No / Raman spectroscopy has been identified as an important first-pass analytical technique for deployment on planetary surfaces as part of a suite of instrumentation in projected remote space exploration missions to detect extant or extinct extraterrestrial life signatures. Aside from the demonstrable advantages of a non-destructive sampling procedure and an ability to record simultaneously the molecular signatures of biological, geobiological and geological components in admixture in the geological record, the interrogation and subsequent interpretation of spectroscopic data from these experiments will be critically dependent upon the recognition of key biomolecular markers indicative of life existing or having once existed in extreme habitats. A comparison made with the characteristic Raman spectral wavenumbers obtained from standards is not acceptable because of shifts that can occur in the presence of other biomolecules and their host mineral matrices. In this paper, we identify the major sources of difficulty experienced in the interpretation of spectroscopic data centring on a key family of biomarker molecules, namely scytonemin and its derivatives; the parent scytonemin has been characterized spectroscopically in cyanobacterial colonies inhabiting some of the most extreme terrestrial environments and, with the support of theoretical calculations, spectra have been predicted for the characterization of several of its derivatives which could occur in novel extraterrestrial environments. This work will form the foundation for the identification of novel biomarkers and for their Raman spectroscopic discrimination, an essential step in the interpretation of potentially complex and hitherto unknown biological radiation protectants based on the scytoneman and scytonin molecular skeletons which may exist in niche geological scenarios in the surface and subsurface of planets and their satellites in our Solar System.

Post-transcriptional control of gene expression in Eukaryotes plays a pivotal role in determining intricated networks defining physiological and pathological conditions among each organism. RNA Binding Proteins (RBPs), by exploiting RNA-protein and protein-protein interactions, have been recognized as the main actors in modulating these processes. As a consequence, RBPs aberrant expression, modulation or mis-localization, leads to the insurgence of complex phenotypes and diseases. Therefore, targeting and modulating the activity of RBPs found associated to different pathologies represents a new promising therapeutic strategy. During my PhD I aimed at identify, characterize and refine inhibitors targeting the RNA binding protein HuR. HuR belongs to the ELAVL protein family, it is ubiquitously expressed in the cells and among tissues and highly conserved throughout mammalian evolution. By binding AU/U rich elements (ARE) in the 3’UTRs of mRNAs, HuR mainly stabilizes its target transcripts, enhancing their translation. ARE sequences are found in 7% of the human mRNAs, coding for protein involved in key cellular processes as: immune response and inflammation, cell division and proliferation, angiogenesis, senescence and apoptosis. Hence, dysregulation in HuR expression and in its subcellular localization have been associated with the insurgence of several pathologies, mostly cancers and inflammation diseases. Notably, malignant transformations and poor prognosis in patients have been found characterized by highly nuclear or cytosolic HuR expression in a significant number of human cancers. Indeed, the majority of HuR regulated transcripts encode for protein responsible for the appearance of several cancerogenic traits. In particular, critical crosstalk established between cancer cells and inflammation processes play a pivotal role in worsening and compromising cancers development and onset. Moreover, considering that 90% of mRNAs coding for cytokines and chemokines contains repeated AREs sites in the 3’UTR, HuR plays a strong regulatory role in immune system (innate and adaptive) development and homeostasis as well as in pathogenic mechanisms. The searching for HuR inhibitors represents a challenging area, in the drug discovery field, due to its pleiotropic functions and its intrinsic structural complexity, which presents unfolded regions and sequences prone to aggregation. HuR disruptors have been reported in the literature, but without systematic studies, thus the identification of a new class of small molecules is still at the beginning. Among the molecules discovered so far, in 2015 our group identified through a High-throughput Screening a natural compound, DHTS, as a bona fide HuR inhibitor. Following that finding, we, me included, ascribed to the molecule a well-defined mechanism of action, identifying the specific binding sites on which HuR:DHTS interaction is based, defining that upon the mRNA binding DHTS interplays with HuR maintaining the protein in a closed conformation, thus inhibiting its function. Furthermore, we demonstrated DHTS anti-cancer activity in vitro, in cellular context and in vivo, in an HuR-dependent manner. In this way, DHTS represented the molecular scaffold, for the generation of a new class of highly potent HuR inhibitors, called Tanshinone Mimics (TMs). A functional oriented approach was applied for the synthesis of new molecules harboring only DHTS chemical elements responsible for HuR targeting, leading to a completely new molecular scaffold, not previously described in the literature, with respect to the ancestor molecule. I have characterized and identified more potent molecules, describing their anticancer properties, through the evaluation of their capabilities of downregulating the total expression level of well-known HuR targets, coding for proteins involved in tumor insurgence and progression, as VEGF, ERBB2 and CTNNB1, and reducing cancer cell migration, cell cycle progression in a minor extent. On the other end, I have explored TMs anti-inflammatory properties, counteracting the inflammatory response mediated by macrophages, directly impairing the binding between HuR and its pro-inflammatory targets, diminishing their expression and related protein secretion. Moreover, I have put evidences on TMs activity in vivo in acute inflammation mouse models. Lastly, I have evaluated TMs activity in affecting T-cells proliferation, on which HuR it is known to play a regulatory role. In conclusion, we identified TMs with Structure-Activity Relationships (SARs) towards HuR inhibition and its biological implications, aimed at ameliorating their specificity and bioavailability suitable for in vivo therapeutic strategies.

