Molecular and Cellular Biology Masters Theses Collection

Downregulation of Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Leads to Improved Biological Conversion Efficiency in Brachypodium distachyon

Trabucco, Gina M. — Fri, 23 Nov 2012 07:53:29 PST

Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. We identified candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon and developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C₃ plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1-silenced and wildtype plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4-downregulated plants exhibited a decrease in total lignin content, a significant reduction of guaiacyl lignin, and a modest reduction of syringyl lignin. Ethanol yield by microbial fermentation was enhanced in both BdCAD1- and BdCOMT4-downregulated plants. These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater biomass yield and bioconversion efficiency.

Investigation of Differential Vector Competence of Bartonella quintana in Human Head and Body Lice

previte, domenic j. — Fri, 23 Nov 2012 07:36:46 PST

Human head and body lice are obligatory hematophagous ectoparasites that belong to a single species Pediculus humanus. Only body lice, however, are vectors of the infections gram-negative bacteria Bartonella quintana. Due to their near identical genomes, yet differential vector competency, head and body lice provide an ideal model system to study an insects ability to gain or lose vector competency. Using our in vitro louse rearing system, we have infected both head and body lice with a blood containing B. quintana in order to detect differences in B. quintana proliferation between head and body lice as well as transcriptional regulation of immune-related genes. B. quintana proliferates rapidly in body lice after 6 days post-infection, but declines in head lice after 4 days post-infection, possibly explaining, in part, the differential vector competence between the two insects.

A transcriptome analysis using whole lice followed by qPCR verification of head and body lice immune-related genes was then conducted using uninfected, versus B. quintana infected lice to identify potential genes involved in vector competence. The immune-related genes Defensin 1, Fibrinogen-related protein and Spaetzle, were differentially regulated between head and body lice and were identified as potential targets for future research.

Previously studied immune-related genes, PGRP, Defensin 1 and Defensin 2 transcription levels were also assessed in body louse midgut using qPCR following B. quintana infection. In this case, B. quintana infection did not result in significant effects on the transcript levels of these genes in midgut tissue. Overall transcriptional profiles of head and body lice genomes were notably different, including difference in the expression of 18.3 % of immune related genes, a finding that strongly supports the contention that immune system differences between head and body lice are the primary reason for difference in vector capacity.

Polymer Prodrug Conjugation to Tumor Homing Mesenchymal Stem Cells

Panzarino, Nick — Fri, 23 Nov 2012 07:36:42 PST

Toxicity resulting from systemic administration continues to limit the effectiveness of modern chemotherapeutics. Administered drugs and biologics damage vital organs via off target effects while in circulation, decreasing the maximum tolerated dose and preventing the use of more effective drug concentrations. Many therapeutic agents are additionally constrained by a narrow therapeutic index, requiring delicate balance between toxicity and loss of efficacy. With dose remaining a critical determinant of efficacy, the inability to utilize increased drug concentrations due to toxicity prevents the full exploitation of the drug’s therapeutic effect.

While exploitation of the enhanced permeability and retention effect has been somewhat successful in targeting nanoparticles to tumors via passive accumulation, elevated tumor pressure and poor drug penetrance prevent a significant improvement in therapeutic effect.

Here we describe the conjugation of polymer prodrugs to tumor homing mesenchymal stem cells as a novel approach to chemotherapeutic drug delivery. The utilization of an actively homing cellular vehicle for effective drug delivery against the pressure gradient has the potential to improve drug penetration of the tumor while reducing systemic toxicity and improving drug efficacy.

RHYTHMIC GROWTH AND VASCULAR DEVELOPMENT IN BRACHYPODIUM DISTACHYON

Matos, Dominick A. — Fri, 23 Nov 2012 07:33:02 PST

Plants reduce inorganic carbon to synthesize biomass that is comprised of mostly polysaccharides and lignin. Growth is intricately regulated by external cues such as light, temperature, and water availability and internal cues including those generated by the circadian clock. While many aspects of polymer biosynthesis are known, their regulation and distribution within the stem are poorly understood. Plant biomass is perhaps the most abundant organic substance on Earth and can be used as feedstock for energy production. Various grass species are under development as energy crops yet several of their attributes make them challenging research subjects. Brachypodium distachyon has emerged as a grass model for food and energy crop research. I studied rhythmic growth, a phenomenon important to understanding how plant biomass accumulates through time, and vascular system development, which has biofuel feedstock conversion efficiency and yield. Growth rate changes within the course of a day in a sinusoidal fashion with a period of approximately 24 hours, a phenomenon known as rhythmic growth. Light and temperature cycles, and the circadian clock determine growth rate and the timing of rate changes. I examined the influences of these factors on growth patterns in B. distachyon using time-lapse photography. Temperature and, to a lesser extent, light influenced growth rate while the circadian clock had no noticeable effect. The vascular system transports important materials throughout the plant and consists of phloem, which conducts photosynthates, and xylem, which conducts water and nutrients. The cell walls of xylem elements and ground tissue sclerenchyma fibers are comprised of cellulose, hemicelluloses, and lignin. These components are important to alternative energy research since cellulose and hemicellulose can be converted to liquid fuel, but lignin is a significant inhibitor of this process. I investigated vascular development of B. distachyon by applying various histological stains to stems from three key developmental. My results described in detail internal stem anatomy and demonstrated that lignification continues after crystalline cellulose deposition ceases. A better understanding of growth cues and various anatomical and cell wall construction features of B. distachyon will further our understanding of plant biomass accumulation processes.

