretano. Inoltre, le distribuzioni dei parametri analizzati erano più strette e piccate in uretano, in corrispondenza di tutte le profondità corticali. Questo comportamento è sicuramente da attribuire al differente meccanismo d'azione dei due anestetici su specifici recettori sinaptici, e quindi nell'elaborazione e nella trasmissione dell'informazione sensoriale lungo tutto il percorso corticale. E' stato inoltre discusso il ruolo della attività basale nella modulazione della risposta evocata. A questo proposito, è stata registrata l'attività spontanea in corrispondenza dei vari layer corticali ed analizzata nel contesto statistico delle 'valanghe neuronali'. Una valanga neuronale è una cascata di attività elettrica in una rete neuronale, la cui distribuzione statistica dei parametri principali (dimensione e vita media) può essere approssimata da una legge di potenza. La distribuzione delle dimensioni di una valanga in una rete neuronale segue una legge di potenza del tipo P(s)=s^-a, con a=1.5. Tale esponente è un riflesso delle correlazioni spaziali a lungo raggio nell'attività neuronale spontanea. Dal momento che i picchi negativi (nLFPs) nelle tracce elettrofisiologiche originano dalla somma di potenziali d'azione sincronizzati generati da neuroni posti nelle vicinanze dell'elettrodo di registrazione, ci siamo chiesti se fosse possibile modellizare i singoli nLFP registrati nell'attività basale tramite un singolo elettrodo come il risultato di valanghe neuronali locali. Pertanto, abbiamo analizzato la distribuzione della dimensione (cioè l'ampiezza in uV) di tali picchi, in modo da identificare una distribuzione power-law appropriata, che potesse descrivere anche le registrazioni a singolo elettrodo. Infine, sono presentate e discusse le prime registrazioni in assoluto degli LFP evocati lungo un'intera colonna corticale ottenute tramite l'ultima generazione di chip impiantabili a tecnologia CMOS. Questi ultimi presentano una matrice di 256 siti di registrazione, organizzata secondo due possibili topologie, 16 x 16 o 4 x 64, e avente una distanza tra gli elettrodi pari a 15 um o 33 um rispettivamente. Una precisa dinamica di propagazione dei potenziali evocati può già essere riconosciuta in questi primissimi profili corticali. Nel prossimo futuro, l'uso di questi dispositivi a semiconduttore potrà aiutare a comprendere il decorso di sindromi neurodegerative come il Parkinson o l'Alzheimer, associando sintomi e comportamenti tipo della malattia a specifiche caratteristiche neuronali. I chip impiantabili potranno anche essere utilizzati come 'electroceuticals', ossia potranno aiutare a rallentare (o addirittura a capovolgere) il decorso delle malattie neurogenerative, costituendo le basi di protesi neuronali in grado di sostenere fisicamente o allenare funzionalmente le popolazioni neuronali danneggiate. L'identificazione e il rilevamento di segnali neuronali ad alta risoluzione aiuterà anche a sviluppare complesse interfacce cervello-macchina, che consentiranno il controllo di protesi intelligenti e che forniranno sofisticati meccanismi di feedback a chi ha perso l'uso di alcune parti del proprio corpo o determinate funzioni cerebrali.

The objective of this work presented here is to extend the capabilities of siloxane waveguide technology in the field of biochemical sensing. Recent advances in the integration of polymeric optical waveguides with electronics onto standard printed circuit boards (PCBs) allow the formation of cost-effective lab-on-achip modules suitable for mass production. This technology has been primarily designed for on-board data communication. The focus of this research is to investigate the possibility of realising a Siloxane polymer based lab-on-chip sensor. Different siloxane-polymer-based optical waveguide sensor structures have been designed and analysed from the aspect of biochemical sensing. An evanescent-wave absorption sensor based on mode-selective asymmetric waveguide junctions is proposed for the first time. The device mitigates the common optical effect of spurious response in absorption sensors due to the analyte transport fluid. Head injury is the leading cause of death in the population of people under 40 years. Currently, 3 out of 5 deaths in emergency rooms are due to severe brain injuries in the developed world. Researchers at the Neurosciences Critical Care Unit (NCCU) at Addenbrooke's Hospital have managed to correlate biochemical changes with the severeness of the injury and the likelihood of patient recovery. Considerable progress has been made to develop a lab-on-chip sensor capable of continuously monitoring glucose, lactate and pyruvate concentrations in the brain fluid, hence the contribution to the current trend in the advancement of portable lab-on-chip technologies for the deployment of point-of-care diagnostic tools. A novel recognition layer has been developed based on porphyrin in combination with glucose, lactate and pyruvate oxidase for measuring all the analytes, enabling fast and reversible chemical reactions to be monitored by optical interrogation. The operational wavelength of the developed recognition layer is 425 nm, which required the formation of polymer features that were beyond the fabrication capabilities at the time. Through considerable process development and the adoption of nanoimprinting lithography, siloxane polymer based optical waveguides were fabricated allowing the realisation of highly sensitive optical sensors. Based on the results that are presented here, it can be concluded the functionalization of siloxane polymer waveguide have a potential for realising biochemical sensors in the future. The new fabrication technique will allow the formation of more robust and complex lab-on-chip sensors based on this material.