The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.

Molecular dynamics of biomolecules, like proteins and nucleic acids dictate essential biological processes allowing life to function. They are involved in a vast number of cellular tasks including DNA replication, genetic recombination, transcription and translation, as well as signalling, translational motion, structure formation, biochemical synthesis, immune response, and many more. Developed over billions of years by evolution they constitute fine-tuned networks modulated by temperature and regulatory mechanisms. A better understanding of the thermodynamic fundamentals of inter- and intramolecular conformational changes can shed light on the underlying processes of diseases and enables the transfer of biological architectures, properties and compositions to nanotechnological applications. Dynamics of biomolecules occur on a wide range of timescales covering more than twelve orders of magnitude. Fluorescence spectroscopy techniques like time-correlated single photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), and immobilized and freely diffusing single-molecule Förster resonance energy transfer (FRET) spectroscopy represent powerful tools monitoring the dynamics at different ranges within this large span of timescales. However, a unified approach covering all biological relevant timescales remains a goal in the field of fluorescence spectroscopy. This would comprise a methodological workflow for qualitative and quantitative analysis of biomolecular dynamics ranging from nanoseconds to hours. In this work, a custom built single-molecule fluorescence spectroscopy set-up was constructed combining confocal single-molecule FRET spectroscopy with TCSPC, FCS and fluorescence anisotropy techniques for multiparameter fluorescence detection (MFD). The set-up allows the complementary observation of single-molecules over an extensive timescale ranging from fast reconfiguration dynamics of polymers (nanoseconds) to slow membrane protein folding (hours) without the need of molecular synchronization. Freely diffusing molecules enable high throughput measurements in heterogeneous membrane-mimetic and denaturing environments. Additionally, routines for data acquisition and processing were developed followed by the elaboration of a methodological workflow for the qualitative and quantitative analysis of biomolecular dynamics. Finally, the applicability was demonstrated on a big diversity of biological systems (DNA hairpin, Holliday junction, soluble and membrane proteins) in aqueous, membrane-mimetic and denaturing environments covering conformational dynamics from nanoseconds to hours.:Chapter 1: Introduction Chapter 2: Dynamics of Biomolecules 2.1 Dynamics of Nucleic Acids 2.1.1 DNA Hairpin Dynamics 2.1.2 Dynamics of Holliday Junctions 2.2 Dynamics of Proteins 2.2.1 Model Systems of Protein Folding Chapter 3: Fundamentals of Fluorescence Spectroscopy 3.1 Basics of Fluorescence 3.2 Förster Resonance Energy Transfer (FRET) Chapter 4: Multiparameter Fluorescence Detection 4.1 Single-Molecule FRET Spectroscopy 4.1.1 Confocal Microscopy 4.1.2 Freely Diffusing Molecules 4.1.3 Fluorescence Spectroscopy 4.2 Time-Correlated Single-Photon Counting (TCSPC) 4.3 Pulsed Interleaved Excitation (PIE) 4.4 Fluorescence Anisotropy 4.5 Fluorescence Correlation Spectroscopy (FCS) 4.6 MFD Setup 4.7 Analysis Software Chapter 5: Analysis of Molecular Dynamics 5.1 Sub-Microseconds – Peptide Chain Dynamics 5.1.1 Identification of Peptide Chain Dynamics 5.1.2 Quantification of Peptide Chain Dynamics 5.1.3 Discussion 5.2 Microseconds – Dynamics of Barrier Crossing 5.2.1 Maximum Likelihood Estimation of the Transition-Path Time 5.2.2 Quantification of the Upper Bound of the Transition-Path Time 5.2.3 Discussion 5.3 Milliseconds – Fast Protein Folding Dynamics 5.3.1 Correlation of the Relative Donor Lifetime (τD(A) / τD(0)) with FRET Efficiency (E) 5.3.2 Burst-Variance Analysis (BVA) 5.3.3 FRET-Two-Channel Kernel-Based Density Distribution Estimator (FRET-2CDE) 5.3.4 Estimation of the Conformational Relaxation Rate using Bin-Time Analysis 5.3.5 Extracting Folding Kinetics using the Three-Gaussian (3G) Approximation 5.3.6 Dynamic Probability Distribution Analysis (dPDA) 5.3.7 Folding and Unfolding Rate Estimation using a Maximum-Likelihood Estimator 5.3.8 Discussion 5.4 Milliseconds to Seconds – Stacking Dynamics of DNA 5.4.1 Identification of Dynamics on the Recurrence Timescale 5.4.2 Quantification of Dynamics on the Recurrence Timescale 5.4.3 