Analyzing the Role of Reactive Oxygen Species in Male-Female Interactions in Arabidopsis thaliana.

Johnson, Eric A. — Fri, 23 Nov 2012 07:23:20 PST

Fertilization, both in plants and animals, is at its core, a study of cell to cell communication. With respect to plants, the male gametophyte, the pollen tube, elongates within the female organ called the pistil, transporting in its cytoplasm two sperm cells. The pollen tube is attracted by signals secreted from the synergid cells that are located at the entrance to the female gametophyte that resides in the ovule. Secondary pollen tube visitors to the ovules are unwanted and repelled presumably by signals emitted by the fertilized female. The final communication between the pollen tube and female gametophyte is the induction of pollen tube rupture upon penetration of the synergid cell, an event that leads to the release of the two sperm cells, which go on to fertilize the central cell and egg cell within the female gametophyte, completing a double fertilization process that is unique to plants. My thesis research is centered on elucidating the mechanism behind the synergid cell-induced pollen tube rupture process. Studies in our laboratory have established that the synergid cell-expressed receptor like kinase, called FERONIA, mediates a highly oxidative environment in the female gematophyte that is necessary for the pollen tube rupture process. Using an in vitro pollen tube culture system, my research showed that reactive oxygen species (ROS) induces pollen tube rupture in a Ca^2+-dependent manner. My results suggests a careful and truly fascinating, though still hypothetical, design of a two molecule, FERONIA and ROS, two step activation system that uses ROS to prime the pollen tube outside the synergid cell, then expose it to calcium within the synergid cell to ensure that pollen tube rupture happens in the synergid cell, enabling fertilization.

The Pyrethroid Deltamethrin, Which Causes Choreoathetosis with Salivation (CS-Syndrome), Enhances Calcium Ion Influx via Phosphorylated CaV2.2 expresssed in Xenopus laevis oocytes

Alves, Anna-Maria — Thu, 12 Apr 2012 01:15:24 PDT

Pyrethroids are insecticides used since the 1970s. They are favored for their low mammalian toxicity, improved environmental stability and insecticidal potency. Voltage-gated sodium channels (VGSCs) are a known target but in vitro evidence indicates that voltage-gated calcium channels (VGCCs) are also targets. Site-directed mutagenesis of Ca_V2.2 (N-type), altering threonine 422 to glutamate (T422E), produces a mutant channel that acts as if permanently phosphorylated. Deltamethrin increases peak current of T422E Ca_V2.2 compared to its antagonistic action on wild type Ca_V2.2 when expressed in Xenopus oocytes. Phosphorylation of wild type Ca_V2.2 is evoked by the phorbol ester PMA by activating endogenous protein kinase C (PKC) in oocytes. Under steady-state conditions, deltamethrin increases transient peak current and slows deactivation kinetics of the PKC phosphorylated channel thereby increasing Ca²⁺ influx and neurotransmitter release. Conversely, deltamethrin treatment resulted in no effect on the deactivation kinetics of the unphosphorylated or T422E channels. Under voltage-dependent conditions, deltamethrin enhances peak current, and causes a hyperpolarizing shift in activation midpoint potential of the PKC phosphorylated channel which is consistent with enhanced Ca²⁺ influx. The hyperpolarizing shift of activation midpoint potential was not observed when deltamethrin was applied to the T422E mutant channels indicating that the other phosphorylation sites on Ca_V2.2 may be playing a role in the differential effects observed in the action of deltamethrin on the unphosphorylated channel, the T422E mutant and the PMA-activated PKC phosphorylation channel.

A Test of the Hypothesis That Environmental Chemicals Interfere With Thyroid Hormone Action in Human Placenta

Geromini, Katherine — Thu, 12 Apr 2012 01:14:41 PDT

Thyroid hormone is essential for normal brain development and recognition of this has led to universal screening of newborns for thyroid function to ensure that circulating levels of thyroid hormone are within a range known to be supportive of normal growth and mental development. Environmental chemicals that interfere with thyroid function are known to inhibit normal growth and mental development. Work from our lab and from labs internationally demonstrates in animal systems that some industrial chemicals such as PCBs, PBDEs, and others may interact with the thyroid hormone receptor(s) in ways that are not predicted by changes in serum thyroid hormone levels. Our work demonstrates that the enzyme CYP1A1 must metabolize some individual PCB congeners before they can interact with the thyroid receptor. In animals, this requirement appears to be manifested in part by a strong correlation between CYP1A1 and TH target gene expression. Here we present that this pattern extends to humans by demonstrating a correlation between increased CYP1A1 mRNA and an abundance of thyroid hormone responsive gene mRNA.