Despite the last 60 years have seen major advances in many scientific and technological inputs of drug Research and Development, the number of new molecules hitting the market per billion US dollars of R&D spending has been declined steadily during the same period. The current scenario highlights the need for new research tools to enable reduce costly animal and clinical trials while providing a better prediction about drug efficacy and security in humans A recent emerging approach to improve the current models is emerging from the field of microfluidics, which studies systems that process or manipulate tiny amounts of fluids using channels with dimensions of tens to hundreds of micrometers. Combining microfluidics with cell culture, scientists gave rise to a new field named “Organ-on-chip” (OOC). Microfluidic OOCs are advanced platforms designed to mimic physiological structures and continuous flow conditions, thus allowing the culture of cells in a friendlier microenvironment. This thesis, titled “Cell culture interfaces for different organ-on-chip applications: from photolithography to rapid-prototyping techniques with sensor embedding”, aims to design, simulate and test new OOC devices to reproduce cell culture interface under flow conditions. The work has a focus on the exploration of novel fabrication techniques which enable rapid prototyping of OOC devices, reducing costs, time and human labor associated to the fabrication process. The final objective is to demonstrate the viability of the devices as research tools for biological problems, applying them to the tubular kidney and the blood brain barrier (BBB). To achieve the objective, at least three device version have been developed: 1) OOCv1, fabricated by multilayer PDMS soft lithography; 2) OOCv2, fabricated in thermoplastic by layered object manufacturing using both a vinyl cutter and a laser cutter, integrating standard fluidic connectors alone (OOCv2.1) or together with embedded electrodes (OOCv2.2); 3) OOCv3 using a mixed technique of laser cut and 3D printing by stereolithography. All devices are fabricated using biocompatible materials with high optical quality and an embedded commercial membrane. The biological experiments with renal tubular epithelial cells, realized on OOCv1 and OOCv2.1 devices, demonstrated the viability of the device for culturing cells under flow conditions. The study realized on fatty acid oxidation and accumulation in cells exposed to physiological and diabetogenic oscillating levels of glucose suggest a possible positive role of shear stress in activation of fatty acid metabolism. The studies were performed using a compact experimental unit with embedded flow control which reduce significatively the complexity and cost of the fluidic experimental setup. The biological experiments on the BBB confirmed viability of OOCv2.1 and OOCv2.2 for compartmentalized co-culturing of endothelial cells and pericytes. The formation and recovery of the barrier after disruptive treatment has been assessed using different techniques, including immunostaining, fluorescence and live phase contrast imaging, and electrical impedance spectroscopy. The repeatability of measurements using electrodes was verified. A model to classify measurements from different timepoints has been developed, resulting in accuracy of 100% in learning and 90% in testing case. Results are confirmed by imaging data, which also suggest a critical role of pericytes in the development, maintenance, and regulation of BBB, in accordance with the literature.
En los últimos años está emergiendo una nueva propuesta para mejorar los modelos actuales en el estudio de nuevos fármacos. Mediante la fusión de cultivos celulares y microfluídica ha nacido un nuevo campo de aplicación denominado “Órgano-en-un-chip” (OOC), donde se recrea un entorno fisiológico capaz de reproducir unidades funcionales mínimas de diversos órganos del cuerpo humano. Un elemento importante para el desarrollo de dispositivos OOC es la reproducción de zonas de interacción entre varios tejidos formados por diferentes tipos celulares. Esta tesis, titulada “Interfaces de cultivo celular para diferentes aplicaciones de OOC: desde fotolitografía a técnicas de prototipado rápido con inclusión de sensores”, tiene como objetivo el diseño, simulación y evaluación de dispositivos OOC capaces de reproducir superficies de contacto de tejidos contiguos expuestos a flujo. El trabajo está enfocado a la exploración de nuevas técnicas de fabricación que permitan el prototipado rápido de dispositivos OOC, reduciendo costes, tiempo y mano de obra asociada a dicha fabricación. El objetivo final es demostrar la utilidad de los dispositivos como herramientas de investigación para problemas biológicos, aplicándolos en esta tesis al estudio del túbulo renal y de la barrera hematoencefálica. Para ello se han fabricado tres versiones de dispositivos: 1) OOCv1 fabricado por litografía suave en múltiples capas de PDMS; 2) OOCv2 fabricado con cortadora de vinilo y cortadora láser en múltiples capas de materiales termoplásticos y con electrodos integrados en la versión OOCv2.2; 3) OOCv3 fabricado mediante impresión 3D por esterolitografía. Todos los dispositivos están hechos de materiales biocompatibles de alta calidad óptica, con conectores fluídicos y una membrana comercial integrada. Los experimentos biológicos sobre túbulo renal, realizados en los dispositivos OOCv1 y OOCv2, han demostrado la viabilidad de los dispositivos, integrados con un sistema de flujo, para estudios de la metabolización de ácidos grasos en el riñón relacionados con condiciones diabetogénicas. Los experimentos biológicos sobre la barrera hematoencefálica han confirmado la viabilidad de OOCv2 para el cocultivo compartimentado de células endoteliales de cerebro y pericitos. La integración de electrodos en el OOCv2.2 ha demostrado ser una técnica fiable para la medición de la integridad de barreras biológicas de modo no-invasivo, libre de etiqueta (“label-free”), y a tiempo real gracias a la espectroscopía de impedancia.

The ionic gating across the neuron membrane generates neuronal activity in the brain. During the last two decades rapid advances in microelectronics and microelectrode technology have provided scientists with many devices enabling them to record extracellularly the transmembrane potentials near the electrode in the brain. These devices that are implanted invasively without causing too much tissue damage, can record from hundreds of neurons, and also simultaneously from a number of channels generating a huge amount of data. Inferring meaningful conclusions by analyzing this massive amount of data often recorded from noisy experimental conditions is a big challenge for the neuroscience and neuroengineering community and sophisticated signal processing and analysis tools are required. But, relatively little work has been done on development of comprehensive signal processing tools operable on different software platforms and that can be easily diffused to the scientific community. Though individual tools are available for signal visualization, spike detection and sorting, spike train analysis, yet analysis of local field potentials (LFPs) are still done manually. Most of these tools are developed by laboratories for their own requirements. Moreover, no software tools are available to date integrating all the signal processing steps under a single platform. This thesis aims at developing a comprehensive tool called ‘SigMate’ for processing and analysis of extracellular potentials; capable of performing operations ranging from signal visualization and basic operations to single sweep analysis and simulation of neuronal activity. The software package is designed to avoid file-type based incompatibility among different acquisition software and works with the neuronal data files in ASCII format. The functionalities of SigMate is described briefly below. • Signal visualization (2D and 3D) and basic operations: This is the starting and home module of the software package that provides connectivity to other functionalities. With signal visualization it includes basic operations like signal averaging, noise estimation, +/- averaging, mean square and root mean square noise estimation. In every module a visualization pane is provided with zooming, panning and data cursor options. • Basic file operations: Usually, incompatibility between acquisition and analysis tools poses a barrier in quick analysis of the recorded signals. However, most of the acquisition tools provide a way to convert the recorded files into ASCII format files and most of the analysis tools require specifically formatted files. To meet this need, the module includes operations like file splitting, file concatenating, and file column rearranging. • Artifact removal: Stimulus artifacts very often obscure the real neuronal response in signals. This module performs artifact removal for both slow and fast stimulus artifacts with an optional baseline correction operation. • Noise characterization: Invasive neuronal recording setups involve sophisticated electronic devices. Due to the wide variety of neural probes used by different labs a unique method for noise analysis is required. This module measures the quality of the recorded signals through noise estimation using detection of steady states. • Latency estimation: Very often neuroscientists use latency information to understand the signal propagation in the brain. This module calculates latency and automatically determines cortical layer activation order using LFPs as well as current source density data by applying current source density analysis on the LFPs. • Spike detection and spike train analysis tool: Neuronal spikes are most widely studied signals. Many tools address spike detection and spike train analysis in the existing literature and this module adapts 'Wave_Clus', a popular tool among them. • Single sweep LFP clustering: LFPs represent cumulative response of neuronal populations around the recording electrode and are studied as an average of many single sweeps. Single sweep LFPs contain response of a neuronal population at a particular time instance and shows a range of shapes. As the shape of an LFP is considered as a fingerprint of the underlying neuronal network generating it, a shape based clustering system is presented in this module to facilitate the study of neuronal circuit activation. • Interface with EEG based robotic system: This module contains an interface with the ‘Simulink’ based EEG acquiring system developed by g.tec medical engineering GmbH. Using this module, it is possible to establish communication with a robotic device for navigation. • Simulations: Neuronal simulations for optimization of stimulation protocol and simulation of calcium based model for flicking-based short-term plasticity. Except the spike detection and spike train analysis tool, the rest of the features are in-house developed algorithms which are tested rigorously with datasets recorded using standard micropipette, implantable and planar EOSFETs from anesthetized rats upon different stimulations. In conclusion, with the growth of neuronal probes, amount of acquired data are increasing and the need of one single software package performing all necessary processing and analysis on the data has become crucial. This thesis is the first step towards meeting that need. As the software has been extensively tested with three possible sources of data, we believe that once it is disseminated to the community (which will happen in the near future), it will serve a good deal in processing and analyzing extracellularly recorded neurophysiological signals.
Sommario 1.1 Motivazioni I segnali neurali registrati con sonde neurali invasive o non invasive richiedono un’elaborazione e un’analisi rigorosa per arrivare a comprendere l’attività generata dalla sottostante rete neurale in risposta a degli stimoli. Nel corso degli ultimi due decenni, il rapido sviluppo della microelettronica e della tecnologia del microelettrodo ha permesso agli scienziati di registrare contemporaneamente segnali provenienti da centinaia di neuroni usando numerosi canali. L’ottenimento di risultati significativi attraverso l’elaborazione e analisi di questa enorme quantità di dati registrati in condizioni sperimentali non ottimali rappresenta una grande sfida per le neuroscienze e la comunità della neuroingegneria. Anche se sono già disponibili singoli software per eseguire l’analisi, ad esempio, di un treno di spike, il sorting e rilevamento del picco dello spike, non sono però ancora stati sviluppati strumenti software che integrino tutti gli step necessari per il processing del segnale EEG, degli spike neurali, e il calcolo dei potenziali di campo (local field potential – LFPs). Pertanto, la comunità della neuroingegneria sente più che mai necessario lo sviluppo di un unico pacchetto software in grado di eseguire tutto il processing e l’analisi standard dei segnali neurali registrati. Questa tesi presenta come risultato finale un pacchetto software, “SigMate”, costruito integrando assieme vari moduli per permettere l’elaborazione e l’analisi di LFP e di segnali EEG per il brain-machine-interface (BMI), la simulazione di un singolo neurone, e la rilevazione, l’ordinamento e l’analisi di un treno di spike. 1.2 Scopi e Obiettivi Il pacchetto software SigMate è sviluppato allo scopo di essere completo, adattabile, robusto e open-source. Per raggiungere questi obiettivi sono stati integrati metodi già disponibili, presenti nella letteratura scientifica del settore e già affermati all’interno di essa, con altri metodi che sono stati sviluppati durante lo svolgimento della tesi. Le capacità di analisi di SigMate permettono di elaborare nello stesso ambiente segnali EEG, spikes, e calcolare LFP. In particolare: • Algoritmi adattabili e robusti: gli algoritmi per l’analisi di segnali neurali registrati usando sonde neurali multicanali devono essere: (i) adattabili per tener conto del numero sempre crescente di siti e canali di registrazione, e (ii), robusti ossia capaci di elaborare calcoli su grandi moli di dati, in modo accurato e veloce, quindi evitando lunghe attese al suo utilizzatore. • Performance: per verificare la performance, l’accuratezza dei risultati, e la giusta integrazione dei moduli, sono stati usati segnali neurali registrati dalla corteccia di topo (in particolare da quella parte sottile della corteccia somatosensoriale (SI) che corrisponde ad una mappatura uno-a-uno dei baffi del naso del ratto) usando tre metodi diversi: (i) con micropipette standard, (ii) con Electrolyte–Oxide–Semiconductor Field Effect Transistor (EOSFET) messi su chip, e (iii) con EOSFET impiantabili. • Open–source: il pacchetto software sarà distribuito come open-source attraverso una GNU–General Public License (GPL) e per questa ragione Matlab è stato selezionato come ambiente di sviluppo. L’utilizzatore è libero di operare proprie modifiche adattando il software alle proprie esigenze. 1.3 Overview della tesi La tesi è organizzata in 5 capitoli. Il primo capitolo contiene l’introduzione, il secondo fornisce gli elementi di base che servono alla comprensione dei vari problemi affrontati e presenta anche una review della letteratura. Il capitolo 3 descrive i metodi per il setup del sistema e l’acquisizione dei segnali. I capitoli 4 e 5 descrivono la ricerca sviluppata durante lo svolgimento della tesi, mentre il capitolo 6 contiene un sommario e un overview sui possibili sviluppi futuri di questo lavoro.

