Chemically-Addressable Protein Entrapment in Silica Sol-Gels

Thesis (Master's)--University of Washington, 2016-06

Redescription of the gnathostome fish fauna from the mid-Palaeozoic Silverband Formation, the Grampians, Victoria

Gnathostome vertebrate remains from fine-grained sandstones of the Silverband Formation in the Grampians, Victoria include dissociated fin spines, scales and teeth. These elements arc assigned herein to the acanthodians Sinacanthus? micracanthus (fin spines) and Radioporacanthodes sp. cf. R. qujingensis (scales and tooth whorls). This fauna indicates a Late Silurian (?late Ludlow) age for the vertebrate-beating Stratum. Under current systematic groupings, the two gnathostome taxa from the Silverband Formation belong to two different families, the Sinacanthidae and the Poracanthodidae. However. the preserved association could indicate that the three element types derived from the same biological species. The possibility that the Sinacanthidae is a sister group to the Climatiidae and the Poracanthodidae is raised by this scenario. The Sinacanthidae is tentatively reassigned to the Acanthodii, as it is considered to lack diagnostic chondrichthyan characters.

Novel hydrogel polymers

Hydrogels are a unique class of polymers which swell, but do not dissolve in water. A range of 2-hydroxyethyl methacrylate based copolymer hydrogels have been synthesised and are described in this thesis. Initially, hydrogels were synthesised containing acryloylmorpholine, N,N-dimethyl acrylamide and N-vinyl pyrrolidone. Variations in structure and composition have been correlated with the sequence distribution, equilibrium water content (EWC) , mechanical and surface properties of the hydrogels. The sequence distribution was found to be dependant on the structure and reactivity of the monomers. The EWC was found to be dependant on the water structuring groups present in the hydrogel, although the water binding abilities were modified by steric effects. The mechanical properties were also investigated and were found to be dependant on the monomer structure, sequence distribution and the amount and nature of water in the hydrogel. The macroscopic surface properties of the hydrogels were probed using surface energy determinations and were found to be a function of the water content and the hydrogel composition. At a molecular level, surface properties were investigated using an in vitro ocular spoilation model and single protein adhesion studies. The results indicate that the sequence distribution and the polarity of the surface affect the adhesion of biological species. Finally, a range of 2-hydroxyethyl methacrylate based copolymer hydrogels containing both charged monomer groups and linear polyethers have been synthesised and described. Although variations in the EWC are observed with the structure of the monomers, it was observed that the EWC increased due to the polar character of the charged monomers and the chain length and hydrophilicity of the polyethers. Investigation of these hydrogel surfaces revealed subtle changes. The molecular surface properties indicate the significance of the effect of charge and molecular mobility of the groups expressed at the hydrogel surface.

Hydrogels containing linear and cyclic polyethers

Hydrogels are a unique class of polymer which swell, but do not dissolve in, water. A range of 2-hydroxyethyl methacrylate based copolymer hydrogels containing both cyclic and linear polyethers have been synthesised and are described in this thesis. Initially, cyclic polyethers were occluded within the polymer matrix and the transport properties investigated. The results indicated that the presence of an ionophore can be used to modulate ion transport and that ion transport is described by a dual-sorption mechanism. However, these studies were limited due to ionophore loss during hydration. Hence, the synthesis of a range of acrylate based crown ether monomers was considered. A pure sample of 4-acryolylaminobenzo-15-crown-5 was obtained and a terpolymer containing this monomer was prepared. Transport studies illustrated that the presence of a `bound' ionophore modulates ion transport in a similar way to the occluded systems. The transport properties of a series of terpolymers containing linear polyethers were then investigated. The results indicated that the dual-sorption mechanism is observed for these systems with group II metal cations while the transport of group I metal cations, with the exception of sodium, is enhanced. Finally, the equilibrium water contents (EWC) surface and mechanical properties of these terpolymers containing linear polyethers were examined. Although subtle variations in EWC are observed as the structure of the polyether side chain varies, generally EWC is enhanced due to the hydrophilicity of the polyether side chain. The macroscopic surface properties were investigated using a sessile drop technique and FTIR spectroscopy. At a molecular level surface properties were probed using an in vitro ocular spoilation model and preliminary cell adhesion studies. The results indicate that the polyethylene oxide side chains are expressed at the polymer surface thus reducing the adhesion of biological species.

