Continuos Flow Single Cell Separation into Open Microwell Arrays

A novel design based on electric field-free open microwell arrays for the automated continuous-flow sorting of single or small clusters of cells is presented. The main feature of the proposed device is the parallel analysis of cell-cell and cell-particle interactions in each microwell of the array. High throughput sample recovery with a fast and separate transfer from the microsites to standard microtiter plates is also possible thanks to the flexible printed circuit board technology which permits to produce cost effective large area arrays featuring geometries compatible with laboratory equipment. The particle isolation is performed via negative dielectrophoretic forces which convey the particles’ into the microwells. Particles such as cells and beads flow in electrically active microchannels on whose substrate the electrodes are patterned. The introduction of particles within the microwells is automatically performed by generating the required feedback signal by a microscope-based optical counting and detection routine. In order to isolate a controlled number of particles we created two particular configurations of the electric field within the structure. The first one permits their isolation whereas the second one creates a net force which repels the particles from the microwell entrance. To increase the parallelism at which the cell-isolation function is implemented, a new technique based on coplanar electrodes to detect particle presence was implemented. A lock-in amplifying scheme was used to monitor the impedance of the channel perturbed by flowing particles in high-conductivity suspension mediums. The impedance measurement module was also combined with the dielectrophoretic focusing stage situated upstream of the measurement stage, to limit the measured signal amplitude dispersion due to the particles position variation within the microchannel. In conclusion, the designed system complies with the initial specifications making it suitable for cellomics and biotechnology applications.

Self assembled materials for solar cell application

In der vorliegenden Arbeit wurden Materialien und Aufbauten für Hybrid Solarzellen entwickelt und erforscht. rnDer Vergleich zweier bekannter Lochleitermaterialien für Solarzellen in einfachen Blend-Systemen brachte sowohl Einsicht zur unterschiedlichen Eignung der Materialien für optoelektronische Bauelemente als auch neue Erkenntnisse in Bereichen der Langzeitstabilität und Luftempfindlichkeit beider Materialien.rnWeiterhin wurde eine Methode entwickelt, um Hybrid Solarzelle auf möglichst unkomplizierte Weise aus kostengünstigen Materialien darzustellen. Die „Eintopf“-Synthese ermöglicht die unkomplizierte Darstellung eines funktionalen Hybridmaterials für die optoelektronische Anwendung. Mithilfe eines neu entwickelten amphiphilen Blockcopolymers, das als funktionelles Templat eingesetzt wurde, konnten mit einem TiO2-Precursor in einem Sol-Gel Ansatz verschiedene selbstorganisierte Morphologien des Hybridmaterials erhalten werden. Verschiedene Morphologien wurden auf ihre Eignung in Hybrid Solarzellen untersucht. Ob und warum die Morphologie des Hybridsystems die Effizienz der Solarzelle beeinflusst, konnte verdeutlicht werden. Mit der Weiterentwicklung der „Eintopf“-Synthese, durch den Austausch des TiO2-Precursors, konnte die Solarzelleneffizienz von 0.15 auf 0.4 % gesteigert werden. Weiterhin konnte die Übertragbarkeit des Systems durch den erfolgreichen Austausch des Halbleiters TiO¬2 mit ZnO bewiesen werden.rn

Synthesis of Biologically Active Small Molecules: Different Approaches to Drug Design