This dissertation is devoted to study of complex systems in human physiology particularly heartbeats and brain dynamics. We have studied the dynamics of heartbeats that has been a subject of investigation of two independent groups. The first group emphasized the multifractal nature of the heartbeat dynamics of healthy subjects, whereas the second group had established a close connection between healthy subjects and the occurrence of crucial events. We have analyzed the same set of data and established that in fact the heartbeats are characterized by the occurrence of crucial and Poisson events. An increase in the percentage of crucial events makes the multifractal spectrum broader, thereby bridging the results of the former group with the results of the latter group. The crucial events are characterized by a power index that signals the occurrence of 1/f noise for complex systems in the best physiological condition. These results led us to focus our analysis on the statistical properties of crucial events. We have adopted the same statistical analysis to study the statistical properties of the heartbeat dynamics of subjects practicing meditation. The heartbeats of people doing meditation are known to produce coherent fluctuations. In addition to this effect, we made the surprising discovery that meditation makes the heartbeat depart from the ideal condition of 1/f noise. We also discussed how to combine the wave-like nature of the dynamics of the brain with the existence of crucial events that are responsible for the 1/f noise. We showed that the anomalous scaling generated by the crucial events could be established by means of a direct analysis of raw data. The efficiency of the direct analysis procedure is made possible by the fact that periodicity and crucial events is the product of a spontaneous process of self-organization. We argue that the results of this study can be used to shed light into the nature of this process of self-organization.

The role of non-neuronal brain cells, called astrocytes, is emerging as crucial in brain function and dysfunction, encompassing the neurocentric concept that was envisioning glia as passive components. Ion and water channels and calcium signalling, expressed in functional micro and nano domains, underpin astrocytes’ homeostatic function, synaptic transmission, neurovascular coupling acting either locally and globally. In this respect, a major issue arises on the mechanism through which astrocytes can control processes across scales. Finally, astrocytes can sense and react to extracellular stimuli such as chemical, physical, mechanical, electrical, photonic ones at the nanoscale. Given their emerging importance and their sensing properties, my PhD research program had the general goal to validate nanomaterials, interfaces and devices approaches that were developed ad-hoc to study astrocytes. The results achieved are reported in the form of collection of papers. Specifically, we demonstrated that i) electrospun nanofibers made of polycaprolactone and polyaniline conductive composites can shape primary astrocytes’ morphology, without affecting their function ii) gold coated silicon nanowires devices enable extracellular recording of unprecedented slow wave in primary differentiated astrocytes iii) colloidal hydrotalcites films allow to get insight in cell volume regulation process in differentiated astrocytes and to describe novel cytoskeletal actin dynamics iv) gold nanoclusters represent nanoprobe to trigger astrocytes structure and function v) nanopillars of photoexcitable organic polymer are potential tool to achieve nanoscale photostimulation of astrocytes. The results were achieved by a multidisciplinary team working with national and international collaborators that are listed and acknowledged in the text. Collectively, the results showed that astrocytes represent a novel opportunity and target for Nanoscience, and that Nanoglial interface might help to unveil clues on brain function or represent novel therapeutic approach to treat brain dysfunctions.

