Positive selection of hearing loss candidate genes,based on multiple microarray platforms experiments and data mining

2006/2007Secondo le stime del World Health Organization, le perdite uditive colpiscono circa 278 milioni di persone in tutto il mondo. Approssimativamente 1 bambino ogni 100, nasce con problemi d’udito. Nonostante l’identificazione negli ultimi 10 anni di più di 100 loci genetici associati a fenotipi di perdita uditiva, non tutti i corrispettivi geni causativi sono stati identificati. Normalmente utilizzando un approccio sperimentale di linkage tradizionale non è sempre possibile identificare un intervallo genomico sufficientemente corto da essere analizzato per la ricerca di mutazioni. Il lavoro presentato in questa tesi ha lo scopo di selezionare un set limitato di geni potenzialmente coinvolti nelle perdite uditive non sindromiche, utilizzando la combinazione di un approccio biologico e bioinformatico. Il punto di partenza dell’analisi è stato il gene GJB2. Il gene GJB2 codifica la Connessina 26, proteina coinvolta nella formazione delle gap junction tra le cellule, ma anche implicata in più del 50% dei casi di perdite uditive non sindromiche. Per questa ragione è stato suggerito un ruolo chiave nella biologia dell’orecchio, che va oltre la sua funzione di proteina canale. In questa tesi è stato esaminato il profilo d’espressione genica di cellule HeLa transfettate con la forma naturale e con delle forme mutate della Connessina26. Le analisi dei dati hanno identificato numerosi geni differenzialmente espressi e si è quindi deciso di passare ad un approccio informatico per ridurne il numero. Questa analisi ha permesso di identificare 19 geni in 11 loci privi di geni causativi selezionandoli in base alla loro espressione rispetto librerie di cDNA prodotte da orecchio. Sono stati quindi identificati i geni omologhi in topo per 5 dei 19 geni, con lo scopo di verificare la loro rilevanza con la perdita uditiva. Per tutti questi 5 geni è stata confermata l’espressione nell’organo di corti in topo e con Real-time RT-PCR nelle linee cellulari transfettate impiegate negli esperimenti di microarray. Il progetto proseguirà ora con lo screening di mutazioni nei geni candidati in famiglie di pazienti selezionate.According to WHO estimates hearing impairment affects 278 million people worldwide. Approximately 1/1000 children are born with a significant hearing impairment. To date approximately 100 genetic loci involved in deafness have been described. Despite the fact that such a large number of genetic locations associated with deafness phenotypes are known, not all the genes involved have been identified yet. Using a traditional linkage approach, however, it is not always possible to map a locus to intervals short enough to be amenable for costly mutation analysis. So far no more than 40 deafness genes have been identified and these encode very heterogeneous proteins. The work presented in this thesis aims to identify a limited set of candidate genes with high potential to be involved in Non-Syndromic Hearing Loss using a combination of biological and bioinformatics approaches. The starting point of the analysis was the GJB2 gene. The GJB2 gene encodes for the gap junction protein Connexin26 and is responsible for more than half of the non-syndromic hearing loss cases. For this reason it has been proposed that this protein might play a wider role in the biology of the ear, beyond its mere channel function. I therefore performed whole genome expression profiles of HeLa cells transfected with the wild type form of the GJB2 gene and compared them to that of cells transfected with mutant forms of this gene to shed light on its function. Initially this experiment yielded a bewildering number of differentially expressed genes (4,984). Thus I devised an in silico strategy to narrow down this number, focusing on genes which were positionally linked to specific non-syndromic hereditary hearing loss conditions, as well as found within human ear cDNA libraries, thus potentially causative of the disease. This further analysis yielded 19 genes within 11 loci. In order to assess their relevance to hearing loss, the mouse homologs of these genes were identified for 5 of them and indeed they were all found to be expressed in the mouse organ of corti. These five genes were also validated by Real-time RT-PCR in the human cell line used for the microarray experiments.197

Emerging Approaches to DNA Data Storage: Challenges and Prospects

With the total amount of worldwide data skyrocketing, the global data storage demand is predicted to grow to 1.75 × 1014GB by 2025. Traditional storage methods have difficulties keeping pace given that current storage media have a maximum density of 103GB/mm3. As such, data production will far exceed the capacity of currently available storage methods. The costs of maintaining and transferring data, as well as the limited lifespans and significant data losses associated with current technologies also demand advanced solutions for information storage. Nature offers a powerful alternative through the storage of information that defines living organisms in unique orders of four bases (A, T, C, G) located in molecules called deoxyribonucleic acid (DNA). DNA molecules as information carriers have many advantages over traditional storage media. Their high storage density, potentially low maintenance cost, ease of synthesis, and chemical modification make them an ideal alternative for information storage. To this end, rapid progress has been made over the past decade by exploiting user-defined DNA materials to encode information. In this review, we discuss the most recent advances of DNA-based data storage with a major focus on the challenges that remain in this promising field, including the current intrinsic low speed in data writing and reading and the high cost per byte stored. Alternatively, data storage relying on DNA nanostructures (as opposed to DNA sequence) as well as on other combinations of nanomaterials and biomolecules are proposed with promising technological and economic advantages. In summarizing the advances that have been made and underlining the challenges that remain, we provide a roadmap for the ongoing research in this rapidly growing field, which will enable the development of technological solutions to the global demand for superior storage methodologies

