Peptide binding to HLA-DP proteins at pH 5.0 and pH 7.0:a quantitative molecular docking study

Background: HLA-DPs are class II MHC proteins mediating immune responses to many diseases. Peptides bind MHC class II proteins in the acidic environment within endosomes. Acidic pH markedly elevates association rate constants but dissociation rates are almost unchanged in the pH range 5.0 - 7.0. This pH-driven effect can be explained by the protonation/deprotonation states of Histidine, whose imidazole has a pKa of 6.0. At pH 5.0, imidazole ring is protonated, making Histidine positively charged and very hydrophilic, while at pH 7.0 imidazole is unprotonated, making Histidine less hydrophilic. We develop here a method to predict peptide binding to the four most frequent HLA-DP proteins: DP1, DP41, DP42 and DP5, using a molecular docking protocol. Dockings to virtual combinatorial peptide libraries were performed at pH 5.0 and pH 7.0. Results: The X-ray structure of the peptide - HLA-DP2 protein complex was used as a starting template to model by homology the structure of the four DP proteins. The resulting models were used to produce virtual combinatorial peptide libraries constructed using the single amino acid substitution (SAAS) principle. Peptides were docked into the DP binding site using AutoDock at pH 5.0 and pH 7.0. The resulting scores were normalized and used to generate Docking Score-based Quantitative Matrices (DS-QMs). The predictive ability of these QMs was tested using an external test set of 484 known DP binders. They were also compared to existing servers for DP binding prediction. The models derived at pH 5.0 predict better than those derived at pH 7.0 and showed significantly improved predictions for three of the four DP proteins, when compared to the existing servers. They are able to recognize 50% of the known binders in the top 5% of predicted peptides. Conclusions: The higher predictive ability of DS-QMs derived at pH 5.0 may be rationalised by the additional hydrogen bond formed between the backbone carbonyl oxygen belonging to the peptide position before p1 (p-1) and the protonated ε-nitrogen of His 79β. Additionally, protonated His residues are well accepted at most of the peptide binding core positions which is in a good agreement with the overall negatively charged peptide binding site of most MHC proteins. © 2012 Patronov et al.; licensee BioMed Central Ltd.

Organic matter dynamics in the Florida coastal Everglades: A molecular marker and isotopic approach

Everglades National Park (ENP) is about to undergo the world's largest wetland restoration with the aim of improving the quality, timing and distribution of water flow. The changes in water flow are hypothesized to alter the nutrient fluxes and organic matter (OM) dynamics within ENP, especially in the estuarine areas. This study used a multi-proxy approach of molecular markers and stable δ 13C isotope measurements, to determine the present day OM dynamics in ENP. ^ OM dynamics in wetland soils/sediments have proved to be difficult to understand using traditional geochemical approaches. These are often inadequate to describe the multitude of OM sources (e.g. higher land plant, emergent vegetation, submerged vegetation) to the soils/sediments and the complex diagenetic processes that can alter the OM characteristics. A multi-proxy approach, however, that incorporates both molecular level and bulk parameter information is ideal to comprehend complex OM dynamics in aquatic environments. Therefore, biomass-specific molecular markers or proxies can be useful in tracing the sources and processing of OM. This approach was used to examine the OM dynamics in the two major drainage basins, Shark River Slough and Taylor River Slough, of ENP. Freshwater to marine transects were sampled in both systems for soils/sediments and suspended particulate organic matter (SPOM) to be characterized through bulk OM analyses, lipid biomarker determinations (e.g. sterols, fatty acids, hydrocarbons and triterpenoids) and compound-specific stable carbon isotope (δ 13C) determinations. ^ One key accomplishment of the research was the assessment of a molecular marker proxy (Paq) to distinguish between emergent/higher plant vegetation from submerged vegetation within ENP. This proxy proved to be quite useful at tracing OM inputs to the soils/sediments of ENP. A second key accomplishment was the development of a 3-way model using vegetation specific molecular markers. This novel, descriptive model was successfully applied to the estuarine areas of Taylor and Shark River sloughs, providing clear evidence of mixing of freshwater, estuarine and marine derived OM in these areas. In addition, diagenetic transformations of OM in these estuaries were found to be quite different between Taylor and Shark Rivers, and are likely a result of OM quality and hydrological differences. ^

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of Nontypeable Haemophilus influenzae Colonization

Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

Programming Molecular Devices using Nucleic Acid Hairpins

Nucleic Acid hairpins have been a subject of study for the last four decades. They are composed of single strand that is

hybridized to itself, and the central section forming an unhybridized loop. In nature, they stabilize single stranded RNA, serve as nucleation

sites for RNA folding, protein recognition signals, mRNA localization and regulation of mRNA degradation. On the other hand,

DNA hairpins in biological contexts have been studied with respect to forming cruciform structures that can regulate gene expression.