Connecting Motors and Membranes: A Quantitative Investigation of Dynein Pathway Components and in vitro Characterization of the NUM1 Coiled Coil Domain

St. Germain, Bryan J. — Mon, 21 Nov 2011 09:36:35 PST

In the budding yeast, Saccharomyces Cerevisiae, dynein, a minus-end directed motor, is involved in nuclear migration and proper orientation of the mitotic spindle during mitosis. Our lab has developed a model that involves the loading of cytoplasmic dynein onto the plus-end of astral microtubules through interactions with Pac1/LIS1 and Bik1/CLIP-170. Dynein is then delivered to the cell cortex and anchored through a cortical receptor protein, Num1. Num1 is a 313KDa protein that localizes to the cell cortex and is an essential component of dynein mediated nuclear migration.

Using quantitative fluorescence techniques I was able to create a molecular inventory of various dynein pathway components. Our results revealed Dyn1, dynein heavy chain, and Pac1/LIS1 associate at the plus end in a 1:1 ratio. Additionally we found that dynein and dynactin associate in a 3:1 ratio at the plus ends and a 2:1 ratio at the cortex. Interestingly, we found that over expression of Pac1/LIS1 augments cortical dynein activity while maintaining the dynein to dynactin ratio and this activity is separate from loss of She1, a negative regulator of dynein-dynactin interaction, which results in a 1:1 ratio of dynein-dynactin at the plus-ends, as well as, the cortex. Our results uncover molecular ratios that enable us to create more defined and detailed model of the dynein pathway.

To elucidate how Num1 attaches dynein to the cortex we created truncations of the Num1 protein. We were able to determine that two coiled-coil (CC) domains in the N-terminus of Num1 are responsible for bright foci formation on the cortex. Cells without these bright foci exhibit a binucleate phenotype similar to that of dyn1∆ implicating that these bright foci are required for the proper function of Num1 in the dynein pathway.

To test the hypothesis that the CC is capable of mediated bright patch assembly through self-association I purified a recombinant CC domain and performed gel filtration analysis, as well as, equilibrium sedimentation. I was able to determine that the CC domain exists as a dimer in solution. However, the mechanism of CC self-assembly may involve a requisite of targeting the CC to the cortex first.

Revealing the Localization of the Class I Formin Family in the Moss Physcomitrella patens Using Gene Targeting Strategies

Pattavina, Kelli — Mon, 21 Nov 2011 09:31:58 PST

Formin proteins, important regulators of a cell's actin cytoskeleton, nucleate actin polymerization and promote filament elongation. Actin dynamics are crucial for a form of polarized growth termed tip growth that is performed by cells involved in reproduction and nutrient uptake in plants. Uncovering the molecular basis of how actin associated proteins like formins control actin dynamics is important to gain a fundamental understanding of plant growth mechanisms. In the moss Physcomitrella patens, there are 9 formin genes that group into three distinct classes (I, II and III). From previous work, we suspect that class I formins may play a role in cytokinesis. Thus, I investigated how class I formins localize in tip-growing protonemal cells to gain further insight into their function. To do this, I tagged class I formins with GFP at the endogenous locus and visualized their subcellular localization using confocal microscopy. I found that Formin 1A, 1D, 1E and 1F localize to punctate spots on the plasma membrane and may concentrate at the cell plate during cell division, while 1B localizes to the cytosol. Overall, these data have shown that class I formins may play a role in cell division and potentially in the secretory pathway.

Examination of Sexually Dimorphic Cell Death in the Pubertal Mouse Brain

Holley, Amanda — Mon, 21 Nov 2011 09:26:43 PST

A period of cell death during a critical period early in life is responsible for causing permanent structural changes to many brain areas, but it is not known whether cell death plays a role in brain organization outside of early postnatal life. Puberty is considered a second sensitive period because the brain is the target organ of gonadal hormones. This study looked at global and regional patterns of cell death during pre-puberty and puberty in the mouse brain. My findings show there is more cell death happening during pre-puberty than during puberty. Cell death does happen during puberty but at adult levels. Furthermore females at P20 have more dying cells than males globally and in the hippocampus, but no other sex differences were observed. Knocking out the Bax gene, which is important for neuronal death, had only a modest effect on cell death during pre-puberty and puberty compared to what has been shown in younger ages. My findings demonstrate that prepubertal animals have more cell death than pubertal animals. Also, since Bax gene deletion only had a modest effect on cell death, cell populations other than neurons may be dying during these periods.