In the past years, genome biology had disclosed an ever-growing kind of biological targets that emerged as ideal points for therapeutic intervention. Nevertheless, the number of new chemical entities (NCEs) translated into effective therapies employed in the clinic, still not observed. Innovative strategies in drug discovery combined with different approaches to drug design should be searched for bridge this gap. In this context organic synthetic chemistry had to provide for effective strategies to achieve biologically active small molecules to consider not only as potentially drug candidates, but also as chemical tools to dissect biological systems. In this scenario, during my PhD, inspired by the Biology-oriented Synthesis approach, a small library of hybrid molecules endowed with privileged scaffolds, able to block cell cycle and to induce apoptosis and cell differentiation, merged with natural-like cores were synthesized. A synthetic platform which joined a Domino Knoevenagel-Diels Alder reaction with a Suzuki coupling was performed in order to reach the hybrid compounds. These molecules can represent either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells. The biological profile expressed by some of these derivatives showed a well defined antiproliferative activity on leukemia Bcr-Abl expressing K562 cell lines. A parallel project regarded the rational design and synthesis of minimally structurally hERG blockers with the purpose of enhancing the SAR studies of a previously synthesized collection. A Target-Oriented Synthesis approach was applied. Combining conventional and microwave heating, the desired final compounds were achieved in good yields and reaction rates. The preliminary biological results of the compounds, showed a potent blocking activity. The obtained small set of hERG blockers, was able to gain more insight the minimal structural requirements for hERG liability, which is mandatory to investigate in order to reduce the risk of potential side effects of new drug candidates.

Design and fabrication of biocompatible scaffolds for the regeneration of tissues

Regenerative medicine and tissue engineering attempt to repair or improve the biological functions of tissues that have been damaged or have ceased to perform their role through three main components: a biocompatible scaffold, cellular component and bioactive molecules. Nanotechnology provide a toolbox of innovative scaffold fabrication procedures in regenerative medicine. In fact, nanotechnology, using manufacturing techniques such as conventional and unconventional lithography, allows fabricating supports with different geometries and sizes as well as displaying physical chemical properties tunable over different length scales. Soft lithography techniques allow to functionalize the support by specific molecules that promote adhesion and control the growth of cells. Understanding cell response to scaffold, and viceversa, is a key issue; here we show our investigation of the essential features required for improving the cell-surface interaction over different scale lengths. The main goal of this thesis has been to devise a nanotechnology-based strategy for the fabrication of scaffolds for tissue regeneration. We made four types of scaffolds, which are able to accurately control cell adhesion and proliferation. For each scaffold, we chose properly designed materials, fabrication and characterization techniques.

Evolution of the structural and fracture design of the ISS payloads due to the new launchers, specifically applied to Biolab IEC-2 projects

The relatively young discipline of astronautics represents one of the scientifically most fascinating and technologically advanced achievements of our time. The human exploration in space does not offer only extraordinary research possibilities but also demands high requirements from man and technology. The space environment provides a lot of attractive experimental tools towards the understanding of fundamental mechanism in natural sciences. It has been shown that especially reduced gravity and elevated radiation, two distinctive factors in space, influence the behavior of biological systems significantly. For this reason one of the key objectives on board of an earth orbiting laboratory is the research in the field of life sciences, covering the broad range from botany, human physiology and crew health up to biotechnology. The Columbus Module is the only European low gravity platform that allows researchers to perform ambitious experiments in a continuous time frame up to several months. Biolab is part of the initial outfitting of the Columbus Laboratory; it is a multi-user facility supporting research in the field of biology, e.g. effect of microgravity and space radiation on cell cultures, micro-organisms, small plants and small invertebrates. The Biolab IEC are projects designed to work in the automatic part of Biolab. In this moment in the TO-53 department of Airbus Defence & Space (formerly Astrium) there are two experiments that are in phase C/D of the development and they are the subject of this thesis: CELLRAD and CYTOSKELETON. They will be launched in soft configuration, that means packed inside a block of foam that has the task to reduce the launch loads on the payload. Until 10 years ago the payloads which were launched in soft configuration were supposed to be structural safe by themselves and a specific structural analysis could be waived on them; with the opening of the launchers market to private companies (that are not under the direct control of the international space agencies), the requirements on the verifications of payloads are changed and they have become much more conservative. In 2012 a new random environment has been introduced due to the new Space-X launch specification that results to be particularly challenging for the soft launched payloads. The last ESA specification requires to perform structural analysis on the payload for combined loads (random vibration, quasi-steady acceleration and pressure). The aim of this thesis is to create FEM models able to reproduce the launch configuration and to verify that all the margins of safety are positive and to show how they change because of the new Space-X random environment. In case the results are negative, improved design solution are implemented. Based on the FEM result a study of the joins has been carried out and, when needed, a crack growth analysis has been performed.