Quantitative Optical Imaging and Sensing by Joint Design of Point Spread Functions and Estimation Algorithms

The joint application of tailored optical Point Spread Functions (PSF) and estimation methods is an important tool for designing quantitative imaging and sensing solutions. By enhancing the information transfer encoded by the optical waves into an image, matched post-processing algorithms are able to complete tasks with improved performance relative to conventional designs. In this thesis, new engineered PSF solutions with image processing algorithms are introduced and demonstrated for quantitative imaging using information-efficient signal processing tools and/or optical-efficient experimental implementations. The use of a 3D engineered PSF, the Double-Helix (DH-PSF), is applied as one solution for three-dimensional, super-resolution fluorescence microscopy. The DH-PSF is a tailored PSF which was engineered to have enhanced information transfer for the task of localizing point sources in three dimensions. Both an information- and optical-efficient implementation of the DH-PSF microscope are demonstrated here for the first time. This microscope is applied to image single-molecules and micro-tubules located within a biological sample. A joint imaging/axial-ranging modality is demonstrated for application to quantifying sources of extended transverse and axial extent. The proposed implementation has improved optical-efficiency relative to prior designs due to the use of serialized cycling through select engineered PSFs. This system is demonstrated for passive-ranging, extended Depth-of-Field imaging and digital refocusing of random objects under broadband illumination. Although the serialized engineered PSF solution is an improvement over prior designs for the joint imaging/passive-ranging modality, it requires the use of multiple PSFs - a potentially significant constraint. Therefore an alternative design is proposed, the Single-Helix PSF, where only one engineered PSF is necessary and the chromatic behavior of objects under broadband illumination provides the necessary information transfer. The matched estimation algorithms are introduced along with an optically-efficient experimental system to image and passively estimate the distance to a test object. An engineered PSF solution is proposed for improving the sensitivity of optical wave-front sensing using a Shack-Hartmann Wave-front Sensor (SHWFS). The performance limits of the classical SHWFS design are evaluated and the engineered PSF system design is demonstrated to enhance performance. This system is fabricated and the mechanism for additional information transfer is identified

Artificial Metabolons: Design of Self-Assembled Bio-Complexes

Protein-protein interactions are vital to every living organism, and it is thought that most, if not all proteins interact in some way with other proteins for purposes including for cellular metabolism, signal transduction and DNA replication. These protein complexes can range in stability from permanent to transient, and they are driven by interactions at the protein-protein interfaces including hydrophobicity, hydrogen bonding, electrostatic interactions, van der Waals interactions and covalent disulfide bonding. Many complexes, such as transient complexes of sequential enzymes called metabolons, are poorly understood. In recent years, there have been many efforts to mimic nature and engineer new protein complexes with defined spatial arrangements with increased stability and more efficient transport of the enzymatic reaction intermediates. There is much to be understood in these complexes, including the role of substrate channeling. In this dissertation, we study a natural metabolon and engineer new protein complexes. In our first study, we construct designed protein aggregates of the single enzyme small laccase (SLAC). SLAC is a multi-copper oxidase that can be easily genetically modified and is used as an oxygen-reduction catalyst on enzymatic bio-cathodes. A new dimeric interface is introduced, which, in combination with the threefold symmetry of the naturally trimeric SLAC, drives the self assembly of SLAC with two disulfide bonds in an oxidative environment. These enzymatically active aggregates form upon the addition of cupric ions to the purified protein, and electron microscopy shows the symmetry of the aggregates to be consistent with the design. We demonstrate improvements over the non-complexed enzyme including an increased resistance to permanent thermal denaturation and a lower reaction overpotential and increased current density when employed on an oxygen-reduction bio-cathode with single-walled carbon nanotubes incorporated into the enzyme aggregates. In our next line of work, we study a natural tricarboxylic acid (TCA) cycle metabolon, focusing on two enzymes: mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS). These enzymes have long been proposed to form a spatially organized complex that facilitates substrate channeling, a process in which a reaction intermediate is transferred directly from one enzyme active site to the next without first diffusing into the bulk through mechanisms such as electrostatic interactions. Structural evidence has been difficult to obtain due to the transient nature of many of these complexes. In Chapter 3, we examine the in vitro complex structure of the recombinant enzymes and find that it is similar to the recently proposed in vivo complex structure. Furthermore, there is evidence of a positively charged electrostatic channel connecting the enzyme active sites along which the oppositely charged reaction intermediate can travel by bounded diffusion. Site-directed mutagenesis along the channel on CS results in inhibited substrate channeling. Finally, we develop a platform to study substrate channeling in engineered multi-enzyme complexes. Efforts to engineer multi-enzyme complexes in recent years have made use of protein and nucleic acid-based scaffolds. Many of these complexes exhibit increased coupled enzymatic activities, but there is a question of what effects are due to substrate channeling and how to apply these strategies to any enzyme pair. In this work, we attach CS and the non-channeling cytosolic malate dehydrogenase to DNA and engineered protein cage scaffolds. These assemblies retain their enzymatic activities, and these methods can be used to study substrate channeling in many enzyme pairs including the naturally channeling and inhibited channeling TCA cycle enzymes

