Universiẗat, Diss., 2004--Düsseldorf.

Στην εποχή της συστημικής βιολογίας, οι υψηλής απόδοσης (-ομικές) τεχνικές βιομοριακής ανάλυσης επέτρεψαν την ολιστική ανάλυση των διαφόρων μοριακών επιπέδων κυτταρικής λειτουργίας μέσω της ταυτόχρονης μέτρησης εκατοντάδων έως χιλιάδων αντιπροσωπευτικών μοριακών ποσοτήτων. Η μεταβολομική αναφέρεται στην ποσοτικοποίηση του (σχετικού) προτύπου συγκέντρωσης των ελεύθερων μικρών μεταβολιτών. Λαμβάνοντας υπ’ όψιν, το ρόλο των μεταβολιτών ως, αντιδρώντα ή/και προϊόντα των μεταβολικών αντιδράσεων, το πρότυπο συγκέντρωσης τους επηρεάζει και επηρεάζεται από την κατανομή των μεταβολικών ροών, αποτελώντας επομένως ένα αποτύπωμα της μεταβολικής κατάστασης ενός βιολογικού συστήματος. Μεταξύ των πλεονεκτημάτων της μεταβολομικής ανάλυσης είναι ότι μπορεί να χρησιμοποιηθεί σε μεταβαλλόμενη κατάσταση φυσιολογίας, ενώ δεν απαιτεί ολοκληρωμένη γνώση του μεταβολικού δικτύου ενός οργανισμού. Αυτά τα χαρακτηριστικά είναι ιδιαίτερα πλεονεκτικά για τη μελέτη της μεταβολικής ενεργότητας του εγκεφάλου, λαμβάνοντας υπ’όψιν την ανατομική, μορφολογική και φαινοτυπική πολυπλοκότητα αυτού του οργάνου και την έως τώρα κατανόηση των μεταβολικών του μηχανισμών. Πιο συγκεκριμένα για την επίδραση του ενήλικου υποθυρεοειδισμού στον μεταβολισμό του εγκεφάλου, η μέχρι σήμερα γνώση παραμένει αποσπασματική, ενώ προέρχεται από διαφορετικά πειράματα και διάφορες εγκεφαλικές περιοχές. Μια ολιστική θεώρηση του μεταβολισμού σε συνθήκες υποθυρεοειδισμού σε συγκεκριμένες εγκεφαλικές περιοχές αναμένεται να αυξήσει σημαντικά τη γνώση μας για την ασθένεια αυτή. Σε μια πρόσφατη μελέτη της ερευνητικής μας ομάδας, που ήταν η πρώτη μεταβολική ανάλυση εγκεφαλικού ιστού σε ζωικό μοντέλο μακρόχρονου υποθυρεοειδισμού, η πολυπαραμετρική στατιστική ανάλυση των μεταβολικών προτύπων της παρεγκεφαλίδας μυός έδειξε διαφορές στη μεταβολική φυσιολογία του ιστού στα ευ- σε σχέση με τα υποθυρεοειδικά ζώα, παρέχοντας ισχυρές ενδείξεις ότι ο μεταβολισμός της παρεγκεφαλίδας θηλαστικών επηρρεάζεται από τον μακρόχρονο υποθυρεοειδισμό. Στην παρούσα εργασία, συγκρίθηκε η επίδραση του μακρόχρονου υποθυρεοειδισμού στη μεταβολική φυσιολογία της παρεγκεφαλίδας μεταξύ αρσενικών και θηλυκών Balb/cJ μυών, αφού επεκτάθηκε για επιπλέον μεταβολίτες η αυτοματοποιημένη μέθοδος προσδιορισμού κορυφών στο χρωματογράφημα. Ο μακρόχρονος υποθυρεοειδισμός επήχθη με χορήγηση 1% υπερχλωρικού καλλίου για 64 μέρες στο πόσιμο νερό των ζώων. Αυτή είναι και η πρώτη μελέτη της επίδρασης του ενήλικου υποθυρεοειδισμού στην μεταβολομική φυσιολογία του 4 εγκεφάλου των θηλυκών μυών. Τα μεταβολικά πρότυπα αναλύθηκαν με το λογισμικό ανοικτού κώδικα ΤΜ4/ MEV(www.tm4.org/MEV) για την πολυπαραμετρική στατιστική ανάλυση των -ομικών δεδομένων. Τα αποτελέσματα συζητήθηκαν στο πλαίσιο ενός κατάλληλα ανακατασκευασμένου μεταβολικού δικτύου για την παρεγκεφαλίδα μυός με βάση τις μεταβολικές βάσεις δεδομένων KEGG και EXPASY και δεδομένα από τη βιβλιογραφία. Η ανάλυση των προτύπων έδειξε ότι η επίδραση της δίμηνης χορήγησης υπερχλωρικού καλλίου στη μεταβολική φυσιολογία της παρεγκεφαλίδας ήταν πιο οξεία στα αρσενικά απ’ότι στα θηλυκά ζώα. Αυτή η παρατήρηση υποστηριζόταν και απο την σημαντικά μικρότερη μείωση του μέσου βάρους των υποθυρεοειδικών σε σχέση με αυτό των ευθυρεοειδικών ζώων στο τέλος της δίμηνης χορήγησης στα θηλυκά σε σχέση με τα αρσενικά. Τέος, σύγκριση των μεταβολικών προτύπων της παρεγκεφαλίδας των ευθυρεοειδικών αρσενικών και θηλυκών μυών έδειξε τους μισούς από τους μεταβολίτες στην παρεγκεφαλίδα των αρσενικών να έχουν σημαντικά μεγαλύτερη συγκέντρωση απ’ ότι