Discussion 5.5 Minutes to Hours – Slow Protein Folding Dynamics 5.5.1 Identification of Slow Protein Folding Dynamics 5.5.2 Quantification of Slow Protein Folding Dynamics 5.5.3 Discussion Chapter 6: Conclusion and Outlook Chapter 7: Appendices 7.1 Derivation of Equation 4.6 (inspired by Daniel Nettels) 7.2 Protein sequences 7.3 Identification of dynamics on the recurrence timescale 7.4 Dependency of psame on the sample concentration 7.5 Effect of fluorescence quenching on MFD parameters Chapter 8: References / Biomoleküle, wie Proteine und Nukleinsäuren, sind essentielle Bausteine des Lebens und permanent an biologischen Prozessen beteiligt. Innerhalb der Zelle nehmen sie eine Vielzahl von Aufgaben wahr, darunter DNA-Replikation, genetische Rekombination, Transkription und Translation, sowie Signalübertragung, Transport, Strukturbildung, biochemische Synthese und Immunreaktion. In Milliarden von Jahren evolutionärer Entwicklung wurden biomolekulare Prozesse immer feiner aufeinander Abgestimmt. Um den zugrundeliegenden Mechanismus von Krankheiten besser zu Verstehen und um die einzigartigen Eigenschaften und Kompositionen biologischer Systeme auf nanotechnologische Anwendungen übertragen zu können, ist es unbedingt notwendig ein besseres Verständnis thermodynamischer Grundlagen inter- und intramolekularer Konformationsänderungen zu erlangen. Dabei finden sich Dynamiken von Biomolekülen über eine Zeitskale von mehr als zwölf Größenordnungen verteilt. Fluoreszenzspektroskopietechniken, wie zeitkorrelierte Einzel-photonenzählung (TCSPC), Fluoreszenzkorrelationsspektroskopie (FCS), und Förster-Resonanzenergietransfer (FRET)–Spektroskopie von immobilisierten und frei diffundierenden Molekülen, stellen leistungsfähige Werkzeuge dar, welche es ermöglichen Dynamiken in der den Techniken entsprechenden Zeitskala aufzulösen. Dennoch, besteht der dringende Bedarf nach einer einheitlichen Methode, der in der Fluoreszenzspektroskopie alle biologisch relevanten Zeitskalen abdeckt. Dies würde einen methodischen Workflow für die qualitative und quantitative Analyse der biomolekularen Dynamik von Nanosekunden bis Stunden bedeuten. In dieser Arbeit wurde ein speziell angefertigter Multiparamter-Fluoreszenzspektroskopie-Aufbau konstruiert, welcher die konfokale Einzelmolekül-FRET-Spektroskopie mit den TCSPC-, FCS- und Fluoreszenz-Anisotropie-Techniken kombiniert. Der Aufbau ermöglicht die Beobachtung komplementärer Eigenschaften von Einzelmolekülen über eine umfangreiche Zeitskala hinweg. Dynamiken von schnell rekonfigurierenden Polymeren (Nanosekunden) bis hin zu langsam faltenden Membranproteinen (Stunden) sind ohne molekulare Synchronisation möglich. Darüber hinaus, ermöglicht der Einsatz frei diffundierender Moleküle einen hohen Messdurchsatz und die Anwendung heterogener membranmimetischer und denaturierender Lösungen. Zusätzlich wurden Routinen zur Datenerfassung und -verarbeitung entwickelt, gefolgt von der Ausarbeitung eines methodischen Workflows zur qualitativen und quantitativen Analyse von biomolekularen Dynamiken. Abschließend wurde die Anwendbarkeit an fünf biologischen Modelsystemen (DNA-Haarnadel, Holliday-Junction, lösliche und Membranproteine) in wässrigen, membranmimetischen und denaturierenden Umgebungen demonstriert und alle biologisch relevanten Zeitskalen von Nanosekunden bis Stunden abgedeckt.:Chapter 1: Introduction Chapter 2: Dynamics of Biomolecules 2.1 Dynamics of Nucleic Acids 2.1.1 DNA Hairpin Dynamics 2.1.2 Dynamics of Holliday Junctions 2.2 Dynamics of Proteins 2.2.1 Model Systems of Protein Folding Chapter 3: Fundamentals of Fluorescence Spectroscopy 3.1 Basics of Fluorescence 3.2 Förster Resonance Energy Transfer (FRET) Chapter 4: Multiparameter Fluorescence Detection 4.1 Single-Molecule FRET Spectroscopy 4.1.1 Confocal Microscopy 4.1.2 Freely Diffusing Molecules 4.1.3 Fluorescence Spectroscopy 4.2 Time-Correlated Single-Photon Counting (TCSPC) 4.3 Pulsed Interleaved Excitation (PIE) 4.4 Fluorescence Anisotropy 4.5 Fluorescence Correlation Spectroscopy (FCS) 4.6 MFD Setup 4.7 Analysis Software Chapter 5: Analysis of Molecular Dynamics 5.1 Sub-Microseconds – Peptide Chain Dynamics 5.1.1 Identification of Peptide Chain Dynamics 5.1.2 Quantification of Peptide Chain Dynamics 5.1.3 Discussion 5.2 Microseconds – Dynamics of Barrier Crossing 5.2.1 Maximum Likelihood Estimation of the Transition-Path Time 5.2.2 Quantification of the Upper Bound of the Transition-Path Time 5.2.3 Discussion 5.3 Milliseconds – Fast Protein Folding Dynamics 5.3.1 Correlation of the