A preliminary investigation into the effect of different storage methods on the keeping quality of smoked Oreochromis niloticus

The study was carried out to asses the nutritional qualities of smoked O. niloticus and to discover the best methods of storage to minimize spoilage and infestation of smoked fish. Result showed that the protein contents in A and D decreased while the protein contents of b and C increased. The lipid content increased only in A while it decreased in B-C and D. The moisture content generally increased over the period of storage and there was an increase in ash content only in C while it decreased in A, B and D. The samples packed in polythene bag suffered about 35% mould infection and a few were attached by rodents with some fouling. Samples packed in jute bag were in good condition but were slightly attached by insect. All samples packed in carton and basket were still in good state but there were insect attack in those packed in carton

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

Effect of locally available spices on the organoleptic and storage periods of Heterotis niloticus

Investigation were carried out on the effect of some locally available species in the enhancement of the organoleptic quality and the storage periods of smoked Heterotis niloticus using Pprosopis africana as common smoke sources. Samples of fresh H. niloticus were bought, cut into chunks while extract juice from pepper, ginger rhizomes, garlic, onion bulb were used as sources of spices. Samples of fish were divided randomly into five (5) batches dipped into spice extract juices for 10 minutes drained and smoked with common firewood. Treatment without spice extract juice served as control. Each batch of fish was smoked for 7 hours on a drum-made smoking kiln products were individually packaged in polythene bag stored at room temperature and used for sensory evaluation and microbial analysis. Results of the sensory evaluation indicated that there was significant difference (P<0.005) for taste, appearance, colour and overall acceptance for the treatments. Ginger juice extract had the best overall acceptance. Similarly there was significant difference (P>0.05) in the microbial analysis. The garlic juice extract had the longest storage period with minimum total plate and mould count after 8 weeks

Coding for information storage

Storage systems are widely used and have played a crucial rule in both consumer and industrial products, for example, personal computers, data centers, and embedded systems. However, such system suffers from issues of cost, restricted-lifetime, and reliability with the emergence of new systems and devices, such as distributed storage and flash memory, respectively. Information theory, on the other hand, provides fundamental bounds and solutions to fully utilize resources such as data density, information I/O and network bandwidth. This thesis bridges these two topics, and proposes to solve challenges in data storage using a variety of coding techniques, so that storage becomes faster, more affordable, and more reliable.

We consider the system level and study the integration of RAID schemes and distributed storage. Erasure-correcting codes are the basis of the ubiquitous RAID schemes for storage systems, where disks correspond to symbols in the code and are located in a (distributed) network. Specifically, RAID schemes are based on MDS (maximum distance separable) array codes that enable optimal storage and efficient encoding and decoding algorithms. With r redundancy symbols an MDS code can sustain r erasures. For example, consider an MDS code that can correct two erasures. It is clear that when two symbols are erased, one needs to access and transmit all the remaining information to rebuild the erasures. However, an interesting and practical question is: What is the smallest fraction of information that one needs to access and transmit in order to correct a single erasure? In Part I we will show that the lower bound of 1/2 is achievable and that the result can be generalized to codes with arbitrary number of parities and optimal rebuilding.

We consider the device level and study coding and modulation techniques for emerging non-volatile memories such as flash memory. In particular, rank modulation is a novel data representation scheme proposed by Jiang et al. for multi-level flash memory cells, in which a set of n cells stores information in the permutation induced by the different charge levels of the individual cells. It eliminates the need for discrete cell levels, as well as overshoot errors, when programming cells. In order to decrease the decoding complexity, we propose two variations of this scheme in Part II: bounded rank modulation where only small sliding windows of cells are sorted to generated permutations, and partial rank modulation where only part of the n cells are used to represent data. We study limits on the capacity of bounded rank modulation and propose encoding and decoding algorithms. We show that overlaps between windows will increase capacity. We present Gray codes spanning all possible partial-rank states and using only ``push-to-the-top'' operations. These Gray codes turn out to solve an open combinatorial problem called universal cycle, which is a sequence of integers generating all possible partial permutations.

On erasure coding for distributed storage and streaming communications

The work presented in this thesis revolves around erasure correction coding, as applied to distributed data storage and real-time streaming communications.

First, we examine the problem of allocating a given storage budget over a set of nodes for maximum reliability. The objective is to find an allocation of the budget that maximizes the probability of successful recovery by a data collector accessing a random subset of the nodes. This optimization problem is challenging in general because of its combinatorial nature, despite its simple formulation. We study several variations of the problem, assuming different allocation models and access models, and determine the optimal allocation and the optimal symmetric allocation (in which all nonempty nodes store the same amount of data) for a variety of cases. Although the optimal allocation can have nonintuitive structure and can be difficult to find in general, our results suggest that, as a simple heuristic, reliable storage can be achieved by spreading the budget maximally over all nodes when the budget is large, and spreading it minimally over a few nodes when it is small. Coding would therefore be beneficial in the former case, while uncoded replication would suffice in the latter case.