στον ιστό των θηλυκών. Αυτή η παρατήρηση καταδεικνύει την ανάγκη της παρεγκεφαλίδας των θηλυκών σε μικρότερες συγκεντρώσεις ελεύθερων μικρών μεταβολιτών για την εύρυθμη λειτουργία της ως ένα πιθανό παράγοντα «προστασίας» του μεταβολισμού της από την επίδραση του μακρόχρονου ενήλικου υποθυρεοειδισμού. Καθώς η παρεγκεφαλίδα των αρσενικών χρειάζεται μεγαλύτερες ποσότητες ελεύθερων μικρών μεταβολιτών, η αναμενώμενη μείωση της συγκέντρωσης των μεταβολιτών που λαμβάνει ο εγκέφαλος μέσω του αιματοεγκεφαλικού φραγμού λόγω του υποθυρεοειδισμού θα την επηρεάσει πιο γρήγορα και πιο δραστικά από αυτή των θηλυκών. Αυτές οι σημαντικές παρατηρήσεις χρειάζονται περαιτέρω διερεύνηση μέσω κατάλληλα σχεδιασμένων αναλύσεων μεταβολομικής και φυσιολογίας και άλλων εγκεφαλικών περιοχών, συνδυάζοντας επίσης μετρήσεις των επιπέδων των θυρεοειδικών ορμονών με μεταβολομική ανάλυση του εγκεφαλικού ιστού με Υγρή Χρωματογραφία- Φασματομετρίας Μάζας, καθώς και μετρήσεις ομικών αναλύσεων από άλλα επίπεδα κυτταρικής λειτουργίας, κυρίως της πρωτεωμικής. / In the systems biology era, the high-throughput “omic” technologies have enabled the holistic analysis of the various molecular levels of cellular function through the simultaneous measurement of hundreds to thousands of relevant molecular quantities. Metabolomics refers to the quantification of the (relative) concentration profile of the free small metabolites. Taking into consideration the role of the metabolites as reactants and products of the metabolic reactions, their concentration profile affects and is affected by the metabolic pathway flux distribution. Thus, the metabolic profile provides a fingerprint of the metabolic state of a biological system. Among the advantages of the metabolomic analysis is that it can be easily used to monitor transient metabolic conditions without requiring extensive knowledge of the structure and regulation of the investigated metabolic networks. This characteristic is especially advantageous for the analysis of brain metabolism, considering the anatomical, morphological and phenotypic complexity of this organ and our current shortages in understanding its metabolic mechanisms. For the effect of adult onset hypothyroidism (AOH) on brain metabolism in particular, the current knowledge remains fragmented, concerning different experimental setups and recovered from various brain regions. A holistic view of metabolism under AOH in particular brain regions is expected to significantly enhance the current knowledge about the disease. In a recent study of our group, which was the first metabolomic analysis of brain tissue in a prolonged AOH mouse model, multivariate statistical analysis of the metabolic profiles of the mouse cerebella indicated differences in the metabolic physiology of the tissue in the eu- compared to the hypo- thyroid animals, providing strong evidence that the mammalian cerebellum is metabolically responsive to prolonged AOH. In the present work, we compared the effect of prolonged AOH on the cerebellar metabolic physiology between male and female Balb/cJ mice, after enhancing the metabolite peak identification method to include additional metabolites. The prolonged AOH was induced by a 64-day treatment with 1% potassium perchlorate in the drinking water of the animals. This is the first reported analysis of the effect of AOH on the brain metabolic physiology of female mice. The raw metabolic profiles were normalized and appropriately filtered. The normalized metabolic profiles were analyzed using the open-source TM4/MeV