Ultrafine Dielectrophoresis-based Technique for Virus and Biofluid Manipulation

abstract: Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported. In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition. DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step.Dissertation/ThesisDoctoral Dissertation Chemistry 201

Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin's lymphoma

Classical Hodgkin's lymphoma (cHL), although originating from B-cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted now a high-throughput pharmacological screening based on more than 28,000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote re-expression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of two of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the anti-leukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked re-expression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell Non-Hodgkin's lymphoma-tailored small compound inhibitors Ibrutinib and Idelalisib. In essence, we report here a conceptually novel, re-differentiation-based treatment strategy for cHL

Detection of Pathogens in Water Using Micro and Nano-Technology

Detection of Pathogens in Water Using Micro and Nano-Technology aims to promote the uptake of innovative micro and nano-technological approaches towards the development of an integrated, cost-effective nano-biological sensor useful for security and environmental assays.  The book describes the concerted efforts of a large European research project and the achievements of additional leading research groups. The reported knowledge and expertise should support in the innovation and integration of often separated unitary processes. Sampling, cell lysis and DNA/RNA extraction, DNA hybridisation detection micro- and nanosensors, microfluidics, together also with computational modelling and risk assessment can be integrated in the framework of the current and evolving European regulations and needs. The development and uptake of molecular methods is revolutionizing the field of waterborne pathogens detection, commonly performed with time-consuming cultural methods. The molecular detection methods are enabling the development of integrated instruments based on biosensor that will ultimately automate the full pathway of the microbiological analysis of water

Application of Medicinal Chemistry Methods on Different Classes of Drugs

The present doctoral thesis is the result of the work carried out during the three years of my PhD scholarship at the Rome Center for Molecular Design laboratory (RCMD, Department of Chemistry and Drug Technologies, Sapienza University of Rome), under the supervision of Prof. Rino Ragno. The research activity was focused mainly on the design, optimization and application of computational strategies to derive quantitative structure-activity relationships (QSAR, 3-D QSAR, and COMBINE) on different molecular classes of current interest, such as: opioid receptor antagonists (OPAs), Hepatitis C Virus NS5B-Polymerase Inhibitors (NS5B-NNIs), Hystone Deacetylase Inhibitors (HDACIs), Anti- tubercular agents, vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors, HSP90 inhibitors, HIV-1 reverse transcriptase inhibitors (NNRTIs), Bovine Serum Amine Oxidase (BSAO) substrates, etc... Moreover two research periods abroad were performed: the first framed in a LLP Erasmus program collaboration, was conducted for six months at the Laboratoire d'Ingénierie et Moléculaire Pharmacologique Biochimie (LIMBP) of the Université de Lorraine Metz (France), directed by Prof. Gilbert Kirsch, and characterized by the application of organic synthesis to obtain new thienopyrimidinone derivatives as potential inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2); the second took place, for three months, at the Department of Biochemistry and Molecular Biophysics in Washington University School of Medicine in Saint Louis (MO, USA), under the supervision of Prof. Garland R. Marshall, investigating the activity profile of new Histone Deacetylases (HDACs) inhibitors by the application of the Mobility Shift Assay Technology. Main purpose of this doctoral thesis is to highlight the activities carried out in the different research projects, the applied methodologies and the obtained results. The text starts describing those studies whose results were published in scientific journals (chapters I-VI): the author decided to omit some procedural details, completely reported in the published papers, that would make the text too long, tedious and redundant; therefore readers who want to delve these aspects can also refer to Chapter XII in which is possible to read the original papers; on the contrary for studies that have not yet been published, as those characterizing the Chapters VII and VIII, discussion is adequately detailed. Chapters IX and X report the scientific activities carried out in France and in USA respectively; Chapter XI summarizes all the scientific activities accomplished during the entire PhD course, whereas Chapter XII, as mentioned, contains the published articles

Detection of Pathogens in Water Using Micro and Nano-Technology

Detection of Pathogens in Water Using Micro and Nano-Technology aims to promote the uptake of innovative micro and nano-technological approaches towards the development of an integrated, cost-effective nano-biological sensor useful for security and environmental assays.  The book describes the concerted efforts of a large European research project and the achievements of additional leading research groups. The reported knowledge and expertise should support in the innovation and integration of often separated unitary processes. Sampling, cell lysis and DNA/RNA extraction, DNA hybridisation detection micro- and nanosensors, microfluidics, together also with computational modelling and risk assessment can be integrated in the framework of the current and evolving European regulations and needs. The development and uptake of molecular methods is revolutionizing the field of waterborne pathogens detection, commonly performed with time-consuming cultural methods. The molecular detection methods are enabling the development of integrated instruments based on biosensor that will ultimately automate the full pathway of the microbiological analysis of water