Solvability and asymptotics of the heat equation with mixed variable lateral conditions and applications in the opening of the exocytotic fusion pore in cells

We investigate a mixed problem with variable lateral conditions for the heat equation that arises in modelling exocytosis, i.e. the opening of a cell boundary in specific biological species for the release of certain molecules to the exterior of the cell. The Dirichlet condition is imposed on a surface patch of the boundary and this patch is occupying a larger part of the boundary as time increases modelling where the cell is opening (the fusion pore), and on the remaining part, a zero Neumann condition is imposed (no molecules can cross this boundary). Uniform concentration is assumed at the initial time. We introduce a weak formulation of this problem and show that there is a unique weak solution. Moreover, we give an asymptotic expansion for the behaviour of the solution near the opening point and for small values in time. We also give an integral equation for the numerical construction of the leading term in this expansion.

Identification of DNA-binding proteins by incorporating evolutionary information into pseudo amino acid composition via the top-n-gram approach

DNA-binding proteins are crucial for various cellular processes and hence have become an important target for both basic research and drug development. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to establish an automated method for rapidly and accurately identifying DNA-binding proteins based on their sequence information alone. Owing to the fact that all biological species have developed beginning from a very limited number of ancestral species, it is important to take into account the evolutionary information in developing such a high-throughput tool. In view of this, a new predictor was proposed by incorporating the evolutionary information into the general form of pseudo amino acid composition via the top-n-gram approach. It was observed by comparing the new predictor with the existing methods via both jackknife test and independent data-set test that the new predictor outperformed its counterparts. It is anticipated that the new predictor may become a useful vehicle for identifying DNA-binding proteins. It has not escaped our notice that the novel approach to extract evolutionary information into the formulation of statistical samples can be used to identify many other protein attributes as well.

Functionalized carbon nanotube nanoelectrodes for biomolecular recognition

The objective of this research is to develop nanoscale ultrasensitive transducers for detection of biological species at molecular level using carbon nanotubes as nanoelectrodes. Rapid detection of ultra low concentration or even single DNA molecules are essential for medical diagnosis and treatment, pharmaceutical applications, gene sequencing as well as forensic analysis. Here the use of functionalized single walled carbon nanotubes (SWNT) as nanoscale detection platform for rapid detection of single DNA molecules is demonstrated. The detection principle is based on obtaining electrical signal from a single amine terminated DNA molecule which is covalently bridged between two ends of an SWNT separated by a nanoscale gap. The synthesis, fabrication, chemical functionalization of nanoelectrodes and DNA attachment were optimized to perform reliable electrical characterization these molecules. Using this detection system fundamental study on charge transport in DNA molecule of both genomic and non genomic sequences is performed. We measured an electrical signal of about 30 pA through a hybridized DNA molecule of 80 base pair in length which encodes a portion of sequence of H5N1 gene of avian Influenza A virus. Due the dynamic nature of the DNA molecules the local environment such as ion concentration, pH and temperature significantly influence its physical properties. We observed a decrease in DNA conductance of about 33% in high vacuum conditions. The counterion variation was analyzed by changing the buffer from sodium acetate to tris(hydroxymethyl) aminomethane, which resulted in a two orders of magnitude increase in the conductivity of the DNA. The fabrication of large array of identical SWNT nanoelectrodes was achieved by using ultralong SWNTs. Using these nanoelectrode array we have investigated the sequence dependent charge transport in DNA. A systematic study performed on PolyG - PolyC sequence with varying number of intervening PolyA - PolyT pairs showed a decrease in electrical signal from 180 pA (PolyG - PolyC) to 30 pA with increasing number of the PolyA - PolyT pairs. This work also led to the development of ultrasensitive nanoelectrodes based on enzyme functionalized vertically aligned high density multiwalled CNTs for electrochemical detection of cholesterol. The nanoelectrodes exhibited selectively detection of cholesterol in the presence of common interferents found in human blood.