software (www.tm4.org/MeV) for the multivariate statistical analysis of “omic” data. The acquired results were interpreted in the context of an appropriately reconstructed metabolic network for the mouse cerebellum based on the metabolic databases, KEGG and Expasy, and a plethora of information mined from the literature. The analysis of the metabolic profiles 6 indicated that the effect of the 2-month potassium perchlorate treatment on the metabolic physiology of the cerebellum is more acute in the male with respect to the female mice. The time profile of the body weight of the female compared to the male mice indicated a significantly smaller decrease in the mean weight of the hypothyroid compared to the euthyroid mice in the female compared to the male population at the end of the KClO4 treatment, an observation that further supports the metabolic profiling results. Finally, comparison between the metabolic profiles of the euthyroid male and female cerebellum indicated a significantly higher concentration in half of the measured free metabolites in the male compared to the female animals. This indicates the “leanness” of the metabolic profile of the female cerebellum as a potential “protective” parameter to the effect of AOH on its metabolic physiology, in the sense that the expected due to AOH decrease in the concentrations of the metabolites that are transferred to the brain through the blood brain barrier may affect more the male cerebellum that requires higher levels of free metabolites for its regular activity. These significant observations are in need of further investigation through appropriately designed physiological and metabolomic studies, integrating also thyroid hormone measurements from Liquid Chromatography-MS metabolomic analysis of brain tissue as well as omic measurements from other molecular levels of cellular function, mainly from proteomics.

Internet applications make use of the services provided by a transport protocol, such as TCP (a reliable, in-order stream protocol). We use this term Transport Service to mean the end-to- end service provided to application by the transport layer.   That service can only be provided correctly if information about the intended usage is supplied from the application. The application may determine this information at the design time, compile time, or run time, and it may include guidance on whether a feature is required, a preference by the application, or something in between. Multipath TCP (MPTCP) adds the capability of using multiple paths to a regular TCP session. Even though it is designed to be totally backward compatible to applications. The data transport differs compared to regular TCP, and there are several additional degrees of freedom that the particular application may want to exploit.   Multipath TCP is particularly useful in the context of wireless networks using both Wi-Fi and a mobile network is a typical use case. In addition to the gains in throughput from inverse multiplexing, links may be added or dropped as the user moves in or out of coverage without disrupting the end-to-end TCP connection. The problem of link handover is thus solved by abstraction in the transport layer, without any special mechanisms at the network or link level.   Handover functionality can then be implemented at the endpoints without requiring special functionality in the sub-networks according to the Internet's end-to-end principle. Multipath TCP can balance a single TCP connection across multiple interfaces and reach very high throughput.