Relative Donor Lifetime (τD(A) / τD(0)) with FRET Efficiency (E) 5.3.2 Burst-Variance Analysis (BVA) 5.3.3 FRET-Two-Channel Kernel-Based Density Distribution Estimator (FRET-2CDE) 5.3.4 Estimation of the Conformational Relaxation Rate using Bin-Time Analysis 5.3.5 Extracting Folding Kinetics using the Three-Gaussian (3G) Approximation 5.3.6 Dynamic Probability Distribution Analysis (dPDA) 5.3.7 Folding and Unfolding Rate Estimation using a Maximum-Likelihood Estimator 5.3.8 Discussion 5.4 Milliseconds to Seconds – Stacking Dynamics of DNA 5.4.1 Identification of Dynamics on the Recurrence Timescale 5.4.2 Quantification of Dynamics on the Recurrence Timescale 5.4.3 Discussion 5.5 Minutes to Hours – Slow Protein Folding Dynamics 5.5.1 Identification of Slow Protein Folding Dynamics 5.5.2 Quantification of Slow Protein Folding Dynamics 5.5.3 Discussion Chapter 6: Conclusion and Outlook Chapter 7: Appendices 7.1 Derivation of Equation 4.6 (inspired by Daniel Nettels) 7.2 Protein sequences 7.3 Identification of dynamics on the recurrence timescale 7.4 Dependency of psame on the sample concentration 7.5 Effect of fluorescence quenching on MFD parameters Chapter 8: References

<p>Microbiological contamination from bacteria such as <em>Escherichia coli</em> and Salmonella is one of the main reasons for waterborne illness. Real time and accurate monitoring of water is needed in order to alleviate this human health concern. Performing multiple and parallel analysis of biomarkers such as DNA and mRNA that targets different regions of pathogen functionality provides a complete picture of its presence and viability in the shortest possible time. These biomarkers are present inside the cell and need to be extracted for analysis and detection. Hence, lysis of these pathogenic bacteria is an important part in the sample preparation for rapid detection. In addition, collecting a small amount of bacteria present in a large volume of sample and concentrating them before lysing is important as it facilitates the downstream assay. Various techniques, categorized as mechanical, chemical, thermal and electrical, have been used for lysing cells. In the electrical method, cells are lysed by exposure to an external electric field. The advantage of this method, in contrast to other methods, is that it allows lysis without the introduction of any chemical and biological reagents and permits rapid recovery of intercellular organelles. Despite the advantages, issues such as high voltage requirement, bubble generation and Joule heating are associated with the electrical method.</p> <p>To alleviate the issues associated with electrical lysis, a new design and associated fabrication process for a microfluidic cell lysis device is described in this thesis. The device consists of a nanoporous polycarbonate (PCTE) membrane sandwiched between two PDMS microchannels with electrodes embedded at the reservoirs of the microchannels. Microcontact printing is used to attach this PCTE membrane with PDMS.</p> <p>By using this PCTE membrane, it was possible to intensify the electric field at the interface of two channels while maintaining it low in the other sections of the device. Furthermore, the device also allowed electrophoretic trapping of cells before lysis at a lower applied potential. For instance, it could trap bacteria such as <em>E. coli</em> from a continuous flow into the intersection between two channels for lower electric field (308 V/cm) and lyse the cell when electric field was increased more than 1000 V/cm into that section.</p> <p>Application of lower DC voltage with pressure driven flow alleviated adverse effect from Joule heating. Moreover, gas evolution and bubble generation was not observed during the operation of this device.</p> <p>Accumulation and lysis of bacteria were studied under a fluorescence microscope and quantified by using intensity measurement. To observe the accumulation and lysis, LIVE/DEAD BacLight Bacterial Viability Kit consisting of two separate components of SYTO 9 and propidium iodide (PI) into the cell suspension in addition to GFP expressed <em>E. coli</em> were used. Finally, plate counting was done to determine the efficiency of the device and it was observed that the device could lyse 90% of bacteria for an operation voltage of 300V within 3 min.</p> <p>In conclusion, a robust, reliable and flexible microfluidic cell lysis device was proposed and analyzed which is useful for sample pretreatment in a Micro Total Analysis System.</p> / Master of Applied Science (MASc)