Effect of Hydrologic Restoration on the Habitat of The Cape Sable Seaside Sparrow, Annual Report of 2004-2005

The major activities in Year 3 on ‘Effect of hydrologic restoration on the habitat of the Cape Sable seaside sparrow (CSSS)’ included presentations, field work, data analysis, and report preparation. During this period, we made 4 presentations, two at the CSSS – fire planning workshops at Everglades National Park (ENP), one at the Society of Wetland Scientists’ meeting in Charleston, SC, and a fourth at the Marl Prairie/CSSS performance measure workshop at ENP. We started field work in the third week of January and continued till June 3, 2005. Early in the field season, we completed vegetation surveys along two transects, B and C (~15.1 km). During April and May, vegetation sampling was completed at 199 census sites, bringing to 608 the total number of CSSS census sites with quantitative vegetation data. We updated data sets from all three years, 2003-05, and analyzed them using cluster analysis and ordination as in previous two years. However, instead of weighted averaging, we used weighted-averaging partial least square regression (WA-PLS) model, as this method is considered an improvement over WA for inferring values of environmental variables from biological species composition. We also validated the predictive power of the WA-PLS regression model by applying it to a sub-set of 100 census sites for which hydroperiods were “known” from two sources, i.e., from elevations calculated from concurrent water depth measurements onsite and at nearby water level recorders, and from USGS digital elevation data. Additionally, we collected biomass samples at 88 census sites, and determined live and dead aboveground plant biomass. Using vegetation structure and biomass data from those sites, we developed a regression model that we used to predict aboveground biomass at all transects and census sites. Finally, biomass data was analyzed in relation to hydroperiod and fire frequency.

(Table 1) Individual codes, haplotype codes, GenBank accession numbers for DNA sequences and sample locality of the analysed Betamorpha fusiformis specimens from the ANDEEP III expedition

Based on our current knowledge about population genetics, phylogeography and speciation, we begin to understand that the deep sea harbours more species than suggested in the past. Deep-sea soft-sediment environment in particular hosts a diverse and highly endemic invertebrate fauna. Very little is known about evolutionary processes that generate this remarkable species richness, the genetic variability and spatial distribution of deep-sea animals. In this study, phylogeographic patterns and the genetic variability among eight populations of the abundant and widespread deep-sea isopod morphospecies Betamorpha fusiformis [Barnard, K.H., 1920. Contributions to the crustacean fauna of South Africa. 6. Further additions to the list of marine isopods. Annals of the South African Museum 17, 319-438] were examined. A fragment of the mitochondrial 16S rRNA gene of 50 specimens and the complete nuclear 18S rRNA gene of 7 specimens were sequenced. The molecular data reveal high levels of genetic variability of both genes between populations, giving evidence for distinct monophyletic groups of haplotypes with average p-distances ranging from 0.0470 to 0.1440 (d-distances: 0.0592-0.2850) of the 16S rDNA, and 18S rDNA p-distances ranging between 0.0032 and 0.0174 (d-distances: 0.0033-0.0195). Intermediate values are absent. Our results show that widely distributed benthic deep-sea organisms of a homogeneous phenotype can be differentiated into genetically highly divergent populations. Sympatry of some genotypes indicates the existence of cryptic speciation. Flocks of closely related but genetically distinct species probably exist in other widespread benthic deep-sea asellotes and other Peracarida. Based on existing data we hypothesize that many widespread morphospecies are complexes of cryptic biological species (patchwork hypothesis).

(Supplementary Material 2) Landmark coordinates of the analyzed specimens with associated metadata

The use of planktonic foraminifera in paleoceanographic studies relies on the assumption that morphospecies represent biological species with ecological preferences that are stable through time and space. However, genetic surveys unveiled a considerable level of diversity in most morphospecies of planktonic foraminifera. This diversity is significant for paleoceanographic applications because cryptic species were shown to display distinct ecological preferences that could potentially help refine paleoceanographic proxies. Subtle morphological differences between cryptic species of planktonic foraminifera have been reported, but so far their applicability within paleoceanographic studies remains largely unexplored. Here we show how information on genetic diversity can be transferred to paleoceanography using Globorotalia inflata as a case study. The two cryptic species of G. inflata are separated by the Brazil-Malvinas Confluence (BMC), a major oceanographic feature in the South Atlantic. Based on this observation, we developed a morphological model of cryptic species detection in core top material. The application of the cryptic species detection model to Holocene samples implies latitudinal oscillations in the position of the confluence that are largely consistent with reconstructions obtained from stable isotope data. We show that the occurrence of cryptic species in G. inflata, can be detected in the fossil record and used to trace the migration of the BMC. Since a similar degree of morphological separation as in G. inflata has been reported from other species of planktonic foraminifera, the approach presented in this study can potentially yield a wealth of new paleoceanographical proxies.