By applying the principles of Darwinian natural selection in the laboratory, directed evolution has become a powerful practical approach to study enzymes and optimize them to catalyze industrially relevant transformations. In this thesis, I applied this strategy to the engineering of amino acid dehydrogenases for biocatalysis of chiral amines, focusing on two crucial features for successful directed evolution experiments. A first key aspect is the development of technologies allowing the screening of large libraries of enzyme variants to explore sequence space efficiently. Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsions has recently led to the development of ultrahigh-throughput screening platforms that allow sorting of more than 106 variants per hour. So far, these microfluidic droplet sorters have relied exclusively on fluorescent readouts. To further extend the range of applications toward enzymes for which no fluorescent assays are available, I successfully developed a sorting module based on absorbance detection. Using this new module, microdroplets could be sorted based on an absorbance readout at rates of up to 1 million droplets per hour. To demonstrate the utility of this module for protein engineering, three rounds of directed evolution were performed to improve a poorly stable NAD+ dependent phenylalanine dehydrogenase (PheDH) toward its native substrate. Five hits showed increased activity (improved up to 10-fold in lysate; kcat increased >3.5-fold), soluble protein expression levels (>2.5-fold) and thermostability (Tm, 8 °C higher). To increase the sensitivity of the device (3–4 orders of magnitude lower than fluorescence assays) for detection of enzymes with limited stability and low turnovers, an extra step of growth in droplets from single cell encapsulation, followed by piconinection of substrates and lysis agents was implemented. As a result, a fivefold signal enhancement over background was achieved, for an amine dehydrogenase (AmDH) reaction shown to be undetectable in a droplet single cell assay. Second, I investigated how mutational robustness may correlate with protein stability and lead to successful hits after mutagenesis and screening. To examine this issue, I initially investigated various approaches (including ancestral resurrection and computational design) to identify stabilized PheDH variants. One such variant (dubbed Pross 4) showed increased expression levels (>3.3-fold) and thermostability (Tm, 13 °C higher) compared to the wild-type PheDH. I further compared the mutational tolerance and the hit rate between PheDH and Pross 4 by generating variant libraries focused on key active site residues and screening them for improved AmDH activity. The Pross 4 background generated 6.4 times more active variants than the PheDH background, the best hits displaying increased activity (up to 2.5-fold in lysate; kcat/KM increased up to 8-fold) compared to previously engineered AmDHs with the PheDH scaffold. In conclusion, this work highlights how directed evolution experiments could be designed for increased success rates, by combining reliable high-throughput screens with careful choice of evolutionary robust starting points.

The human genome is highly heterogeneous in its GC composition. How codon usage affects translation rates has been extensively studied and exploited to increase protein expression. Although effects on virtually all other steps in gene expression have been reported as well, so far no systematic approach has been taken to quantitatively measure the contribution of each to overall protein levels in human cells. Here, I utilise a library of several hundred synonymous variants of the Green fluorescent protein (GFP) to characterise the influence of codon usage on gene expression in human cells. In an initial small-scale screen, I show that protein levels are largely correlated with codon-usage and particularly GC-content. Additionally, I demonstrate that these changes can already be seen on the RNA level, confirming more broadly previously published data from our lab (Kudla et al., 2006). In order to assess the consequences of randomised codon usage on a larger scale, I established and validated a high-throughput approach for the phenotypic profiling of reporter genes. Using a pool of cells stably expressing >200 GFP variants, I measured multiple parameters simultaneously, such as protein levels, translational state, RNA levels, stability and export. Data from these experiments confirm a strong relationship between GC-content, protein levels, as well as RNA export, reproducibly in two cell lines. Low expression of especially GC-poor variants could not be rescued by splicing, but increased nuclear-to-cytoplasmic RNA ratio, suggesting further mechanisms important for efficient gene expression. These effects are even more pronounced when the distribution of GC is spread evenly along the coding sequence. Interestingly, our data also suggests that high GC within the first 200nt is more predictive of efficient gene expression, contrasting studies performed on bacteria, in which strong secondary folding near the ribosomal binding site was shown to be non-permissive for translation (Kudla et al., 2009). By relating experimentally derived parameters to sequence features known to inhibit expression, I demonstrate that cryptic splicing is a major factor leading to decreased levels of particularly GC-poor GFP variants. An attempt to quantitatively assess the relative contribution of several sequence features (e.g. tAI, GC3, CpG) using multiple regression analysis lead to inconclusive results, leaving the requirement for the exploration of alternative approaches in order to dissect the role of individual parameters, as well as to identify novel determinants of gene expression.