Application of Medicinal Chemistry Methods on Different Classes of Drugs

The present doctoral thesis is the result of the work carried out during the three years of my PhD scholarship at the Rome Center for Molecular Design laboratory (RCMD, Department of Chemistry and Drug Technologies, Sapienza University of Rome), under the supervision of Prof. Rino Ragno. The research activity was focused mainly on the design, optimization and application of computational strategies to derive quantitative structure-activity relationships (QSAR, 3-D QSAR, and COMBINE) on different molecular classes of current interest, such as: opioid receptor antagonists (OPAs), Hepatitis C Virus NS5B-Polymerase Inhibitors (NS5B-NNIs), Hystone Deacetylase Inhibitors (HDACIs), Anti- tubercular agents, vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors, HSP90 inhibitors, HIV-1 reverse transcriptase inhibitors (NNRTIs), Bovine Serum Amine Oxidase (BSAO) substrates, etc... Moreover two research periods abroad were performed: the first framed in a LLP Erasmus program collaboration, was conducted for six months at the Laboratoire d'Ingénierie et Moléculaire Pharmacologique Biochimie (LIMBP) of the Université de Lorraine Metz (France), directed by Prof. Gilbert Kirsch, and characterized by the application of organic synthesis to obtain new thienopyrimidinone derivatives as potential inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2); the second took place, for three months, at the Department of Biochemistry and Molecular Biophysics in Washington University School of Medicine in Saint Louis (MO, USA), under the supervision of Prof. Garland R. Marshall, investigating the activity profile of new Histone Deacetylases (HDACs) inhibitors by the application of the Mobility Shift Assay Technology. Main purpose of this doctoral thesis is to highlight the activities carried out in the different research projects, the applied methodologies and the obtained results. The text starts describing those studies whose results were published in scientific journals (chapters I-VI): the author decided to omit some procedural details, completely reported in the published papers, that would make the text too long, tedious and redundant; therefore readers who want to delve these aspects can also refer to Chapter XII in which is possible to read the original papers; on the contrary for studies that have not yet been published, as those characterizing the Chapters VII and VIII, discussion is adequately detailed. Chapters IX and X report the scientific activities carried out in France and in USA respectively; Chapter XI summarizes all the scientific activities accomplished during the entire PhD course, whereas Chapter XII, as mentioned, contains the published articles

Obstacles and opportunities in the functionalanalysis of extracellular vesicle RNA – an ISEVposition paper

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data

Evaluation of fast spectroscopic analysis techniques for freeze-dried live, attenuated virus vaccines

Freeze-drying is today the preferred method for stabilizing live, attenuated virus vaccines. Despite its wide use in the pharmaceutical industry, freeze-drying formulation and process development of live, attenuated virus vaccines are still performed by trial-and-error experimental work resulting in sub-optimal processes. The stabilization and destabilization mechanisms of the live, attenuated viruses during freeze-drying, are still not fully understood. The molecular complexity of live, attenuated viruses, the multiple destabilization pathways and the lack of analytical techniques allowing the measurement of physicochemical changes in the antigen’s structure during and after lyophilization partly explain this lack of knowledge. Therefore, the possibility to apply spectroscopic process analytical tools, in particular NIR and FTIR spectroscopy, to (i) evaluate live, attenuated viruses and their interactions with the stabilizer(s) in freeze-dried samples, (ii) understand virus destabilization mechanisms during storage and (iii) develop practical applications using chemometric models to predict critical end product quality parameters, has been evaluated in this thesis

Dietary Selenium Supplementation: Effects on Neurodegeneration Following Traumatic Brain and Spinal Cord Injury

Traumatic brain and spinal cord injury continue to be substantial clinical problems with few available treatment strategies. Individuals who are at a greater risk for sustaining a central nervous system (CNS) injury, such as professional athletes and military personnel, may benefit from a prophylactic supplement that would intervene in the neurodegenerative pathways immediately following injury. The high demand for selenium within the central nervous system, as well as the synthesis of selenoproteins by neurons and astrocytes suggests a critical role of selenium within the brain and spinal cord. Studies were designed to test the efficacy of enriched dietary selenium status in providing neuroprotective benefits in rodent models of spinal cord and traumatic brain injury. Levels of selenium storage within the CNS are increased relative to the amount of selenium present in the diet, indicating that selenium compounds effectively cross the blood brain barrier. In a model of moderate severity spinal cord contusion injury, dietary selenium supplementation reduced the number of days until recovery of independent bladder function following injury. These benefits did not translate to improvements in locomotor function during open field testing or reduction in overall lesion volume in the injured animal groups. Examination of gene expression changes 24 hours after spinal cord injury revealed that dietary selenium enrichment increased expression of genes involved in DNA repair, mitochondrial respiration, and transcriptional regulation. By expanding the scope of these studies to include models of traumatic brain injury, these data show the importance of selenium in the cortex as well. In particular, when compared to diets deficient in selenium, higher levels of dietary selenium improve spatial memory performance and mitochondrial respiration. The results of this dietary study show modest improvements following both traumatic brain and spinal cord injury and suggest that while selenium enrichment may not have a profound effect on the secondary injury cascade immediately following injury, the presence of adequate dietary selenium is critical for mitochondrial respiration. Together the results of these studies suggest that dietary supplementation may play a subtle role in injury mechanisms within the CNS and warrant further investigation