Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6480. Adviser: Carol A. Gross.

Thesis (Ph. D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2533. Advisers: Bradford W. Gibson; Francis Szoka.

Thesis (Ph. D.)--Indiana University, Dept. of Biology, 2007. / Title from dissertation home page (viewed Sept. 29, 2008). Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 1002. Adviser: Carl E. Bauer.

Thesis (Ph. D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-12, Section: B, page: 7300. Adviser: Jeffery S. Cox.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006. / Source: Dissertation Abstracts International, Volume: 67-04, Section: B, page: 1764. Adviser: Roger W. Innes. "Title from dissertation home page (viewed June 20, 2007)."

<p> <i>Aspergillus flavus</i> infection of agriculturally important crops such as tree nuts, maize, peanuts, and cotton has decreased crop value. Researchers have identified three major approaches to combat <i>A. flavus </i> growth and aflatoxin accumulation: identifying natural resistance in crops, genetically engineering crops for enhanced resistance, and introducing an atoxigenic fungal strain as a competitor. In this dissertation, I investigated two of the three means to control <i>A. flavus</i> growth and infection: genetically engineered crops and identification of natural resistance. My studies of natural resistance in cotton crop show that Sa 1595, a <i> Gossypium hirsutum</i> cultivar, is significantly more susceptible to <i> A. flavus</i> infection; however, no significantly resistant cultivars were observed, but I did observe a trend of diminished susceptibility in A2 186 and Tamcot Sp 23. I then examined synthetic antimicrobial peptide, D4E1, as a means to increase resistance in crops. My research shows that D4E1 effectively increases reactive oxygen species (ROS), an apoptosis precursor at concentrations as low as 1 &micro;M. Breaches in the membrane that allow infiltration and subsequent fluorescence from Sytox^&reg; green occur at higher concentrations. Finally, genetically engineered tobacco plants were examined for D4E1 localization. My research shows that the HA-D4E1 construct was present in the most abundance in the chloroplast of plastid transformed plants, while nuclear transformed plants had nuclear localization. All of my findings suggest that cotton crops do not exhibit any significant enhanced natural resistance to <i>A. flavus</i> infection and growth; however, engineering crops with D4E1 will exhibit enhanced crop resistance.</p><p>

The molecular mechanism of transmembrane signaling is unknown. Investigations have been hampered by the limits of current biophysical methods. Recently developed solid-state nuclear magnetic resonance (NMR) techniques provide a new approach for selective distance measurements probing structure and function of membrane proteins including ligand interactions. The environmental signal transduced by the serine receptor of bacterial chemotaxis is initiated by the attractant molecule serine. We initially demonstrated the feasibility of direct internuclear distance measurements between the 15N-amino labeled serine ligand and phenylalanine 13C-carbonyl labeled receptor. The two 4.0 ± 0.2 Å distances measured from the serine receptor to the ligand match the distances observed in the crystal structure of the ligand-binding domain fragment of the homologous aspartate receptor. Demonstration of the structural similarity between the aspartate receptor and serine receptor instigated further investigations into the mechanism of ligand specificity. To probe transmembrane signaling we developed a new constant time version of the rotational resonance solid-state NMR technique with improved reliability and efficiency. Combined with a site-directed strategy, this is a valuable and general tool for probing structure in large membrane proteins. A CO( i) to Cβ(i + 3) distance measurement along the periplasmic edge of the second membrane-spanning helix, provides a structural constraint for an unmapped region of the serine receptor. The 5.4 Å internuclear distance measured in the presence and in the absence of serine shows that any signaling mechanism must conserve the helical pitch of the second transmembrane domain. To test abundant and conflicting models of transmembrane signaling we measured an inter-helical distance across the dimer interface in the transmembrane region of the receptor. Computer modeling of this distance predicted sensitivity to proposed long-range ligand-induced conformational changes including piston, scissors, and rotational motions. Ile measured 5.0 Å distance provides a valuable structural constraint of tertiary structure. Both ligand-free and -bound signaling states of the receptor show the same inter-helical distance, suggesting that conformational changes are not propagated into the transmembrane domain. This approach provides a means for testing proposed mechanisms and mapping conformational changes involved in transmembrane signaling.