La plupart des constructeurs de systèmes d'imagerie par résonance magnétique (IRM) proposent un large choix d'applications de post-traitement sur les données IRM reconstruites a posteriori, mais très peu de ces applications peuvent être exécutées en temps réel pendant l'examen. Mises à part certaines solutions dédiées à l'IRM fonctionnelle permettant des expériences relativement simples ainsi que d'autres solutions pour l'IRM interventionnelle produisant des scans anatomiques pendant un acte de chirurgie, aucun outil n'a été développé pour l'IRM pondérée en diffusion (IRMd). Cependant, comme les examens d'IRMd sont extrêmement sensibles à des perturbations du système hardware ou à des perturbations provoquées par le sujet et qui induisent des données corrompues, il peut être intéressant d'investiguer la possibilité de reconstruire les données d'IRMd directement lors de l'examen. Cette thèse est dédiée à ce projet innovant. La contribution majeure de cette thèse a consisté en des solutions de débruitage des données d'IRMd en temps réel. En effet, le signal pondéré en diffusion peut être corrompu par un niveau élevé de bruit qui n'est plus gaussien, mais ricien ou chi non centré. Après avoir réalisé un état de l'art détaillé de la littérature sur le bruit en IRM, nous avons étendu l'estimateur linéaire qui minimise l'erreur quadratique moyenne (LMMSE) et nous l'avons adapté à notre cadre de temps réel réalisé avec un filtre de Kalman. Nous avons comparé les performances de cette solution à celles d'un filtrage gaussien standard, difficile à implémenter car il nécessite une modification de la chaîne de reconstruction pour y être inséré immédiatement après la démodulation du signal acquis dans l'espace de Fourier. Nous avons aussi développé un filtre de Kalman parallèle qui permet d'appréhender toute distribution de bruit et nous avons montré que ses performances étaient comparables à celles de notre méthode précédente utilisant un filtre de Kalman non parallèle. Enfin, nous avons investigué la faisabilité de réaliser une tractographie en temps-réel pour déterminer la connectivité structurelle en direct, pendant l'examen. Nous espérons que ce panel de développements méthodologiques permettra d'améliorer et d'accélérer le diagnostic en cas d'urgence pour vérifier l'état des faisceaux de fibres de la substance blanche
Most magnetic resonance imaging (MRI) system manufacturers propose a huge set of software applications to post-process the reconstructed MRI data a posteriori, but few of them can run in real-time during the ongoing scan. To our knowledge, apart from solutions dedicated to functional MRI allowing relatively simple experiments or for interventional MRI to perform anatomical scans during surgery, no tool has been developed in the field of diffusion-weighted MRI (dMRI). However, because dMRI scans are extremely sensitive to lots of hardware or subject-based perturbations inducing corrupted data, it can be interesting to investigate the possibility of processing dMRI data directly during the ongoing scan and this thesis is dedicated to this challenging topic. The major contribution of this thesis aimed at providing solutions to denoise dMRI data in real-time. Indeed, the diffusion-weighted signal may be corrupted by a significant level of noise which is not Gaussian anymore, but Rician or noncentral chi. After making a detailed review of the literature, we extended the linear minimum mean square error (LMMSE) estimator and adapted it to our real-time framework with a Kalman filter. We compared its efficiency to the standard Gaussian filtering, difficult to implement, as it requires a modification of the reconstruction pipeline to insert the filter immediately after the demodulation of the acquired signal in the Fourier space. We also developed a parallel Kalman filter to deal with any noise distribution and we showed that its efficiency was quite comparable to the non parallel Kalman filter approach. Last, we addressed the feasibility of performing tractography in real-time in order to infer the structural connectivity online. We hope that this set of methodological developments will help improving and accelerating a diagnosis in case of emergency to check the integrity of white matter fiber bundles

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is considered the master circadian pacemaker which coordinates circadian rhythms in the central nervous system (CNS) and across the entire body. The SCN receives light input from the eyes through the retinohypothalamic tract and then it synchronizes other clocks in the CNS and periphery, thus orchestrating rhythms throughout the body. However, little is known about how so many cellular clocks within and across brain circuits can be effectively synchronized to entrain the coordinated expression of clock genes in cells distributed all over the brain. In this work I investigated the possible implication of two possible pathways: i) paracrine factors-mediated synchronization and ii) astrocytes-mediated synchronization. To study these pathways, I adopted an in vitro research model that I developed based on a lab-on-a-chip microfluidic device designed and realized in our laboratory. This device allows growing and compartmentalizing distinct neural populations connected through a network of astrocytes or through a cell-free channel in which the diffusion of paracrine factors is allowed. By taking advantage of this device, upon its validation, I synchronized neural clocks in one compartment and analyzed, in different experimental conditions, the induced expression of clock genes in a distant neural network grown in the second compartment. Results show that both pathways can be involved, but might have different roles. Neurons release factors that can diffuse to synchronize a neuronal population. The same factors can also synchronize astrocytes that, in turn, can transmit astrocyte-mediated molecular clocks to more distant neuronal populations. This is supported by experimental data obtained using microfluidic devices featuring different channel lengths. I found that paracrine factors-mediated synchronization occurs only in the case of a short distance between neuronal populations. On the contrary, interconnecting astrocytes define an active channel that can transfer molecular clocks to neural populations also at long distances. The study of possibly involved signaling factors indicate that paracrine factors-mediated synchronization occurs through GABA signaling, while astrocytes-mediated synchronization involves both GABA and glutamate. These findings strength the importance of the synergic regulation of clock genes among neurons and astrocytes, and identify a previously unknown role of astrocytes as active cells in distributing signals to regulate the expression of clock genes in the brain. Preliminary results also show a correlation between astrocyte reactivity and local alterations in neuronal synchronization, thus opening a new scenario for future studies in which disease-induced astrocyte reactivity might be linked to alterations in clock gene expression.
Three-dimensional (3D) brain models hold great potential for the generation of functional in vitro models to advance studies on human brain development, diseases and possible therapies. The routine exploitation of such models, however, is hindered by the lack of technologies to chronically monitor the activity of neural aggregates in three dimensions. A promising new approach consists in growing bio-artificial 3D brain model systems with seamless tissue-integrated biosensing artificial microdevices. Such devices could provide a platform for in-tissue sensing of diverse biologically relevant parameters. To date there is very little information on how to control the extracellular integration of such microscale devices into neuronal 3D cell aggregates. In this direction, in the present work I contributed to investigated the growth of hybrid neurospheroids obtained by the aggregation of silicon sham microchips (100x100x50μm3) with primary cortical cells. Interestingly, by coating microchips with different adhesion-promoting molecules, we reveal that surface functionalization can tune the integration and final 3D location of self-standing microdevices into neurospheroids. Morphological and functional characterization suggests that the presence of an integrated microdevice does not alter spheroid growth, cellular composition, nor network activity and maturation. Finally, we also demonstrate the feasibility of separating cells and microchips from formed hybrid neurospheroids for further single-cell analysis, and quantifications confirm an unaltered ratio of neurons and glia. These results uncover the potential of surface-engineered self-standing microdevices to grow untethered three-dimensional brain-tissue models with inbuilt bioelectronic sensors at predefined sites.