abstract: Interference constitutes a major challenge for communication networks operating over a shared medium where availability is imperative. This dissertation studies the problem of designing and analyzing efficient medium access protocols which are robust against strong adversarial jamming. More specifically, four medium access (MAC) protocols (i.e., JADE, ANTIJAM, COMAC, and SINRMAC) which aim to achieve high throughput despite jamming activities under a variety of network and adversary models are presented. We also propose a self-stabilizing leader election protocol, SELECT, that can effectively elect a leader in the network with the existence of a strong adversary. Our protocols can not only deal with internal interference without the exact knowledge on the number of participants in the network, but they are also robust to unintentional or intentional external interference, e.g., due to co-existing networks or jammers. We model the external interference by a powerful adaptive and/or reactive adversary which can jam a (1 &#8722; &#949;)-portion of the time steps, where 0 < &#949; &#8804; 1 is an arbitrary constant. We allow the adversary to be adaptive and to have complete knowledge of the entire protocol history. Moreover, in case the adversary is also reactive, it uses carrier sensing to make informed decisions to disrupt communications. Among the proposed protocols, JADE, ANTIJAM and COMAC are able to achieve &#920;(1)-competitive throughput with the presence of the strong adversary; while SINRMAC is the first attempt to apply SINR model (i.e., Signal to Interference plus Noise Ratio), in robust medium access protocols design; the derived principles are also useful to build applications on top of the MAC layer, and we present SELECT, which is an exemplary study for leader election, which is one of the most fundamental tasks in distributed computing. / Dissertation/Thesis / Ph.D. Computer Science 2012

Internet applications make use of the services provided by a transport protocol, such as TCP (a reliable, in-order stream protocol). We use the term Transport Service to mean the endtoend service provided to application by the transport layer. That service can only be provided correctly if information about the intended usage is supplied from the application. The application may determine this information at the design time, compile time, or run time, and it may include guidance on whether a feature is required, a preference by the application, or something in between. Multipath TCP (MPTCP) adds the capability of using multiple paths to a regular TCP session. Even though it is designed to be totally backward compatible to applications. The data transport differs compared to regular TCP, and there are several additional degrees of freedom that the particular application may want to exploit. Multipath TCP is particularly useful in the context of wireless networks using both Wi-Fi and a mobile network is a typical use case. In addition to the gains in throughput from inverse multiplexing, links may be added or dropped as the user moves in or out of coverage without disrupting the end-to-end TCP connection. The problem of link handover is thus solved by abstraction in the transport layer, without any special mechanisms at the network or link level. Handover functionality can then be implemented at the endpoints without requiring special functionality in the sub-networks according to the Internet's end-to-end principle. Multipath TCP can balance a single TCP connection across multiple interfaces and reach very high throughput.

Apresentamos um novo Protocolo de Controle de Transporte fim-a-fim, implementado somente do lado do transmissor, chamado TCP HolyWood ou, abreviadamente, TCP-HW. Em um ambiente de rede cabeada simulada, TCP HolyWood supera em vazão media três dos mais importantes protocolos TCPs já elaborados. Estamos falando de TCP Reno, TCP Westwood, e TCP Vegas; e em variação de retardo media ao TCP Reno bem como ao TCP Vegas. Alem disso, de acordo com o índice de Jain, nossa proposta e tão imparcial quanto o padrão, TCP Reno. / We introduce a new end-to-end, sender side Transport Control Protocol called TCP HolyWood or in short TCP-HW. In a simulated wired environment, TCP HolyWood outperforms in average throughput, three of the more important TCP protocols ever made, we are talking about TCP Reno, TCP Westwood, and TCP Vegas; and in average jitter to TCP Reno and TCP Vegas too. In addition, according to Jain’s index, our proposal is as fair as TCP Reno, the Standard.