HIV-1 protease inhibitor drugs are important components of current antiretroviral therapy (cART). The cART treatment regimens dramatically improved life expectancy and mortality of patients with HIV-1 infection and AIDS. However, new and improved protease inhibitor drugs are essential for future treatment options. To this end, syntheses of optically active (3a<i>S</i>,4<i>S</i>,7a<i>R</i>)-hexahydro-4<i>H</i>-furo[2,3-<i>b</i>]pyran-4-ol, (3a<i>R</i>,4<i>R</i>,7a<i>S</i>)-hexahydro-4<i>H</i>-furo[2,3-b]pyran-4-ol, and (3<i>R</i>,3a<i>S</i>,6a<i>R</i>)-hexahydrofuro[2,3-6]furan-3-ol have been accomplished. These stereochemically defined heterocyclic derivatives are important high-affinity P2 ligands for a variety of highly potent HIV-1 protease inhibitors. The key steps for the synthesis hexehydrofuropyranol involve an efficient Paternò-Büchi [2+2] photocycloaddition, catalytic hydrogenation, acid-catalyzed cyclization to form the racemic ligand alcohol, and enzymatic resolution with immobilized Amano Lipase PS-30. Optically active ligand alcohols were obtained with high enantiomeric purity. Enantiomer (-)-ligand alcohol has been converted to potent HIV-1 protease inhibitors. <div><br></div><div>(3<i>R</i>,3a<i>S</i>,6a<i>R</i>)-Hexahydrofuro[2,3-<i>b</i>]furan-3-ol(<i>bis</i>-tetrahydrofuran) is a key subunit of darunavir, an FDA approved HIV-1 protease inhibitor drug which is widely used for the treatment of HIV/AIDS patients. This stereochemically defined bicyclic heterocycle is also embedded in a variety of highly potent HIV-1 protease inhibitors. The synthesis of optically active <i>bis</i>-tetrahydrofuran was achieved in optically pure form utilizing commercially available and inexpensive 1,2-<i>O</i>-isopropylidene-α-D-xylofuranose or 1,2-O-isopropylidene-α-D-glucofuranose as the starting material. The key steps involve a highly stereoselective substrate-controlled hydrogenation of ethyl 2-(dihydrofuran-3(2H)-ylidene)acetate, a Lewis acid-catalyzed anomeric reduction of a 1,2-<i>O</i>-isopropylidene-protected glycofuranoside, and a Baeyer-Villiger oxidation of a tetrahydrofuranyl-2-aldehyde derivative. Optically active (3<i>R</i>,3a<i>S</i>,6a<i>R</i>)-hexahydrofuro[2,3-<i>b</i>]furan-3-ol ligand was converted to darunavir efficiently. Furthermore, both furopyranol and bis-tetrahydrofuran ligand alcohols have been converted into a variety of potent HIV-1 protease inhibitors including inhibitors containing P2'-boronic acid ligands.<br></div><div><br></div><div>Diabetes mellitus is a chronic, progressive metabolic disorder that seriously threatens human health worldwide, particularly in developing countries. The prevalence of diabetes has been increasing steadily, especially in developing countries. Carambolaflavone A is a natural flavonoid isolated from the leaves of starfruit tree, <i>Averrhoacarambola</i>, in 2005. Carambolaflavone A possesses a <i>C</i>-aryl glycosidic linkage. Carambolaflavone A exhibited significant antihyperglycemic properties. More detailed biological studies reveal that it can lower acute blood glucose. The biology and chemistry of carambolaflavone A attracted our interest in synthesis and further design of interesting structural variants. A convergent total synthesis of carambolaflavone A has been accomplished. The synthesis highlights a bismuth triflate-catalyzed stereoselective C-aryl glycosylation of flavan and an appropriately protected D-fucose derivative as the key step. The glycosylation partners were synthesized from commercially available (±)-naringenin and D-(+)-galactose, respectively. An oxidative bromination and elimination reaction sequence was utilized to construct the flavone. The natural product is obtained in 10 steps (longest linear sequence) from D-(+)-galactose.<br></div>

Hydrogels are popular materials for biological applications since they exhibit properties like that of natural soft tissue and have tunable properties. Biodegradable hydrogels provide an added advantage in that they degrade in an aqueous environment thereby avoiding the need for removal after the useful lifetime. In this work, we investigated poly(β-amino ester) (PBAE) biodegradable hydrogel systems. To begin, the factors affecting the macromer synthesis procedure were studied to optimize the reproducibility of the resulting hydrogels made and create new methods of tuning the properties. Hydrogel behavior was then tuned by altering the hydrophilic/hydrophobic balance of the chemicals used in the synthesis to develop systems with linear and two-phase