The experimental work presented in this Thesis involves new strategies focused during my Ph.D. activity to improve drug oral bioavailability, avoid side effects or promote the targeting of therapeutic agents to their action site. The co-crystallization strategy was applied to increase the dissolution rate and the permeability across the intestinal barrier of nitrofurantoin (NITRO), an antibiotic characterized by low aqueous solubility and low oral bioavailability. In particular, NITRO dissolution rate and permeability were compared with those of its cocrystals containing isoniazid (ISO), bipyridyl (BIP), or phenanthroline (PHE) as coformers, and their parent mixtures. NITRO dissolution profiles were evaluated via High-Performance Liquid Chromatography (HPLC), whereas permeation studies were performed by using an in vitro model of the small intestine based on rat intestine epithelial cells (IEC-6). The research activity was then focused on D-limonene, eugenol and cinnamaldehyde, natural compounds derived from essential oils promising in the prevention and protection of neurodegenerative diseases. In particular, I contributed to perform a systematic in vivo study to elucidate their pharmacokinetic profile, oral bioavailability, and aptitude to permeate the central nervous system from the bloodstream. Based on obtained data, eugenol was recruited for in vitro studies of viability and time/dose-dependent dopamine release in neuronal differentiated PC12 cells, a recognized cellular model mimicking dopaminergic neurons. The prodrug approach was considered to study self-assemble nanomicelles consisting in amphiphilic inulin-D-α-tocopherol succinate bioconjugates (INVITE) and loaded with an antioxidant compound, curcumin (INVITE C), to enhance curcumin biopharmaceutical properties and induce its targeting to the retina from the bloodstream. Transport experiments on polarized monolayers of human retinal pigment epithelium (HRPE) cells were performed, evaluating the transepithelial electrical resistance of the HRPE monolayer in physiologic and diabetic conditions. The prodrug approach for nasal formulations and brain targeting was then focused on ferulic acid (Fer), known for its antioxidant and anti-inflammatory activities, potentially useful against neurodegenerative diseases. A prodrug of Fer (methyl ferulate, Fer-Me) was synthesised and loaded in tristearin or stearic acid solid lipid microparticles (SLMs) as sustained delivery and targeting systems for Fer. In vitro pharmacokinetic studies were performed via HPLC to evaluate the prodrug behaviour of Fer-Me. The ability of SLMs to control the prodrug release and the dissolution rate were observed via dissolution and release from SLMs studies and quantification via HPLC. The prodrug approach on Fer was further developed and a conjugate of Fer itself methylated on the carboxylic moiety, without the use of linkers, was synthesised (Fer-Fer-Me). Fer-Fer-Me and its potential hydrolysis products, namely the non-methylated homologous (Fer-Fer-OH), Fer-Me and Fer, after appropriate purification of blood samples, were quantified via HPLC. The prodrug behaviour of Fer-Fer-Me was evidenced by in vitro pharmacokinetic studies, then it was loaded in tristearin and stearic acid SLMs. The results obtained by their characterization allowed to select the stearic acid SLMs loaded with Fer-Fer-Me for a nasal administration in rats, quantifying the prodrug in the cerebrospinal fluid of rats (CSF). A further approach related to nasal administration and brain targeting was studied considering the use of cyclodextrins focusing on geraniol (GER), a natural compound derived from essential oils that may exert anti-inflammatory effects in neurodegenerative diseases. Inclusion complexes with β-cyclodextrin (βCD) and its hydrophilic derivative hydroxypropyl-β-cyclodextrin (HPβCD) were formulated and the biocompatibility with nasal mucosae and drug bioavailability into CSF were studied in rats.
Nuove strategie per migliorare la biodisponibilità orale dei farmaci, limitare effetti collaterali o promuovere il direzionamento di agenti terapeutici al loro sito di azione sono state presentate in questa Tesi. I cocristalli sono stati sfruttati per aumentare la velocità di dissoluzione e la permeabilità attraverso la barriera intestinale della nitrofurantoina (NITRO), antibiotico caratterizzato da bassa solubilità acquosa e biodisponibilità orale. NITRO è stata confrontata, in termini di velocità di dissoluzione e permeazione, con i cocristalli contenenti isoniazide, bipiridile o fenantrolina come coformeri e con le miscele fisiche. I profili di dissoluzione della NITRO sono stati valutati via cromatografia liquida ad alta prestazione (HPLC), gli studi di permeazione sono stati eseguiti su un modello in vitro basato su cellule epiteliali di intestino di ratto. Ho contribuito ad effettuare uno studio in vivo per valutare il profilo farmacocinetico, biodisponibilità orale e tendenza a permeare nel sistema nervoso centrale dal sangue di D-limonene, eugenolo e cinnamaldeide, composti naturali derivati da oli essenziali promettenti nella prevenzione e protezione di patologie neurodegenerative. In base ai risultati, l’eugenolo è stato selezionato per studi in vitro su vitalità e rilascio tempo/dose-dipendente della dopamina in cellule PC12 differenziate a fenotipo neuronale, un modello di neuroni dopaminergici. Sono state studiate nanomicelle self-assemblanti costituite da bioconiugati anfifilici di inulina-D-α-tocoferolo succinato caricate con un composto antiossidante (INVITE C), la curcumina, per migliorarne le proprietà biofarmaceutiche e indurne il direzionamento alla retina. Sono stati effettuati esperimenti di trasporto su monostrati polarizzati di cellule di epitelio pigmentato umano, valutandone la resistenza elettrica transepiteliale e il beneficio di INVITE C in condizioni diabetiche simulate. Sono stati progettati e sintetizzati profarmaci dell’acido ferulico (Fer), noto per le attività antiossidanti e antinfiammatorie, potenzialmente utili contro patologie neurodegenerative. È stato sintetizzato un profarmaco di Fer (metil ferulato, Fer-Me). Fer-Me è stato caricato in microparticelle solide lipidiche (SLM) di tristearina o acido stearico come sistema di trasporto e di direzionamento per Fer. Studi farmacocinetici in vitro sono stati condotti via HPLC per valutare se Fer-Me fosse un profarmaco. La capacità delle SLM di controllare il rilascio del profarmaco e la velocità di dissoluzione sono stati osservati attraverso studi di dissoluzione e rilascio dalle SLM, quantificando via HPLC. Inoltre, è stato sintetizzato un coniugato di Fer con se stesso senza l’uso di linkers, metilato sul carbossile (Fer-Fer-Me). Fer-Fer-Me e i suoi potenziali prodotti di idrolisi, ovvero l’omologo non metilato (Fer-Fer-OH), Fer-Me e Fer, sono stati quantificati via HPLC in seguito ad appropriate procedure di estrazione da fluidi fisiologici. Studi farmacocinetici in vitro hanno dimostrato che Fer-Fer-Me è un profarmaco di Fer, ed è stato caricato in SLM di tristearina e acido stearico. I risultati ottenuti dalla caratterizzazione hanno permesso di selezionare le SLM di acido stearico per una somministrazione nasale a ratti, quantificando il profarmaco nel liquido cerebrospinale (CSF) per dimostrare la capacità della formulazione di indurre il direzionamento nel sistema nervoso centrale. È stato studiato un ulteriore approccio relativo alla somministrazione nasale e al direzionamento centrale utilizzando ciclodestrine e geraniolo (GER), un composto naturale derivato dagli oli essenziali che potrebbe esercitare effetti antinfiammatori in patologie neurodegenerative. Sono stati formulati complessi di inclusione con β-ciclodestrina (β-CD) e il suo derivato idrofilico idrossipropil-β-ciclodestrina (HP-β-CD), studiando la biocompatibilità con la mucosa nasale e la biodisponibilità di GER nel CSF nei ratti.

