Seminars

• Semianr: Chagas Disease: Many problems, but are there solutions?
  Rick L. Tarleton, Ph.D.
  Center for Tropical and Emerging Global Diseases
  University of Georgia, Athens, GA
  (December 8, 2006)
  
  Recently Chagas disease has received some renewed attention as the “most neglected of the neglected diseases”. This presentation will outline some of the roadblocks to making progress in Chagas disease. In brief, mechanisms to control transmission of Trypanosoma cruzi infection are quite limited and are failing, effective drugs to treat the infection are few and have substantial side-effects, and diagnosis is relatively difficult. Drawing upon data from both experimental animals and human populations, potential solutions to some of these problems will be discussed.
  
  
• Seminar: The rodent malaria parasite Plasmodium berghei: a malaria model for all seasons?
  Andrew P. Waters, PhD,
  Leiden University Medical Centre
  (January 26, 2006)
  
  Gametocytes, the precursor cells of malaria parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. Substantial proteome coverage for both male and female gametocytes has been generated and confirmed the highly differentiated nature of gametocytes from other parasite forms and the sexes from each other. Subsequent analyses of two sex specific kinases demonstrated the existence of sex-specific regulatory pathways. One clear lesson from analysis of the proteomes is that the gametocytes contain much of the protein and signalling networks necessary both to respond to external stimuli and implement the early (sex specific) developmental programmes necessary not only for gamete formation but also for subsequent development of the zygote and the ookinete. Amongst the categories of proteins enriched in gametocytes are the RNA binding proteins (RBP). We have initiated a systematic analysis of the functional role of these proteins in gametocyte development, the RNA elements to which RBP bind, the mRNA species that bind such proteins and the function of the proteins encoded by mRNA bound by stage specific RBP. Data will be presented that demonstrated the central role of RBP-containing protein complexes in the homeostasis of subsets of gametocyte mRNA populations. Furthermore the RNA elements responsible for such interactions are identified and characterised. The therapeutic possibilities of a thorough understanding of gametocyte mRNA homeostasis will be discussed.
  
  
• Seminar: Persistence of Helicobacter pylori in the human gastric mucosa
  Martin J. Blaser, MD,
  University of New York
  (December 15, 2005).
  
  
• Seminar: Neisseria gonorrhoeae pili and porin cooperatively induce Ca2+ transients during infection
  Blanca P. Ayala, PhD,
  Oregon Health & Sciences University
  (November 18, 2005)
  
  Purified pili and porin from Neisseria quickly mobilize calcium (Ca2+) stores in monocytes and epithelial cells, ultimately influencing host cell viability as well as bacterial intracellular survival [Kallstrom et al. (1998) J. Biol. Chem. 273,21777-21782; Muller et al. (1999) EMBO J. 18, 339-352; Ayala et al. (2001) Cell. Microbiol. 3, 265-275; Ayala et al. (2002) Infect. Immun. 70, 5965-5971]. We have examined the Ca2+ transients induced in human epithelial cells during infection by live, piliated N. gonorrhoeae. Porin induced an influx of Ca2+ from the extracellular medium less than 60 seconds post-infection. The porin-induced transient is followed by a pilus-induced release of Ca2+ from intracellular stores. The timing of these events is similar to that observed using purified proteins. Interestingly, the porin-induced Ca2+ flux is required for the pilus-induced transient, indicating that the pilus-induced Ca2+ release is, itself, Ca2+ dependent. Several lines of evidence indicate that porin is present on pili. Moreover, pilus retraction strongly influences the porin- and pilus-induced Ca2+ fluxes. These and other results, strongly suggest that the pilus and porin cooperate to modulate calcium signaling in epithelial cells, and propose a model to explain how N. gonorrhoeae triggers Ca2+ transients in the initial stages of pilus-mediated attachment.
  
  
• Seminar: Functional Studies of Trypanosoma brucei Purine Transporters
  Marco Sanchez, PhD,
  Oregon Health & Sciences University
  (November 16, 2005)
  
  Purine transporters play a primary role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purines de novo and must salvage them from their hosts. Moreover, these transporters have been implicated in the permeation of antiparasitic drugs and experimental drugs in these and other parasites. The molecular and functional analyses of all purine transporters from T. brucei reveals that purine transport in this parasite is much more complex than studies using intact parasites suggested. This underscores the importance of molecular studies of individual transporters.
  
  
• Seminar: Orquídeas y Hongos: Belleza y Decepción
  Paul Bayman, PhD,
  University of Puerto Rico,
  Rio Piedras campus
  (November 1, 2005)
  
  Dr. Paul Bayman discussed the combination of phylogenetic studies of orchid mycorrhizal fungi with functional tests of the interaction.
  
  
• Seminar: Changing substrate specificity by remodeling domain interfaces in Glutathione Transferases
  Hugh Nicholas, Jr., PhD,
  Pittsburg Supercomputing Center
  (August 19, 2005.)
  