The use of DNA hairpins as fuel for synthetic molecular devices, including locomotion, was proposed and experimental demonstrated in 2003. They

were interesting because they bring to the table an on-demand energy/information supply mechanism.

The energy/information is hidden (from hybridization) in the hairpin’s loop, until required.

The energy/information is harnessed by opening the stem region, and exposing the single stranded loop section.

The loop region is now free for possible hybridization and help move the system into a thermodynamically favourable state.

The hidden energy and information coupled with

programmability provides another functionality, of selectively choosing what reactions to hide and

what reactions to allow to proceed, that helps develop a topological sequence of events.

Hairpins have been utilized as a source of fuel for many different DNA devices. In this thesis, we program four different

molecular devices using DNA hairpins, and experimentally validate them in the

laboratory. 1) The first device: A

novel enzyme-free autocatalytic self-replicating system composed entirely of DNA that operates isothermally. 2) The second

device: Time-Responsive Circuits using DNA have two properties: a) asynchronous: the final output is always correct

regardless of differences in the arrival time of different inputs.

b) renewable circuits which can be used multiple times without major degradation of the gate motifs

(so if the inputs change over time, the DNA-based circuit can re-compute the output correctly based on the new inputs).

3) The third device: Activatable tiles are a theoretical extension to the Tile assembly model that enhances

its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly.

4) The fourth device: Controlled Amplification of DNA catalytic system: a device such that the amplification

of the system does not run uncontrollably until the system runs out of fuel, but instead achieves a finite

amount of gain.

Nucleic acid circuits with the ability

to perform complex logic operations have many potential practical applications, for example the ability to achieve point of care diagnostics.

We discuss the designs of our DNA Hairpin molecular devices, the results we have obtained, and the challenges we have overcome

to make these truly functional.

Molecular Mechanisms of Airway Epithelial Progenitor Cell Maintenance and Repair.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Resonance Energy Transfer-Based Molecular Switch Designed Using a Systematic Design Process Based on Monte Carlo Methods and Markov Chains

A RET network consists of a network of photo-active molecules called chromophores that can participate in inter-molecular energy transfer called resonance energy transfer (RET). RET networks are used in a variety of applications including cryptographic devices, storage systems, light harvesting complexes, biological sensors, and molecular rulers. In this dissertation, we focus on creating a RET device called closed-diffusive exciton valve (C-DEV) in which the input to output transfer function is controlled by an external energy source, similar to a semiconductor transistor like the MOSFET. Due to their biocompatibility, molecular devices like the C-DEVs can be used to introduce computing power in biological, organic, and aqueous environments such as living cells. Furthermore, the underlying physics in RET devices are stochastic in nature, making them suitable for stochastic computing in which true random distribution generation is critical.

In order to determine a valid configuration of chromophores for the C-DEV, we developed a systematic process based on user-guided design space pruning techniques and built-in simulation tools. We show that our C-DEV is 15x better than C-DEVs designed using ad hoc methods that rely on limited data from prior experiments. We also show ways in which the C-DEV can be improved further and how different varieties of C-DEVs can be combined to form more complex logic circuits. Moreover, the systematic design process can be used to search for valid chromophore network configurations for a variety of RET applications.

We also describe a feasibility study for a technique used to control the orientation of chromophores attached to DNA. Being able to control the orientation can expand the design space for RET networks because it provides another parameter to tune their collective behavior. While results showed limited control over orientation, the analysis required the development of a mathematical model that can be used to determine the distribution of dipoles in a given sample of chromophore constructs. The model can be used to evaluate the feasibility of other potential orientation control techniques.

A Molecular-scale Programmable Stochastic Process Based On Resonance Energy Transfer Networks: Modeling And Applications

While molecular and cellular processes are often modeled as stochastic processes, such as Brownian motion, chemical reaction networks and gene regulatory networks, there are few attempts to program a molecular-scale process to physically implement stochastic processes. DNA has been used as a substrate for programming molecular interactions, but its applications are restricted to deterministic functions and unfavorable properties such as slow processing, thermal annealing, aqueous solvents and difficult readout limit them to proof-of-concept purposes. To date, whether there exists a molecular process that can be programmed to implement stochastic processes for practical applications remains unknown.

In this dissertation, a fully specified Resonance Energy Transfer (RET) network between chromophores is accurately fabricated via DNA self-assembly, and the exciton dynamics in the RET network physically implement a stochastic process, specifically a continuous-time Markov chain (CTMC), which has a direct mapping to the physical geometry of the chromophore network. Excited by a light source, a RET network generates random samples in the temporal domain in the form of fluorescence photons which can be detected by a photon detector. The intrinsic sampling distribution of a RET network is derived as a phase-type distribution configured by its CTMC model. The conclusion is that the exciton dynamics in a RET network implement a general and important class of stochastic processes that can be directly and accurately programmed and used for practical applications of photonics and optoelectronics. Different approaches to using RET networks exist with vast potential applications. As an entropy source that can directly generate samples from virtually arbitrary distributions, RET networks can benefit applications that rely on generating random samples such as 1) fluorescent taggants and 2) stochastic computing.