degradation profiles. The goal of the research was to better understand methods of controlling hydrogel properties to develop systems for several biomedical applications. Several systems with a range of properties were synthesized, and their in vitro behavior was characterized (degradation, mechanical properties, cellular response, etc.). From these studies, materials were chosen to serve as porogen materials and an outer matrix material to create a composite scaffold for tissue engineering. In most cases, a porous three dimensional scaffold is ideal for cellular growth and infiltration. In this work, a composite with a slow degrading outer matrix PBAE with fast degrading PBAE microparticles was created. First, a procedure for developing porogen particles of controlled size from a fast-degrading hydrogel material was developed. Porogen particles were then entrapped in the outer hydrogel matrix during polymerization. The resulting composite systems were degraded and the viability of these systems as tissue engineering scaffolds was studied. In a second area of work, two polymer systems, one PBAE hydrogel and one sol-gel material were altered through the addition of iron oxide nanoparticles to create materials with remote controlled properties. Iron oxide nanoparticles have the ability to heat in an alternating magnetic field due to the relaxation processes. The incorporation of these nanoscale heating sources into thermosensitive polymer systems allowed remote actuation of the physical properties. These materials would be ideal for use in applications where the system can be changed externally such as in remote controlled drug delivery.

The function of biomacromolecules is controlled by their structure and conformational flexibility. Investigating the structure of biologically important macromolecules can, therefore, yield information that could explain their complex biological function. In addition to X ray crystallography and nuclear magnetic resonance (NMR) methods, pulsed electron paramagnetic resonance (EPR) methods, in particular the pulsed electron electron double resonance (PELDOR) technique has, during the last decade, become a valuable tool for structural determination of macromolecules. Long range distance constraints obtained from pulsed EPR measurements, make it possible to carry out structural refinements on structures from NMR and X ray methods. In addition, EPR yields distance distributions that give information about structural flexibility. The use of EPR for structural studies of biomacromolecules requires in most cases site specific incorporation of paramagnetic centres known as spin labelling. To date, spin labelling nucleic acids has required complex spin labelling chemistry. The first application of a site directed and noncovalent spin labelling method for distance measurements on DNA is described. It is demonstrated that noncovalent spin labelling with a rigid spin label can afford detailed information on internal DNA dynamics using PELDOR. Furthermore, it is shown that noncovalent spin labelling can be used to study DNA protein complexes. PELDOR can also yield information about spin label orientation. Therefore, spin labels with limited flexibility can be used to measure the relative orientation of the spin labelled sites. Although information on orientation can be obtained from 9.7 GHz PELDOR measurements in selected applications, measurements at 97 GHz or higher, increases orientation selection. It is shown that PELDOR measurements on semi rigid and rigid nitroxide biradicals using a home built high power 97 GHz EPR spectrometer (Hiper) and model based simulations yield quantitative information on spin label orientations and dynamics. The most widely used spin labels for EPR studies on biomacromolecules are the aminoxyl (nitroxide) radicals. The major drawbacks of nitroxide spin labels include low sensitivity for distance measurements, fast spin spin relaxation in solution and limited stability in reducing environments. Carbon centered triarylmethyl (trityl) radicals have properties that could eliminate some of the limitations of nitroxide spin labels. To evaluate the use of trityl spin labels for nanometer distance measurements, models systems with trityl and nitroxide spin labels were measured using PELDOR and Double Quantum Coherence (DQC). This study shows that trityl spin labels yield reliable information on interlabel distances and dynamics, establishing the trityl radical as a viable spin label for structural studies on biomacromolecules.