Computer aided design and finite element modeling to predict bulk mechanical properties of bone scaffolds using triply periodic minimal surfaces (progettazione assistita al calcolatore ed analisi con il metodo degli elementi finiti per predire il comportamento meccanico di scaffolds ossei creati con l'uso di superfici minime periodiche in tre direzioni)

The aim of Tissue Engineering is to develop biological substitutes that will restore lost morphological and functional features of diseased or damaged portions of organs. Recently computer-aided technology has received considerable attention in the area of tissue engineering and the advance of additive manufacture (AM) techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. To regenerate tissues more efficiently, an ideal scaffold should have appropriate porosity and pore structure. More sophisticated porous configurations with higher architectures of the pore network and scaffolding structures that mimic the intricate architecture and complexity of native organs and tissues are then required. This study adopts a macro-structural shape design approach to the production of open porous materials (Titanium foams), which utilizes spatial periodicity as a simple way to generate the models. From among various pore architectures which have been studied, this work simulated pore structure by triply-periodic minimal surfaces (TPMS) for the construction of tissue engineering scaffolds. TPMS are shown to be a versatile source of biomorphic scaffold design. A set of tissue scaffolds using the TPMS-based unit cell libraries was designed. TPMS-based Titanium foams were meant to be printed three dimensional with the relative predicted geometry, microstructure and consequently mechanical properties. Trough a finite element analysis (FEA) the mechanical properties of the designed scaffolds were determined in compression and analyzed in terms of their porosity and assemblies of unit cells. The purpose of this work was to investigate the mechanical performance of TPMS models trying to understand the best compromise between mechanical and geometrical requirements of the scaffolds. The intention was to predict the structural modulus in open porous materials via structural design of interconnected three-dimensional lattices, hence optimising geometrical properties. With the aid of FEA results, it is expected that the effective mechanical properties for the TPMS-based scaffold units can be used to design optimized scaffolds for tissue engineering applications. Regardless of the influence of fabrication method, it is desirable to calculate scaffold properties so that the effect of these properties on tissue regeneration may be better understood.

Ribosome-inactivating proteins and their immunotoxins for cancer therapy: insights into the mechanism of cell death

Ribosome-inactivating proteins (RIPs) are a family of plant toxic enzymes that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death involving different and still not completely understood pathways. The high cytotoxic activity showed by many RIPs makes them ideal candidates for the production of immunotoxins (ITs), chimeric proteins designed for the selective elimination of unwanted or malignant cells. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively employed to construct anticancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. Here we investigated the anticancer properties of two saporin-based ITs, anti-CD20 RTX/S6 and anti-CD22 OM124/S6, designed for the experimental treatment of B-cell NHLs. Both ITs showed high cytotoxicity towards CD20-positive B-cells, and their antitumor efficacy was enhanced synergistically by a combined treatment with proteasome inhibitors or fludarabine. Furthermore, the two ITs showed differencies in potency and ability to activate effector caspases, and a different behavior in the presence of the ROS scavenger catalase. Taken together, these results suggest that the different carriers employed to target saporin might influence saporin intracellular routing and saporin-induced cell death mechanisms. We also investigated the early cellular response to stenodactylin, a recently discovered highly toxic type 2 RIP representing an interesting candidate for the design and production of a new IT for the experimental treatment of cancer.