Microcantilever sensors for biochemical detection and analysis

Biochemical agents, including bacteria and toxins, are potentially dangerous and responsible for a wide variety of diseases. Reliable detection and characterization of small samples is necessary in order to reduce and eliminate their harmful consequences. Microcantilever sensors offer a potential alternative to the state of the art due to their small size, fast response time, and the ability to operate in air and liquid environments. At present, there are several technology limitations that inhibit application of microcantilever to biochemical detection and analysis, including difficulties in conducting temperature-sensitive experiments, material inadequacy resulting in insufficient cell capture, and poor selectivity of multiple analytes. This work aims to address several of these issues by introducing microcantilevers having integrated thermal functionality and by introducing nanocrystalline diamond as new material for microcantilevers. Microcantilevers are designed, fabricated, characterized, and used for capture and detection of cells and bacteria. The first microcantilever type described in this work is a silicon cantilever having highly uniform in-plane temperature distribution. The goal is to have 100 μm square uniformly heated area that can be used for thermal characterization of films as well as to conduct chemical reactions with small amounts of material. Fabricated cantilevers can reach above 300C while maintaining temperature uniformity of 2−4%. This is an improvement of over one order of magnitude over currently available cantilevers. The second microcantilever type is a doped single crystal silicon cantilever having a thin coating of ultrananocrystalline diamond (UNCD). The primary application of such a device is in biological testing, where diamond acts as a stable, electrically isolated reaction surface while silicon layer provides controlled heating with minimum variations in temperature. This work shows that composite cantilevers of this kind are an effective platform for temperature-sensitive biological experiments, such as heat lysing and polymerase chain reaction. The rapid heat-transfer of Si-UNCD cantilever compromised the membrane of NIH 3T3 fibroblast and lysed the cell nucleus within 30 seconds. Bacteria cells, Listeria monocytogenes V7, were shown to be captured with biotinylated heat-shock protein on UNCD surface and 90% of all viable cells exhibit membrane porosity due to high heat in 15 seconds. Lastly, a sensor made solely from UNCD diamond is fabricated with the intention of being used to detect the presence of biological species by means of an integrated piezoresistor or through frequency change monitoring. Since UNCD diamond has not been previously used in piezoresistive applications, temperature-denpendent piezoresistive coefficients and gage factors are determined first. The doped UNCD exhibits a significant piezoresistive effect with gauge factor of 7.53±0.32 and a piezoresistive coefficient of 8.12×10^−12 Pa^−1 at room temperature. The piezoresistive properties of UNCD are constant over the temperature range of 25−200C. 300 μm long cantilevers have the highest sensitivity of 0.186 m-Ohm/Ohm per μm of cantilever end deflection, which is approximately half that of similarly sized silicon cantilevers. UNCD cantilever arrays were fabricated consisting of four sixteen-cantilever arrays of length 20–90 μm in addition to an eight-cantilever array of length 120 μm. Laser doppler vibrometry (LDV) measured the cantilever resonant frequency, which ranged as 218 kHz−5.14 MHz in air and 73 kHz−3.68 MHz in water. The quality factor of the cantilever was 47−151 in air and 18−45 in water. The ability to measure frequencies of the cantilever arrays opens the possibility for detection of individual bacteria by monitoring frequency shift after cell capture.

Research, STEM Education, & Native American Programs in Montana

Oscillating electroosmotic flows in microchannels can be used as a separating mechanism for biological species. This MEMS related work specifically studies the separation of different sized DNA on the microscale as a pre-cursor to lab on a chip systems. In addition to this research, Dr. Thomas will present programs and activities that enable Native American students to obtain their STEM degrees. This involves working with middle school through graduate students at the 4 year institutions, Tribal colleges, and K-12 schools in Montana.