All-optical interrogation of neural circuits during behaviour

This thesis explores the fundamental question of how patterns of neural activity encode information and guide behaviour. To address this, one needs three things: a way to record neural activity so that one can correlate neuronal responses with environmental variables; a flexible and specific way to influence neural activity so that one can modulate the variables that may underlie how information is encoded; a robust behavioural paradigm that allows one to assess how modulation of both environmental and neural variables modify behaviour. Techniques combining all three would be transformative for investigating which features of neural activity, and which neurons, most influence behavioural output. Previous electrical and optogenetic microstimulation studies have told us much about the impact of spatially or genetically defined groups of neurons, however they lack the flexibility to probe the contribution of specific, functionally defined subsets. In this thesis I leverage a combination of existing technologies to approach this goal. I combine two-photon calcium imaging with two-photon optogenetics and digital holography to generate an “all-optical” method for simultaneous reading and writing of neural activity in vivo with high spatio-temporal resolution. Calcium imaging allows for cellular resolution recordings from neural populations. Two-photon optogenetics allows for targeted activation of individual cells. Digital holography, using spatial light modulators (SLMs), allows for simultaneous photostimulation of tens to hundreds of neurons in arbitrary spatial locations. Taken together, I demonstrate that this method allows one to map the functional signature of neurons in superficial mouse barrel cortex and to target photostimulation to functionally-defined subsets of cells. I develop a suite of software that allows for quick, intuitive execution of such experiments and I combine this with a behavioural paradigm testing the effect of targeted perturbations on behaviour. In doing so, I demonstrate that animals are able to reliably detect the targeted activation of tens of neurons, with some sensitive to as few as five cortical cells. I demonstrate that such learning can be specific to targeted cells, and that the lower bound of perception shifts with training. The temporal structure of such perturbations had little impact on behaviour, however different groups of neurons drive behaviour to different extents. In order to probe which characteristics underly such variation, I tested whether the sensory response strength or correlation structure of targeted ensembles influenced their behavioural salience. Whilst these final experiments were inconclusive, they demonstrate their feasibility and provide us with some key actionable improvements that could further strengthen the all-optical approach. This thesis therefore represents a significant step forward towards the goal of combining high resolution readout and perturbation of neural activity with behaviour in order to investigate which features of the neural code are behaviourally relevant

Particle trapping with functionalized hybrid optical fibers

Understanding processes on sub-micron scales that are obscured from the observer’s naked eye represents a long cherished desire of mankind. Unfortunately, single particle studies are time demanding and suffer from Brownian motion, which thus limits their practicability and range of applications. Optical and electrical trapping, however, both awarded with a Nobel prize, represent two sophisticated and widely applied solutions allowing for controlled access to individual particles via almost the entire room angle. Particle trapping via optical fibers in principle provides a flexible and low-cost photonic platform enabling remotely operable applications within difficult to reach environments, including in situ and in vivo scenarios. The microtechnologically functionalized tip of a hybrid optical fiber (HOF), in particular, which in contrast to conventional optical fibers incorporates additional materials, offers a unique platform for implementing electromagnetic, i.e., optical and electrical, fields that are essentially required for the trapping of particles and unavailable by standard fibers alone. Within the scope of this work, three unique implementations of HOF tip-based particle traps, which in detail rely on integrating a liquid channel, a pure silica section and metallic wires for functionalizing the fibers, are demonstrated, discussed, and compared to state-of-the-art concepts. First, the principles of optical phenomena, the motion of microscopic objects and influences of confinements including different particle trapping mechanisms, as well as required methods for analyzing and characterizing fiber-based particle traps are introduced. Subsequently, three unique concepts, which in detail consist of a dual fiber focus trap, a single meta-fiber trap and a fiber point Paul trap, and effectively represent two optical and one electrical trap, are discussed and compared with respect to current implementations. ..