Second, we study how distributed storage allocations affect the recovery delay in a mobile setting. Specifically, two recovery delay optimization problems are considered for a network of mobile storage nodes: the maximization of the probability of successful recovery by a given deadline, and the minimization of the expected recovery delay. We show that the first problem is closely related to the earlier allocation problem, and solve the second problem completely for the case of symmetric allocations. It turns out that the optimal allocations for the two problems can be quite different. In a simulation study, we evaluated the performance of a simple data dissemination and storage protocol for mobile delay-tolerant networks, and observed that the choice of allocation can have a significant impact on the recovery delay under a variety of scenarios.

Third, we consider a real-time streaming system where messages created at regular time intervals at a source are encoded for transmission to a receiver over a packet erasure link; the receiver must subsequently decode each message within a given delay from its creation time. For erasure models containing a limited number of erasures per coding window, per sliding window, and containing erasure bursts whose maximum length is sufficiently short or long, we show that a time-invariant intrasession code asymptotically achieves the maximum message size among all codes that allow decoding under all admissible erasure patterns. For the bursty erasure model, we also show that diagonally interleaved codes derived from specific systematic block codes are asymptotically optimal over all codes in certain cases. We also study an i.i.d. erasure model in which each transmitted packet is erased independently with the same probability; the objective is to maximize the decoding probability for a given message size. We derive an upper bound on the decoding probability for any time-invariant code, and show that the gap between this bound and the performance of a family of time-invariant intrasession codes is small when the message size and packet erasure probability are small. In a simulation study, these codes performed well against a family of random time-invariant convolutional codes under a number of scenarios.

Finally, we consider the joint problems of routing and caching for named data networking. We propose a backpressure-based policy that employs virtual interest packets to make routing and caching decisions. In a packet-level simulation, the proposed policy outperformed a basic protocol that combines shortest-path routing with least-recently-used (LRU) cache replacement.

Identification of fucose-α(1-2)-galactose binding proteins in the mammalian brain

Fucose-α(1-2)-galactose (Fucα(1-2)Gal) carbohydrates have been implicated in cognitive functions. However, the underlying molecular mechanisms that govern these processes are not well understood. While significant progress has been made toward identifying glycoconjugates bearing this carbohydrate epitope, a major challenge remains the discovery of interactions mediated by these sugars. Here, we employ the use of multivalent glycopolymers to enable the proteomic identification of weak affinity, low abundant Fucα(1-2)Gal-binding proteins (i.e. lectins) from the brain. End-biotinylated glycopolymers containing photoactivatable crosslinkers were used to capture and enrich potential Fucα(1-2)Gal-specific lectins from rat brain lysates. Candidate lectins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for one such candidate, SV2a, using a knock-out mouse system. Our results suggest an important role for this glycan-lectin interaction in facilitating synaptic changes necessary for neuronal communication. This study highlights the use of glycopolymer mimetics to discover novel lectins and identify functional interactions between fucosyl carbohydrates and lectins in the brain.

Direct visualization of the dynamics of membrane-anchor proteins in living cells

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane-anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane.

Engineering multi-step electron tunneling systems in proteins

Multi-step electron tunneling, or “hopping,” has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.

The protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan “wire.” The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Revealing, illuminating, and modifying proteins in human diseases using noncanonical amino acids

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Electrochemical and electron transfer investigations of copper proteins

A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

Evaluation of two alternative carbon capture and storage technologies

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The Get3 ATPase drives unidirectional targeting of tail-anchored membrane proteins

Efficient and accurate localization of membrane proteins is essential to all cells and requires a complex cascade of interactions between protein machineries. This is exemplified in the recently discovered Guided Entry of Tail-anchored protein pathway, in which the central targeting factor Get3 must sequentially interact with three distinct binding partners (Get4, Get1 and Get2) to ensure the targeted delivery of Tail-anchored proteins to the endoplasmic reticulum membrane. To understand the molecular and energetic principles that provide the vectorial driving force of these interactions, we used a quantitative fluorescence approach combined with mechanistic enzymology to monitor the effector interactions of Get3 at each stage of Tail-anchored protein targeting. We show that nucleotide and membrane protein substrate generate a gradient of interaction energies that drive the cyclic and ordered transit of Get3 from Get4 to Get2 and lastly to Get1. These data also define how the Get3/Tail-anchored complex is captured, handed over, and disassembled by the Get1/2 receptor at the membrane, and reveal a novel role for Get4/5 in recycling Get3 from the endoplasmic reticulum membrane at the end of the targeting reaction. These results provide general insights into how complex cascades of protein interactions are coordinated and coupled to energy inputs in biological systems.