Branchial vascular pathways in two species of Tetraodontiformes and the concept of secondary vessels and nutrient arteries

The vascular organisation of the branchial basket was examined in two Tetraodontiform fishes; the three-barred porcupinefish, Dicotylichthys punctulatus and the banded toadfish, Marylina pleurosticta by scanning electron microscopy of vascular casts and standard histological approaches. In D. punctulatus, interarterial anastomoses (iaas) originated at high densities from the efferent filamental and branchial arteries, subsequently re-anastomosing to form progressively larger secondary vessels. Small branches of this system entered the filament body, where it was interspersed between the intrafilamental vessels. Large-bore secondary vessels ran parallel with the efferent branchial arteries, and were found to constitute an additional arterio-arterial pathway, in that these vessels exited the branchial basket in company with the mandibular, the carotid and the afferent and efferent branchial arteries, from where they gave rise to capillary beds after exit. Secondary vessels were not found to supply filament muscle; rather these tissues were supplied by single specialised vessels running in parallel between the efferent and afferent branchial arteries in both species examined. Although the branchial vascular anatomy was generally fairly similar for the two species examined, iaas were not found to originate from any branchial component in the banded toadfish, M. pleurosticta, which instead showed a moderate frequency of iaas on other vessels in the cephalic region. It is proposed that four independent vascular pathways may be present within the teleostean gill filament, the conventional arterio-arterial pathway across the respiratory lamellae; an arterio-arterial system of secondary vessels supplying the filament and non-branchial tissues; a system of vessels supplying the filament musculature; and the intrafilamental vessels (central venous sinus). The present study demonstrates that phylogenetic differences in the arrangement of the branchial vascular system occur between species of the same taxon.

Practical implementation of species' recovery plans - Lessons from the White-backed Woodpecker Action Plan in Sweden

Successful biodiversity conservation requires safeguarding viable populations of species. To work with this challenge Sweden has introduced a concept of Action Plans, which focus on the recovery of one or more species; while keeping in mind the philosophy of addressing ecosystems in a more comprehensive way, following the umbrella concept. In this paper we investigate the implementationprocess of the ActionPlanfor one umbrella species, the White-backed Woodpecker (WBW) Dendrocopos leucotos. We describe the plan's organisation and goals, and investigate its implementation and accomplishment of particular targets, based on interviewing and surveying the key actors. The achievement of the targets in 2005-2008 was on average much lower than planned, explained partially by the lack of knowledge/data, experienced workers, and administrative flexibility. Surprisingly, the perceived importance of particular conservation measures, the investment priority accorded to them, the money available and various practical obstacles all failed to kg? explain the target levels achieved. However qualitative data from both the interviews and the survey highlight possible implementation obstacles: competing interests with other conservation actions and the level of engagement of particular implementing actors. Therefore we suggest that for successful implementation of recovery plans, there is aneed for initial and inclusive scoping prior to embarking on the plan, where not only issues like ecological knowledge and practical resources are considered, but also possible conflicts and synergies with other conservation actions. An adaptive approach with regular review of the conservation process is essential, particularly in the case of such complex action plans as the one for the WBW.

Molecular data extend Australian Cricotopus midge (Chironomidae) species diversity, and provide a phylogenetic hypothesis for biogeography and freshwater monitoring

Resolving species relationships and confirming diagnostic morphological characters for insect clades that are highly plastic, and/or include morphologically cryptic species, is crucial for both academic and applied reasons. Within the true fly (Diptera) family Chironomidae, a most ubiquitous freshwater insect group, the genera CricotopusWulp, 1874 and ParatrichocladiusSantos-Abreu, 1918 have long been taxonomically confusing. Indeed, until recently the Australian fauna had been examined in just two unpublished theses: most species were known by informal manuscript names only, with no concept of relationships. Understanding species limits, and the associated ecology and evolution, is essential to address taxonomic sufficiency in biomonitoring surveys. Immature stages are collected routinely, but tolerance is generalized at the genus level, despite marked variation among species. Here, we explored this issue using a multilocus molecular phylogenetic approach, including the standard mitochondrial barcode region, and tested explicitly for phylogenetic signal in ecological tolerance of species. Additionally, we addressed biogeographical patterns by conducting Bayesian divergence time estimation. We sampled all but one of the now recognized Australian Cricotopus species and tested monophyly using representatives from other austral and Asian locations. Cricotopus is revealed as paraphyletic by the inclusion of a nested monophyletic Paratrichocladius, with in-group diversification beginning in the Eocene. Previous morphological species concepts are largely corroborated, but some additional cryptic diversity is revealed. No significant relationship was observed between the phylogenetic position of a species and its ecology, implying either that tolerance to deleterious environmental impacts is a convergent trait among many Cricotopus species or that sensitive and restricted taxa have diversified into more narrow niches from a widely tolerant ancestor.