Radiotherapy is second only to surgery in the curative management of patients with cancer, and yet the molecular mechanisms that determine the sensitivity of tumours to radiation remain largely unclear. A high-throughput radiosensitivity screening method based on clonogenicity was developed and a siRNA library against kinase targets was screened. The gold standard colony formation endpoint was chosen for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitisation. TPK1 knockdown caused significant radiosensitisation in cancer but not normal tissue cell lines. Other means of blocking this pathway such as knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumour specific radiosensitisation. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitisation target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumour specific radiosensitisation. Three additional genes, signal recognition particle-72 kDa (SRP72), glycogen synthase 3-beta (GSK3&beta;) and MAP/Microtubule Affinity-Regulating Kinase 2 (MARK2) were also investigated. Knockdown of these genes radiosensitised both tumour and normal tissue cell lines and expression of two of them, GSK3&beta; and SRP72 were found to be associated with poor recurrence-free survival in early breast cancer patients.

Cloud computing enables the delivery of remote computing, software and storage services through web browsers following pay-as-you-go model. In addition to successful commercial applications, many research efforts including DOE Magellan Cloud project focus on discovering the opportunities and challenges arising from the computing and data-intensive scientific applications that are not well addressed by the current supercomputers, Linux clusters and Grid technologies. The elastic resource provision, noninterfering resource sharing and flexible customized configuration provided by the Cloud infrastructure has shed light on efficient execution of many scientific applications modeled as Directed Acyclic Graph (DAG) structured workflows to enforce the intricate dependency among a large number of different processing tasks. Meanwhile, the Cloud environment poses various challenges. Cloud providers and Cloud users pursue different goals. Providers aim to maximize profit by achieving higher resource utilization and users want to minimize expenses while meeting their performance requirements. Moreover, due to the expanding Cloud services and emerging newer technologies, the ever-increasing heterogeneity of the Cloud environment complicates the challenges for both parties. In this thesis, we address the workflow scheduling problem from different applications and various objectives. For batch applications, due to the increasing deployment of many data centers and computer servers around the globe escalated by the higher electricity price, the energy cost on running the computing, communication and cooling together with the amount of CO2 emissions have skyrocketed. In order to maintain sustainable Cloud computing facing with ever-increasing problem complexity and big data size in the next decades, we design and develop energy-aware scientific workflow scheduling algorithm to minimize energy consumption and CO2 emission while still satisfying certain Quality of Service (QoS) such as response time specified in Service Level Agreement (SLA). Furthermore, the underlying Cloud hardware/Virtual Machine (VM) resource availability is time-dependent because of the dual operation modes namely on-demand and reservation instances at various Cloud data centers. We also apply techniques such as Dynamic Voltage and Frequency Scaling (DVFS) and DNS scheme to further reduce energy consumption within acceptable performance bounds. Our multiple-step resource provision and allocation algorithm achieves the response time requirement in the step of forward task scheduling and minimizes the VM overhead for reduced energy consumption and higher resource utilization rate in the backward task scheduling step. We also evaluate the candidacy of multiple data centers from the energy and performance efficiency perspectives as different data centers have various energy and cost related parameters. For streaming applications, we formulate scheduling problems with two different objectives, namely one is to maximize the throughput under a budget constraint while another is to minimize execution cost under a minimum throughput constraint. Two different algorithms named as Budget constrained RATE (B-RATE) and Budget constrained SWAP (B-SWAP) are designed under the first objective; Another two algorithms, namely Throughput constrained RATE (TP-RATE) and Throughput constrained SWAP (TP-SWAP) are developed under the second objective.