I sistemi di veicolazione del farmaco sono largamente studiati come tra i più efficienti metodi per migliorare l’efficacia dei farmaci. Negli ultimi vent’anni un’enorme attenzione è stata focalizzata sui sistemi nanometrici di veicolazione che sono in grado di interagire selettivamente con organismi patogeni, cellule o tessuti. Tra i gli eccipienti utilizzabili nella preparazione di questi sistemi, lipidi e oligosaccaridi mostrano una elevata biocompatibilità, biodegradabilità e idoneità per la somministrazione di farmaci attraverso varie vie. Lo scopo della tesi è stato lo sviluppo di sistemi di veicolazione nanometrici progettati specificatamente per aumentare l’efficacia e il direzionamento di determinati composti attivi. Nel primo progetto nanoparticelle lipidiche in grado di auto-assemblarsi (SALNs) sono state sviluppate per incorporare a loro volta nanoparticelle di ossido di ferro ricoperte da eparina (Fe@hepa) al fine di ottenere un sistema “teranostico” per via orale assorbibile mediante la circolazione linfatica. Le SALNs sono state caratterizzate e testate in vitro su modelli cellulari (CaCo-2) di assorbimento intestinale. I risultati hanno dimostrato la capacità delle SALNs di veicolare le Fe@hepa in cellule CaCo-2 senza indurre tossicità. Nel secondo progetto, sono stati sviluppati liposomi co-caricati con due farmaci anti-tubercolosi di prima scelta, isoniazide (INH) e rifampicina (RIF) somministrabili per via inalatoria. I liposomi sono stati caratterizzati mediante la tecnica di scattering di neutroni a piccolo angolo (SANS). Le analisi hanno evidenziato che il co-caricamento di RIF e INH induce una stabilizzazione sulla struttura dei liposomi, confermata con l’aumento del loro caricamento. Nel contesto della tubercolosi polmonare, anche nanoparticelle solido-lipidiche in cluster (SLNas) sono state sviluppate, caratterizzate e somministrate in vivo su topi. SLNas sono state preparate mediante un tensioattivo mannosilato di neo-sintesi (SLNas/MS) per il direzionamento attivo ai macrofagi alveolari (AM). Dopo la somministrazione inalatoria, SLNas/MS hanno dimostrato di raggiungere gli alveoli e di localizzarsi nei polmoni senza diffondersi nel resto del corpo. Inoltre, è stata dimostrata l’internalizzazione delle particelle da parte dei AM (raccolti dopo il trattamento) mediante microscopia a fluorescenza. Tutti i risultati suggeriscono la reale capacità delle SLNas di agire sui AM. Nel terzo progetto, sono state studiate due strategie basate su sistemi nanometrici per una efficiente e sicura veicolazione di geraniolo (GER) per il trattamento del Morbo di Parkinson mediante la via “nose-to-brain”. Nella prima strategia nanoparticelle polimeriche(NP) e lipidiche (SLN) sono state preparate, liofilizzate per aumentarne la stabilità e ne è stato valutato il contenuto in GER-. I risultati hanno indicato che, durante la liofilizzazione, il GER non è trattenuto dalle particelle, probabilmente a causa della sua volatilità. Pertanto NP e SLN cariche del coniugato GER-acido ursodesossicolico (GER-UDCA, profarmaco di GER) sono state sviluppate e caratterizzate in termini di contenuto, rilascio in vitro, morfologia, e infine somministrate in vivo. I risultati hanno dimostrato che le SLN garantiscono alte concentrazioni fino a 3 ore del profarmaco nel cervello, senza recare nessun danno alla mucosa nasale. Per la seconda strategia, complessi di inclusione tra GER e ciclodestrine (GER-CD) sono stati preparati usando la 2-hydroxypropyl-β-CD (HPβCD) e la β-CD. I complessi GER-CD sono stati caratterizzati e i risultati hanno dimostrato la reale inclusione di GER nella cavità delle CD. La somministrazione in vivo dei complessi è attualmente in corso.
Drug delivery systems (DDS) are widely investigated as one of the main tools in medicine due to their potential to treat diseases. During the last two decades, great attention has been focused on nanostructured DDS able to selectively interact with pathogens, cells or tissues. Among all the exploitable materials in formulating DDS, lipids and oligosaccharides exhibit high biocompatibility, biodegradability, and suitability for the administration of drugs through several routes. The aim of this thesis was the development of specific nanostructured DDS designed to enhance efficacy and targeting of active compounds. In the first project, self-assembled lipid nanoparticles (SALNs) were developed for the encapsulation of heparin-coated iron oxide nanoparticles (Fe@hepa) in order to obtain a nanotheranostic tool able to be absorbed orally through the lymphatic route. SALNs were fully characterized and tested in vitro on cell models (CaCo-2 cell line) for intestinal absorption. The results demonstrated the suitability of SALNs in efficiently delivering Fe@hepa into CaCo-2 cells without causing cytotoxicity. In the second project, co-loaded liposomes with two first-line antituberculosis drugs, isoniazid (INH) and rifampicin (RIF), were developed for inhaled therapy. Liposomes were characterized in-depth by small-angle neutron scattering technique (SANS). The analysis highlighted that the RIF-INH co-loading elicited a stabilizing effect on the liposome structure, confirmed by the increment of the drug loading capacity. In a pulmonary tuberculosis context, RIF-loaded solid lipid nanoparticles assemblies (SLNas) were also developed, fully characterized in vitro and administered in vivo on mice. SLNas were formulated with the employment of a newly synthesized mannosylated surfactant (SLNas/MS) for the active targeting to the alveolar macrophages (AM). After administration, SLNas/MS demonstrated the ability to reach the alveolar region and to be retained in the lungs without broad distribution in the body. Furthermore, fluorescence microscopy analysis was performed on AM (collected after the treatment) showing cell internalization of the particles. All the results suggested the suitability of SLNas/MS in efficiently targeting AM. In the third project, two different strategies based on nanostructured DDS were investigated for an efficient and safe delivery of Geraniol (GER) via nose-to-brain for the treatment of Parkinson’s Disease. In the first strategy, polymeric (NP) and lipid-based (SLN) nanoparticles were prepared. In order to obtain long-term stable formulations, the samples were freeze-dried and characterized regarding GER loading. The results indicated that no GER was retained in the nanoparticles, probably due to its volatility during the freeze-drying process. Therefore, GER-ursodeoxycholic acid conjugate (GER-UDCA, a GER prodrug) was used instead of GER. NP and SLN were developed, characterized regarding drug content, in vitro release and morphology, and finally administered in vivo. The results demonstrated the suitability of GER-UDCA-loaded SLN for the in vivo administration, which guaranteed high concentrations of the prodrug up to 3 hours in the brain without causing any damage to the nasal mucosa. For the second strategy, inclusion complexes between GER and cyclodextrins (CD) were prepared by using 2-hydroxypropyl-β-CD (HP-βCD) and β-CD. The inclusion complexes were characterized in-depth and the results confirmed the real inclusion of GER into CD cavities. In vivo administration of both the inclusion complexes will be further investigated.