Biomolecules on cell surfaces play critical roles in diverse biological and physiological processes. However, conventional bulk scale techniques are unable to clarify the density and distribution of specific biomolecules in situ on single, living cell surfaces at the micro or nanoscale. In this work, a single cell analysis technique based on Atomic Force Microscopy (AFM) is developed to spatially identify biomolecules and characterize nanomechanical properties on single cell surfaces. The unique advantage of these AFM-based techniques lies in the ability to operate in situ (in a non-destructive fashion) and in real time, under physiological conditions or controlled micro-environments. First, AFM-based force spectroscopy was developed to study the fundamental biophysics of the heparin/thrombin interaction at the molecular level. Based on force spectroscopy, a force recognition mapping strategy was developed and optimized to spatially detect single protein targets on non-biological surfaces. This platform was then translated to the study of complex living cell surfaces. Specific carbohydrate compositions and changes in their distribution, as well as elasticity change were obtained by monitoring Bacillus cells sporulation process. The AFM-based force mapping technique was applied to different cellular systems to develop a cell surface biomolecule library. Nanoscale imaging combined with carbohydrate mapping was used to evaluate inactivation methods and growth temperatures effects on Yersinia pestis surface. A strategy to image cells in real time was coupled with hydrophobicity mapping technique to monitor the effect of antimicrobials (antimicrobial polymer and copper) on Escherichia coli and study their killing mechanisms. The single spore hydrophobicity mapping was used to localize the exosporium structure and potentially reconstruct culture media. The descriptions of cell surface DNA on single human epithelial cells potentially form a novel tool for forensic identification. Overall, these nanoscale tools to detect and assess changes in cell behavior and function over time, either as a result of natural state changes or when perturbed, will further our understanding of fundamental biological processes and lead to novel, robust methods for the analysis of individual cells. Real time analysis of cells can be used for the development of lab-on-chip type assays for drug design and testing or to test the efficacy of antimicrobials.

O trabalho descrito nesta tese mostra a aplicação de um sistema de detecção condutométrica sem contato acoplado capacitivamente (C4D) para monitorar interações biomoleculares em microssistemas analíticos. Inicialmente, o desempenho analítico de microssistemas fabricados em vidro, poli(dimetilsiloxano) (PDMS) e poliéster-toner (PT) foi avaliado de modo a escolher o melhor material (em termos de facilidades de fabricação, custo e repetibilidade) para os ensaios biomoleculares. Dentre os materiais estudados, os dispositivos fabricados em PT mostraram-se mais adequados para testes rápidos, onde a repetibilidade analítica não é o parâmetro mais importante. Os dispositivos fabricados em PDMS e selados contra uma placa de vidro apresentaram os melhores resultados em termos de repetibilidade e o desempenho analítico foi similar aos dispositivos de vidro. Dessa maneira, os dispositivos fabricados em PDMS/vidro foram escolhidos para a demonstração dos objetivos da tese. Por outro lado, os dispositivos fabricados em PT foram explorados para estudar a configuração geométrica do sistema de C4D. A instrumentação para monitoramento dos ensaios de ligação foi composta basicamente de dois sistemas de C4D, um software escrito em LabVIEW e um sistema de bombeamento das soluções. De modo a encontrar a configuração ideal da cela de detecção, geometrias contendo três, quatro e cinco eletrodos foram avaliadas em dispositivos de PT. A configuração ótima foi composta de três eletrodos, espaçados simetricamente. Nesta geometria, um eletrodo é utilizado para aplicar o sinal senoidal de excitação e os outros dois são utilizados para capturar o sinal resultante. As dimensões dos eletrodos (largura e espaçamento entre eles) foram otimizados usando ferramentas quimométricas. O complexo avidina-biotina foi utilizado como modelo de ligação para mostrar a aplicabilidade do sistema proposto. Para os microssistemas biomoleculares, os eletrodos (com geometria otimizada) foram fabricados sobre a superfície de uma placa de vidro por fotolitografia, sputtering e lift-off. Os eletrodos de detecção foram isolados com uma camada de óxido de silício com espessura de 50 nm, depositada pelo processo de deposição química em fase de vapor assistida por plasma. A camada de SiO2 foi modificada quimicamente com solução de 3-amino-propil-trietóxi-silano em etanol. Para imobilização covalente de biotina, uma alíquota de 10 ?L de fotobiotina dissolvida em água (0,1 mg/mL) foi