Electrochemical imaging of living cell metabolism: investigation on Warburg effect in cancer

Cancer is one of the principal causes of death in the world; almost 8.2 million of deaths were counted in 2012. Emerging evidences indicate that most of the tumors have an increased glycolytic rate and a detriment of oxidative phosphorylation to support abnormal cell proliferation; this phenomenon is known as aerobic glycolysis or Warburg effect. This switching toward glycolysis implies that cancer tissues metabolize approximately tenfold more glucose to lactate in a given time and the amount of lactate released from cancer tissues is much greater than from normal ones. In view of these fundamental discoveries alterations of the cellular metabolism should be considered a crucial hallmark of cancer. Therefore, the investigation of the metabolic differences between normal and transformed cells is important in cancer research and it might find clinical applications. The aim of the project was to investigate the cellular metabolic alterations at single cell level, by monitoring glucose and lactate, in order to provide a better insight in cancer research. For this purpose, electrochemical techniques have been applied. Enzyme-based electrode biosensors for lactate and glucose were –ad hoc- optimized within the project and used as probes for Scanning Electrochemical Microscopy (SECM). The UME biosensor manufacturing and optimization represented a consistent part of the work and a full description of the sensor preparation protocols and of the characterization methods employed is reported. This set-up (SECM used with microbiosensor probes) enabled the non-invasive study of cellular metabolism at single cell level. The knowledge of cancer cell metabolism is required to design more efficient treatment strategies.

Automatic Design of Boolean Networks for Modelling Differentiation Trees

Real living cell is a complex system governed by many process which are not yet fully understood: the process of cell differentiation is one of these. In this thesis work we make use of a cell differentiation model to develop gene regulatory networks (Boolean networks) with desired differentiation dynamics. To accomplish this task we have introduced techniques of automatic design and we have performed experiments using various differentiation trees. The results obtained have shown that the developed algorithms, except the Random algorithm, are able to generate Boolean networks with interesting differentiation dynamics. Moreover, we have presented some possible future applications and developments of the cell differentiation model in robotics and in medical research. Understanding the mechanisms involved in biological cells can gives us the possibility to explain some not yet understood dangerous disease, i.e the cancer. Le cellula è un sistema complesso governato da molti processi ancora non pienamente compresi: il differenziamento cellulare è uno di questi. In questa tesi utilizziamo un modello di differenziamento cellulare per sviluppare reti di regolazione genica (reti Booleane) con dinamiche di differenziamento desiderate. Per svolgere questo compito abbiamo introdotto tecniche di progettazione automatica e abbiamo eseguito esperimenti utilizzando vari alberi di differenziamento. I risultati ottenuti hanno mostrato che gli algoritmi sviluppati, eccetto l'algoritmo Random, sono in grado di poter generare reti Booleane con dinamiche di differenziamento interessanti. Inoltre, abbiamo presentato alcune possibili applicazioni e sviluppi futuri del modello di differenziamento in robotica e nella ricerca medica. Capire i meccanismi alla base del funzionamento cellulare può fornirci la possibilità di spiegare patologie ancora oggi non comprese, come il cancro.

Mining tissue microarray data to uncover combinations of biomarker expression patterns that improve intermediate staging and grading of clear cell renal cell cancer

PURPOSE: Tumor stage and nuclear grade are the most important prognostic parameters of clear cell renal cell carcinoma (ccRCC). The progression risk of ccRCC remains difficult to predict particularly for tumors with organ-confined stage and intermediate differentiation grade. Elucidating molecular pathways deregulated in ccRCC may point to novel prognostic parameters that facilitate planning of therapeutic approaches. EXPERIMENTAL DESIGN: Using tissue microarrays, expression patterns of 15 different proteins were evaluated in over 800 ccRCC patients to analyze pathways reported to be physiologically controlled by the tumor suppressors von Hippel-Lindau protein and phosphatase and tensin homologue (PTEN). Tumor staging and grading were improved by performing variable selection using Cox regression and a recursive bootstrap elimination scheme. RESULTS: Patients with pT2 and pT3 tumors that were p27 and CAIX positive had a better outcome than those with all remaining marker combinations. A prolonged survival among patients with intermediate grade (grade 2) correlated with both nuclear p27 and cytoplasmic PTEN expression, as well as with inactive, nonphosphorylated ribosomal protein S6. By applying graphical log-linear modeling for over 700 ccRCC for which the molecular parameters were available, only a weak conditional dependence existed between the expression of p27, PTEN, CAIX, and p-S6, suggesting that the dysregulation of several independent pathways are crucial for tumor progression. CONCLUSIONS: The use of recursive bootstrap elimination, as well as graphical log-linear modeling for comprehensive tissue microarray (TMA) data analysis allows the unraveling of complex molecular contexts and may improve predictive evaluations for patients with advanced renal cancer.