In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.   Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

Multiresistenta gramnegativa bakterier (MRGN) som Escherichia coli och Pseudomonas aeruginosa utgör ett globalt hälsoproblem och på grund av resistensutveckling hos bakterierna behövs nya behandlingsalternativ. Extended Spectrum β-Lactamase (ESBL) är vanligt förekommande hos MRGN och det finns olika typer av ESBL (exempelvis ESBLA och ESBLCARBA). Gemensamt är att det finns få behandlingsalternativ för ESBL-producerande MRGN. Syftet med studien var att undersöka effekten av potentiellt nya behandlingsalternativ mot MRGN i form av cykloserin och ceftazidim tillsammans med β-laktamasinhibitorn avibaktam. För att undersöka minimum inhibitory concentration (MIC) för cykloserin (CYK) mot E. coli (n=26) användes en egenutvecklad buljongspädningsmetod. Isolat av P. aeruginosa med och utan karbapenemasproduktion (n=25) undersöktes avseende MIC för ceftazidim/avibaktam (CZA) med två kommersiella buljongspädningsmetoder.  För CYK låg medianen för E. coli-stammarnas MIC-värden vid 32 mg/L (16 – 64 mg/L) vilket ligger kring det epidemiologiska cut-off värdet för Mycobacterium tuberculosis (32 mg/L), för vilken CYK idag används som behandling.  För CZA låg medianen för P. aeruginosa-stammarnas MIC-värden vid 8 mg/L (&lt;1 - ≥8 mg/L), och 40% (2/5) av de karbapenemasproducerande isolaten var känsliga enligt kliniska brytpunkter (S≤8 mg/L). Sammanfattningsvis visar studien att CYK har MIC-värden mot non-ESBL- och ESBL-producerande E. coli i nivå med andra patogener där preparatet används. Studien visar också att CZA kan ha effekt mot isolat med nedsatt känslighet mot meropenem eller ESBLCARBA-producerande isolat av P. aeruginosa, men isolaten måste resistensbestämmas innan behandling sätts in. / Multiresistant gramnegative bacteria (MRGN) like Escherichia coli and Pseudomonas aeruginosa, constitute a global health issue. Due to the resistance development among bacteria, new options for treatment are needed. Extended Spectrum β-Lactamase (ESBL) is common among MRGN, and there are different types of ESBLs (as ESBLA and ESBLCARBA). The increasing lack of treatment alternatives is mutual for the different ESBLs. The purpose of this study was to examine cycloserine and ceftazidime with the β-lactamase-inhibitor avibactam, two potentially new options for treatments effective against MRGN. To examine the minimum inhibitory concentration (MIC) for cycloserine (CYK) against E. coli (n=26) a in-house broth microdilution-method was used. Using two commercial broth microdilution-methods, isolates of P. aeruginosa with and without carbapenemaseproduction (n=23) were examined regarding MIC for ceftazidime/avibactam (CZA).  Regarding CYK, the median MIC-value of the E. coli-strains was 32 mg/L (16 - 64 mg/L), which is around the epidemiological cut-off-value (32 mg/L) for Mycobacterium tuberculosisfor which CYK is being used as treatment. The median of the MIC-values for CZA was 8 mg/L (&lt;1 - ≥8 mg/L) for all P. aeruginosa-strains and 40% (2/5) of the carbapenemaseproducing isolates were sensitive according to the clinical breakpoint (S≤8 mg/L). In summary, this study shows that CYK has MIC-values against non-ESBL- and ESBL-producing E. coli at the same level as other pathogens where CYK is being used. Further, CZA may have effect against the isolates with reduced susceptibility against meropenem and ESBLCARBA-producing P. aeruginosa, although the isolates need to be susceptibility-determined before treatment.