adicionada à superfície e exposta a radiação ultravioleta (365 nm, 10 mW/cm2) durante 15 min. A detecção foi realizada aplicando um sinal senoidal, a partir de um gerador de funções, ao eletrodo de excitação registrando o sinal resultante nos dois eletrodos receptores. Para minimizar a captura de ruído elétrico, os experimentos foram realizados em uma gaiola de Faraday. O controle e a aquisição de dados foi feito mediante um software escrito em LabVIEW monitorando os sensorgramas de condutividade em tempo real. Os canais microfluídicos foram fabricados em PDMS por litografia suave e selados irrevesivelmente contra a placa de vidro contendo os eletrodos isolados e modificados quimicamente. As soluções (tampão e amostra) foram manuseadas com auxílio de uma bomba peristáltica ou duas bombas seringas. Soluções contendo tampão e avidina foram introduzidas nos microcanais e as mudanças de conductividade foram monitoradas em função do tempo. As soluções contendo avidina permaneceram em contato com a superfície modificada até o sinal de condutividade atingir um patamar de equilíbrio. Depois disso, solução tampão foi introduzida no microcanal para remover os analitos adsorvidos à superfície. Duas válvulas solenóides foram utilizadas para permitir um controle automático da distribuição das soluções nos microcanais. O limite de detecção obtido para a interação entre avidina e biotina foi de 75 nmol L-1. / The study reported in this thesis shows the application of a capacitively coupled contactless conductivity detection (C4D) for monitoring biomolecular interactions on analytical microsystems. Initially, the analytical performance of the microsystems fabricated in glass, poly(dimethylsiloxane) (PDMS) and polyester-toner (PT) was investigated in order to choose the best material (in terms of fabrication facilities, costs and repeatability) for the biomolecular assays. Among all substrate materials studied, devices fabricated in PT showed suitability for quick experiments, in which the analytical repeatability is not the most important parameter. The devices fabricated in PDMS and sealed against a glass plate presented the best results in terms of repeatability and the analytical performance was similar to that one of glass devices. For this reason, PDMS/glass devices were chosen for showing the goals of this thesis. On the other hand, PT devices were employed to study the geometrical design of the C4D system. The instrumentation for monitoring binding assays was basically composed of two C4D systems, a software written in LabVIEW and a solution pumping system. In order to find the suitable detection cell configuration for this dual-C4D system, designs containing three, four and five electrodes were evaluated on PT devices. The optimal design was composed of three electrodes symmetrically spaced. In this configuration, one electrode is used for applying an excitation sinusoidal wave and the other two for picking up the resulting signal. The dimensions of the electrodes (width and gap) were optimized by chemometric tools. The avidin-biotin complex was used as a binding model for showing the feasibility of the proposed system. For the biomolecular microsystems, electrodes were fabricated on glass surface using photolithographic, sputtering and lift-off processes. Detection electrodes were insulated with a 50-nm silicon oxide layer deposited by plasmaenhanced chemical vapor deposition. The SiO2 layer was functionalized by immersing the cleaned surface in a 3-aminopropyltriethoxy-silane solution in ethanol for 3 h. For biotinylation of the amino-silane layer, 10 ?L of photobiotin dissolved in deionized water (0.1 mg/mL) was dropped on the modified glass surface and exposed to a 365 nm UV radiation at intensity of 10 mW/cm2 for 15 min. Detection was carried out by passing a sinusoidal excitation signal from the function signal generator to the first electrode and picking up the resulting signal at the two receiver electrodes. To reduce electrical noise pickup, all measurements were carried out in a Faraday cage. The data acquisition was obtained in a software written in LabVIEW and the conductivity sensorgrams were recorded in real-time. The microfluidic network was fabricated in PDMS by soft lithography and irreversibly sealed against the electrodes plate. Solutions were handled into microfluidic channels using a peristaltic pump or two syringe pumps. Buffer and avidin-containing solution was injected into the microchannels and conductivity changes were monitored over time. Avidin solutions were allowed to remain in contact with the surface until a stable conductivity had reached equilibrium. Avidin-free buffer solutions were then injected to rinse off non-specifically bound analytes. Two solenoid valves were used to allow an automatic dispensing of the sample/buffer solution into microchannels. The limit of detection found for avidin-biotin system was 75 nmol L-1.