Synthesis and biological activity of new functionalized epothilones for prodrug design and tumor targeting

Epothilones are potent antiproliferative agents, which have served as successful lead structures for anticancer drug discovery. However, their therapeutic efficacy would benefit greatly from an increase in their selectivity for tumor cells, which may be achieved through conjugation with a tumor-targeting moiety. Three novel epothilone analogs bearing variously functionalized benzimidazole side chains were synthesized using a strategy based on palladium-mediated coupling and macrolactonization. The synthesis of these compounds is described and their in vitro biological activity is discussed with respect to their interactions with the tubulin/microtubule system and the inhibition of human cancer cell proliferation. The additional functional groups may be used to synthesize conjugates of epothilone derivatives with a variety of tumor-targeting moieties.

Computational Design and Experimental Discovery of an Anti-estrogenic Peptide Derived from Alpha-Fetoprotein

Breast cancer is the most common cancer among women, and tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment. Many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Consequently, there is an ongoing need for breast cancer drugs that have different molecular targets. Previous work has shown that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat models, interacting with an unsolved receptor, while peptides smaller than eight amino acids did not. We show that the use of replica exchange molecular dynamics predicts the structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. Simulations identified smaller peptide analogues with the same conserved reverse turn demonstrated in the larger peptides. These analogues were synthesized and shown to inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition.

Computational approaches for the design of peptides with anti-breast cancer properties

Background: Breast cancer is the most common cancer among women. Tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment, yet many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Therefore, scientists are searching for breast cancer drugs that have different molecular targets. Methodology: Recently, a computational approach was used to successfully design peptides that are new lead compounds against breast cancer. We used replica exchange molecular dynamics to predict the structure and dynamics of active peptides, leading to the discovery of smaller bioactive peptides. Conclusions: These analogs inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition. We outline the computational methods that were tried and used along with the experimental information that led to the successful completion of this research.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Cell-specific expression of human HGF by alveolar type II cells induces remodeling of septal wall tissue in the lung: a morphometric study

Hepatocyte growth factor (HGF) is involved in development and regeneration of the lungs. Human HGF, which was expressed specifically by alveolar epithelial type II cells after gene transfer, attenuated the bleomycin-induced pulmonary fibrosis in an animal model. As there are also regions that appear morphologically unaffected in fibrosis, the effects of this gene transfer to normal lungs is of interest. In vitro studies showed that HGF inhibits the formation of the basal lamina by cultured alveolar epithelial cells. Thus we hypothesized that, in the healthy lung, cell-specific expression of HGF induces a remodeling within septal walls. Electroporation of a plasmid of human HGF gene controlled by the surfactant protein C promoter was applied for targeted gene transfer. Using design-based stereology at light and electron microscopic level, structural alterations were analyzed and compared with a control group. HGF gene transfer increased the volume of distal air spaces, as well as the surface area of the alveolar epithelium. The volume of septal walls, as well as the number of alveoli, was unchanged. Volumes per lung of collagen and elastic fibers were unaltered, but a marked reduction of the volume of residual extracellular matrix (all components other than collagen and elastic fibers) and interstitial cells was found. A correlation between the volumes of residual extracellular matrix and distal air spaces, as well as total surface area of alveolar epithelium, could be established. Cell-specific expression of HGF leads to a remodeling of the connective tissue within the septal walls in the healthy lung, which is associated with more pronounced stretching of distal air spaces at a given hydrostatic pressure during instillation fixation.