This PhD thesis debates, mostly, on two main topics: - Drug delivery to brain - Nanosuspensions for different applications The objective of the first topic was the development of liposomes to which anti-TfR-monoclonal antibodies (Ox26) or lactoferrin was bounded to transport the selective NK3 receptor agonist senktide to CNS across the BBB. NK3 receptors are widely expressed in the CNS and their stimulation by senktide (ICV) increase extracellular DA. Liposomes were prepared using the film hydration method. In vivo microdialysis studies were performed to estimate the responsiveness of NAc shell DA to senktide as a consequence of its CNS delivery. Senktide given ICV or loaded into Ox26/lactoferrin-liposome (0.5 μg/kg iv) elicited a significant increase of dialysate DA in the NAc shell of rats whilst senktide given iv (0.1 mg/kg) or loaded in control stealth liposomes did not affect NAc shell DA. Liposomes formulation here described represent an effective way of CNS delivering of senktide following intravenous administration the TfR-transport system. On the other hand, three different types of nanosuspensions were formulated and studied: - Tretinoin nanosuspensions for topical delivery - Piroxicam nanosuspensions loaded in oral disintegrating tablet (ODT) for oral delivery - Quercetin nanosuspensions loaded in fast dissolving films for oral delivery The aims of the first work were to improve cutaneous targeting and photostability of tretinoin by using nanosuspension formulation. Tretinoin is a drug widely used in the topical treatment of various dermatological diseases. The tretinoin nanosuspension was prepared by precipitation method and then characterized by photo correlation spectroscopy for mean size and size distribution, and by transmission electron microscopy for morphological studies. An oil in water tretinoin nanoemulsion was also prepared and used as a control. Dermal and transdermal delivery of both tretinoin nanosuspension and nanoemulsion were tested in vitro by using Franz diffusion cells and newborn pig skin. Photodegradation studies were carried out by UV irradiation (1 h, λ=366 nm) of the tretinoin nanosuspension in comparison with the nanoemulsion and a methanolic solution of the drug. During 8 h percutaneous experiments, no permeation of tretinoin through the whole skin thickness was detected but the drug was deposited into the skin layers, mainly in the stratum corneum, similarly to the nanoemulsion. UV irradiation of the nanosuspension showed a great improvement of tretinoin stability in comparison with both controls. Overall results show that nanosuspension might be a useful formulation for improving tretinoin dermal delivery and stability. Piroxicam (PRX) is a non-steroidal anti-inflammatory drug characterized by a poorly water solubility and consequently by a low oral bioavailability. Different nanocrystals orally disintegrating tablets (ODT) were prepared to enhance piroxicam dissolution velocity and saturation solubility. Nanosuspensions were prepared using high pressure homogenization technique. Different ODT formulations were prepared using the same nanosuspension but changing different excipients in order to optimize dissolution properties. PRX nanocrystals size and zeta potential were determined by photon correlation spectroscopy (PCS). Characterization of PRX nanocrystals ODT was carried out by infrared spectroscopy (FTIR), X-ray powder diffractometry (XRPD), differential scanning calorimetry. Dissolution study of PRX ODT was performed in distilled water (pH 5.5) and was compared with PRX coarse suspension ODT, PRX/poloxamer 188 physical mixture, bulk PRX samples and a piroxicam commercial ODT (Feldene). All PRX nanocrystals ODT formulations showed a higher drug dissolution rate than coarse PRX ODT. PRX nanocrystals ODT prepared using gelatin or croscaramellose as excipient showed a higher PRX dissolution rate compared with the commercial formulation and with ODT prepared using xanthan gum. The improvement in PRX dissolution rate is mainly caused by the increased surface-to-volume ratio due to the submicron dimension of the drug nanocrystal, however, also the presence of the correct excipients (as disgregant) seem to play an essential role. Finally, quercetin nanosuspensions loaded fast dissolving films were formulated and studied. The aim of this work was to investigate the possible use of maltodextrin IT6 (MDX) to prepare fast-dissolving films, loaded by quercetin nanocrystals. Quercetin nanosuspensions were prepared using an high pressure homogenizer, meanwhile drug loaded films were obtained drying in a siliconized polyester sheet quercetin nanosuspensions with the others compounds in a oven at 60 °C. Films were finally cut and packed within sealed aluminium pouches. Quercetin nanocrystals were characterized by photo correlation spectroscopy for mean size and size distribution and by transmission electron microscopy for morphological studies. On the other hand, quercetin nanosuspensions loaded films were characterized in term of flexibility, tensile strength and thickness. Finally, dissolution studies in distilled water were performed, comparing release profiles of quercetin loaded films, quercetin raw material and quercetin nanosuspensions.

Notre génome est constamment attaqué par des facteurs endogènes et exogènes qui menacent son intégrité et conduisent à différents types de dommages. Les cassures double brins (CDBs) font partie des dommages les plus nuisibles car elles peuvent entraîner la perte d'information génétique, des translocations chromosomiques et la mort cellulaire. Tous les processus de réparation se déroulent dans le cadre d'une chromatine hautement organisée et compartimentée. Cette chromatine peut être divisée en un compartiment ouvert transcriptionnellement actif (euchromatine) et un compartiment compacté transcriptionnellement inactif (hétérochromatine). Ces différents degrés de compaction jouent un rôle dans la régulation de la réponse aux dommages à l’ADN. L'objectif de mon premier projet était de comprendre l'influence de l'organisation 3D du génome sur la réparation de l'ADN. Pour cela, j’ai utilisé deux approches complémentaires dans le but d’induire et de cartographier les CDBs dans le génome de souris. Mes résultats ont mis en évidence un enrichissement de γH2AX, facteur de réparation des dommages à l’ADN, sur différentes régions du génome de cellules souches embryonnaires de souris, et ont également montré que les dommages persistent dans l’hétérochromatine, contrairement à l’euchromatine qui est protégée des dommages. Pour mon deuxième projet, j'ai cartographié l'empreinte génomique de 53BP1, facteur impliqué dans la réparation des CDBs, dans des cellules U2OS asynchrones et des cellules bloquées en G1 afin d’identifier de nouveaux sites de liaison de 53BP1. Mes résultats ont permis d’identifier de nouveaux domaines de liaison de 53BP1 couvrant de larges régions du génome, et ont montré que ces domaines de liaison apparaissent dans des régions de réplication moyenne et tardive
Our genome is constantly under attack by endogenous and exogenous factors which challenge its integrity and lead to different types of damages. Double strand breaks (DSBs) constitute the most deleterious type of damage since they maylead to loss of genetic information, translocations and cell death. All the repair processes happen in the context of a highly organized and compartmentalized chromatin. Chromatin can be divided into an open transcriptionally active compartment (euchromatin) and a compacted transcriptionally inactive compartment (heterochromatin). These different degrees of compaction play important roles in regulating the DNA damage response. The goal of my first project was to understand the influence of 3D genome organization on DNA repair. I used two complementary approaches to induce and map DSBs in the mouse genome. My results have shown that enrichment of the DNA damage repair factor γH2AX occurs at distinct loci in the mouse embryonic stem cell genome and that the damage persists in the heterochromatin compartment while the euchromatin compartment is protected from DNA damage. For my second project, I mapped the genomic footprint of 53BP1, a factor involved in DSBs repair, in asynchronous and G1 arrested U2OS cells to identify novel 53BP1 binding sites. My results have identified novel 53BP1 binding domains which cover broad regions of the genome and occur in mid to late replicating regions of the genome