By using RET networks between chromophores to implement fluorescent taggants with temporally coded signatures, the taggant design is not constrained by resolvable dyes and has a significantly larger coding capacity than spectrally or lifetime coded fluorescent taggants. Meanwhile, the taggant detection process becomes highly efficient, and the Maximum Likelihood Estimation (MLE) based taggant identification guarantees high accuracy even with only a few hundred detected photons.

Meanwhile, RET-based sampling units (RSU) can be constructed to accelerate probabilistic algorithms for wide applications in machine learning and data analytics. Because probabilistic algorithms often rely on iteratively sampling from parameterized distributions, they can be inefficient in practice on the deterministic hardware traditional computers use, especially for high-dimensional and complex problems. As an efficient universal sampling unit, the proposed RSU can be integrated into a processor / GPU as specialized functional units or organized as a discrete accelerator to bring substantial speedups and power savings.

Enzymatic Polymerization of High Molecular Weight DNA

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.

The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.

Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.

Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.

Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.

In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.

Bulk and molecular proxies of sediment core GeoB6518-1

A 20 kyr long sediment sequence from the Congo deep sea fan (core GeoB 6518-1), one of the world's largest deep sea river fans, has been analysed for bulk and molecular proxies in order to reconstruct the marine, soil and plant organic carbon (OC) contributions to these sediments since the last glacial maximum. The bulk proxies applied, C/N ratio and d13Corg, ranged from 10 to 12.5 and from -24.5 to -21 per mill VPDB, respectively. As molecular proxies, concentrations of marine derived alkenones and terrestrial derived odd-numbered n-alkanes were used, which varied between 0.2 and 4 µg/g dry weight sediment. In addition, the branched vs. isoprenoid tetraether (BIT) index, a proxy for soil organic matter input, was used, which varied from 0.3 to 0.5 in this core. Application of binary mixing models, based on the different individual proxies, showed estimates for terrestrial OC input varying by up to 50% due to the heterogeneous nature of the OC. Application of a three end-member mixing model using the d13Corg content, the C/N ratio and the BIT index, enabled the distinction of soil and plant organic matter as separate contributors to the sedimentary OC pool. The results show that marine OC accounts for 20% to 40% of the total OC present in the deep sea fan sediments over the last 20 kyr and that soil OC accounts for about half (45% on average) of the OC present. This suggests that soil OC represents the majority of the terrestrial OC delivered to the fan sediments. Accumulation rates of the plant and soil OC fractions over the last 20 kyr varied by a factor of up to 5, and are strongly related to sediment accumulation rates. They showed an increase starting at ca. 17 kyr BP, a decline during the Younger Dryas, peak values during the early Holocene and lower values in the late Holocene. This pattern matches with reconstructions of past central African humidity and Congo River discharge from the same core and revealed that central African precipitation patterns exert a dominant control on terrestrial OC deposition in the Congo deep sea fan. Marine OC accumulation rates are only weakly related to sediment accumulation rates and vary only little over time compared to the terrigenous fractions. These variations are likely a result of enhanced preservation during times of higher sedimentation rates and of relative small fluctuations in primary production due to wind-driven upwelling.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

New Method for the Estimation of Viscosity of Pure and Mixtures of Ionic Liquids Based on the UNIFAC–VISCO Model

A modified UNIFAC–VISCO group contribution method was developed for the correlation and prediction of viscosity of ionic liquids as a function of temperature at 0.1 MPa. In this original approach, cations and anions were regarded as peculiar molecular groups. The significance of this approach comes from the ability to calculate the viscosity of mixtures of ionic liquids as well as pure ionic liquids. Binary interaction parameters for selected cations and anions were determined by fitting the experimental viscosity data available in literature for selected ionic liquids. The temperature dependence on the viscosity of the cations and anions were fitted to a Vogel–Fulcher–Tamman behavior. Binary interaction parameters and VFT type fitting parameters were then used to determine the viscosity of pure and mixtures of ionic liquids with different combinations of cations and anions to ensure the validity of the prediction method. Consequently, the viscosities of binary ionic liquid mixtures were then calculated by using this prediction method. In this work, the viscosity data of pure ionic liquids and of binary mixtures of ionic liquids are successfully calculated from 293.15 K to 363.15 K at 0.1 MPa. All calculated viscosity data showed excellent agreement with experimental data with a relative absolute average deviation lower than 1.7%.