Alzheimer’s disease is a chronic progressive neurodegenerative disease and is the most common form of dementia (estimated 50−60% of all cases), associated with loss of memory (in particular episodic memory), cognitive decline, and behavioural and physical disability, ultimately leading to death. Alzheimer’s disease is a complex disease, mostly occurring sporadically with no apparent inheritance and being the age the main risk factor. The production and accumulation of amyloid-beta peptide in the central nervous system is a key event in the development of Alzheimer’s disease. This project is devoted to the synthesis of amyloid-beta ligands, fluorophores and blood brain barrier-transporters for diagnosis and therapy of Alzheimer’s disease. Different amyloid-beta ligands will be synthesized and their ability to interact with amyloid-beta plaques will be studied with nuclear magnetic resonance techniques and a process of lead optimization will be performed. Many natural and synthetic compounds able to interact as amyloid-beta ligands have been identified. Among them, a set of small molecules in which aromatic moieties seem to play a key role to inhibit amyloid-beta aggregation, in particular heteroaromatic polycyclic compounds such as tetracyclines. Nevertheless tetracyclines suffer from chemical instability, low water solubility and possess, in this contest, undesired anti-bacterial activity. In order to overcome these limitations, one of our goals is to synthesize tetracyclines analogues bearing a polycyclic structure with improved chemical stability and water solubility, possibly lacking antibacterial activity but conserving the ability to interact with amyloid-beta peptides. Known tetracyclines have in common a fourth cycle without an aromatic character and with different functionalisations. We aim to synthesize derivatives in which this cycle is represented by a sugar moiety, thus bearing different derivatisable positions or create derivatives in which we will increase or decrease the number of fused rings. In order to generate a potential drug-tool candidate, these molecules should also possess the correct chemical-physical characteristics. The glycidic moiety, not being directly involved in the binding, it assures further possible derivatizations, such as conjugation to others molecular entities (nanoparticles, polymeric supports, etc.), and functionalization with chemical groups able to modulate the hydro/lipophilicity. In order to be useful such compounds should perform their action within the brain, therefore they have to be able to cross the blood brain barrier, and to be somehow detected for diagnostic purposes.

The effects of sorghum bran addition on table tortillas and tortilla chip properties were evaluated. Texture, phenol content, antioxidant activity, and sensory characteristics were evaluated. Texture was measured by objective and subjective tests. Products were analyzed for phenols following the Folin-Ciocalteu procedure and for antioxidant potential following the ABTS (2,2'-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) method. Sensory properties were evaluated using a nine point hedonic scale. Bran from two specialty sorghums: sumac (high tannin) and black (high anthocyanins) was added at 0, 5, and 10% to table tortillas and tortilla chips. For table tortillas the interaction of sorghum bran with an antistaling formula containing guar gum, carboxymethylcellulose and maltogenic alpha-amylase was assessed. Tortillas containing sorghum bran had a more friable structure than the control. This detrimental effect was overcome by the antistaling formula. Additives made fluffier tortillas with improved texture and appearance. Tortillas containing sorghum bran and the antistaling formula were acceptable to panelists. At 5% sorghum bran inclusion, there was no significant difference in sensory attributes from the control aside from appearance. Tortillas containing sorghum bran had a dark natural color comparable to that of blue corn tortillas. Tortilla chip texture was not significantly affected by addition of bran to the formula. As in table tortillas, addition of sorghum bran produced minor changes in the texture and flavor of the product, but a significant change in appearance acceptability. Tortilla chips had a dark color, comparable to the one of blue corn tortilla chips. Sumac bran yielded larger amounts of phenols and antioxidant activity than black bran. Levels of phenols and antioxidant potential increased with increased bran. Although processing caused a measurable loss of sorghum bran antioxidants, table tortilla and tortilla chips were still a significant source of phenols and antioxidant activity. The addition of sorghum bran produced tortillas and tortilla chips with increased levels of dietary fiber and antioxidants, without adversely affecting other sensory properties.

ChIP-chip is a technology originally developed to determine the binding sites of proteins in chromatin on a genome wide scale. Its uses have since been expanded to analyse other genome features, such as epigenetic modifications and, in our laboratory, DNA damage. Datasets comprise many thousands of data points and therefore require bioinformatic tools for their analysis. Currently available tools are limited in their applications and lack the ability to normalise data so as to allow relative comparisons between different datasets. This has limited the analyses of multiple ChIP-chip datasets from different experimental conditions. The first part of the study presented here is bioinformatic, presenting a selection of tools written in R for ChIP-chip data analysis, including a novel normalisation procedure which allows datasets from different conditions to be analysed together, permitting comparisons of values between different experiments and opening up a new dimension of analysis of these datasets. A novel enrichment detection procedure is presented, suited to many formats of data, including protein binding (which forms peaks) and epigenetic modifications (which can form extended regions of enrichment). Graphical tools are also presented, to facilitate the analysis of these large datasets. A method of predicting the output of a ChIP-chip dataset is presented, which has been used to show that ChIP-chip is capable of detecting sequence dependent damage events. All functions work together, using a common data format, and are effcient and easy to use. The second part of this study applies these bioinformatic tools in a biological context. An analysis of Abf1 protein binding datasets has been undertaken, revealing many more binding sites than had previously been identified. Analysis of the sequences at these binding sites identifed the previously determined consensus binding motif in only a subset, with no novel motif identifiable in the remainder, suggesting binding may be in uenced by factors other than sequence.

Many high throughput sequencing protocols for RNA and DNA require that the polynucleic acid is fragmented so that the identity of a limited number of nucleic acids of one or both of the ends of the fragments can be determined by sequencing. The nucleic acid sequence allows the fragment to be located within the genome, and the fragment distribution can then be used for a variety of different purposes. In the case of DNA this includes identifying the locations where specific proteins are bound to the genome. In the case of RNA this includes quantifying the expression levels of different gene variants or transcripts. If the locations of the polynucleic acid fragments are partly determined by the underlying nucleic acid sequence this could bias any results derived from the data. Unfortunately, such sequence dependencies have already been observed in the distribution of both RNA and DNA fragments. Previous analyses of such data in order to reduce the bias have examined the role of regional characteristics such as GC bias, or the bias towards a specific sequence at the start of the fragments. This thesis introduces a new method for modelling the bias which considers the degree to which the nucleotide sequence affects the likelihood of a fragment originating at that location. This shows that there is often not a single bias characteristic, but multiple, alternative sequence biases that coexist within a single dataset. This also shows that the nucleotide sequence immediately proximal to the fragment also has a significant effect on the fragment likelihood. This new approach highlights characteristics that were previously hidden and provides a more powerful basis for correcting such bias. Multiple alternative sequence biases are observed when both RNA and DNA are fragmented, but the more detailed information provided by the new technique shows in detail how the characteristics are different for RNA and DNA and indicates that very different molecular mechanisms are responsible for the biases in the two processes. This thesis also shows how removing the effect of this bias in ChIP-seq experiments can reveal more subtle features of the distribution of the fragments. This can provide information on the nature of the binding between proteins and the DNA with per-nucleotide precision, revealed through the change in likelihood of the DNA fragmenting at each position in the binding site. It is also shown how the model fitting technique developed to analyse sequence bias can also be used to obtain additional information from the results of ChIP-chip experiments. The approach is used to find the nucleotide sequence preference of DNA binding proteins, and also the cooperative effects associated with binding at multiple binding sites in close proximity.

This paper discusses the challenges and the trade-offs in the design of laser drivers for very-short distance optical communications. A prototype integrated circuit is designed and fabricated in 28 nm super-low-power CMOS technology. The power consumption of the transmitter is 17.2 mW excluding the VCSEL that in our test has a DC power consumption of 10 mW. The active area of the driver is only 0.0045 mm². The driver can achieve an error-free (