  I present the analysis of the major isozyme subfamilies of the cytoplasmic Glutathione Transferases that predicts the amino acids responsible for reorienting the glutathione binding domain and the substrate binding domain relative to each other. This reorientation allows different isozyme groups to accommodate different categories of substrates. The analysis uses a symmetric cross entropy measure to identify the residues within each isozyme group that are most diagnostic of group membership. These residues are, in effect, a distributed sequence motif that serves to uniquely identify the isozyme family to which a sequence belongs and hence greatly reduces the ambiguity in determining wether homologous sequences are paralogs or orthologs. The cross entropy analysis is integrated with other analyses to create a comprehensive picture of the evolution of the cytoplasmic Glutathione Transferase isozyme families and their adaptation to binding new classes of substrates. The cytoplasmic Glutathione Tranferases are medically important in eliminating drugs and chemotherapeutic agents from the body and play an important role in resistance to drug or xenobiotics in other species.
  
  
• Seminar: Do bacterial species exist? Genomic, phylogenetic, and populational perspectives
  Pablo Vinuesa, PhD,
  Universidad Nacional Autónoma de México
  (April 15, 2005)
  
  
• Seminar: BioPlex™ Suspension Array System Multiplexing Cytokine and Phosphoprotein Arrays Using xMAP Bead Technology
  Frank Bizouarn,
  Bio-Plex™ Application Specialist
  (April 20th, 2005)
  
  The Bio-Plex™ System allows you to profile up to 17 human, 18 mouse or 9 rat cytokines from a 12 ml sample in less than 2 hours with outstanding sensitivity and dynamic range. No more ELISAs or Western Blots are needed. Multiplex phosphoproteins for cell signaling studies. Antibodies exclusively developed and validated by Cell Signaling Technology, Inc.
  
  
• Seminar: Microevolution and virulence of Dengue viruses
  Rebeca Rico-Hesse, PhD, MPH,
  Scientist,
  Southwest Foundation for Biomedical Research,
  (April 11, 2005)
  
  Dr. Rebeca Rico discussed the evolution of dengue viruses using phylogenetic and epidemiologic analysis.
  
  
• Seminar: Immune Control and Disease Development in Chagas Disease
  Rick L. Tarleton, PhD,
  Center for Tropical & Emerging Global Diseases
  University of Georgia
  (March 14, 2005)
  
  Immune control of Trypanosoma cruzi, the causative agent of Chagas disease, involves multiple immune mechanisms, including the generation of potent cytolytic T cell responses by CD8+ T cells. Capitalizing on the recent sequencing of the T. cruzi genome, we have conducted a whole organism proteome analysis of T. cruzi. Using genome and proteome data, and algorithms for the prediction of epitopes capable of binding to class I MHC molecules, we have conducted a broad search for the primary targets of CD8+ T cells in T. cruzi infection. This analysis reveals that the trans-sialidase family of genes encodes the major targets of CD8+ T cell responses. In one case, more than 20% of the entire CD8+ T cell population in mice during the acute phase of T. cruzi infection are specific for a single trans-sialidase peptide. In contrast, screening of epitopes from other genes and gene families failed to reveal additional epitopes outside of the trans-sialidase family. Trans-sialidase encoded peptides are also major targets of CD8+ T cells in human chagasic patients. These results demonstrate the utility of genome and proteome data, combined with computation and experimental tools, for the identification of immune system targets and potential vaccine candidates in complex parasitic infections.
  
  
• Seminar: Physical and functional connections between RNA polymerase II and the pre-messenger RNA splicing machinery
  Mariano García-Blanco, PhD,
  Duke University
  (January 18, 2005)
  
  Human and viral genes are complex and tightly regulated genetic units of information. The products of these genes, the nascent transcripts contain coding information in short exons, which are identified and ligated together in the process known as pre-messenger RNA splicing. The complex nature of genes provides for versatility of expression because one gene can encode for many proteins by altering the selection of exons to be included in the messenger. This process is known as alternative splicing and it is the major engine of proteome diversity in humans. Alternative splicing is tightly regulated in normal development and is deregulated in disease. Although the importance of connections between RNA processing and synthesis (transcription) has been appreciated for some time, the molecular nature of these connections remains unresolved. The laboratory investigates these connections, physical and functional, between RNA polymerase II transcript elongation and alternative splicing. This work is focused on a protein found to be a repressor of HIV-1 transcription elongation, CA150. CA150 appears to physically and functionally link the elongating RNAP II complex with the splicing machinery. We will discuss the data that supports these statements and will also show results that indicated that CA150 can alter alternative splicing decisions. We have also developed an in vitro system that recapitulates the connections observed in vivo and we believe this system will instrumental in unraveling the molecular underpinnings of the functional coupling. We will present results obtained with this system. We will also present a model of exon definition that takes into account the functional coupling of transcription and splicing.
  
  
• Seminar: Caracterización de la respuesta de expresión genética en las células hospederas del virus de Dengue
  Dr. Irene Bosch
  Assistant Professor
  Center for Infectious Disease and Vaccine Research
  University of Massachussets Medical School
  (September 29, 2004)
  
  
• Seminar: Lost in Translation: Novel mechanisms of translation inhibition and induction of apoptosis by Reaper and viral Reaper-like proteins
  Dr. Daniel A. Colón Ramos
  Post Doctoral Fellow
  Stanford University
  (September 3, 2004)
  Reaper, a Drosophila apoptotic regulator, induces programmed cell death, at least in part, by inhibiting the activity of Inhibitor of Apoptosis (IAP) proteins. We have previously shown that Reaper can decrease IAP levels by accelerating IAP autoubiquitination and degradation, and by inhibiting general protein synthesis. This previously unrecognized ability of Reaper to repress total protein translation resides in a motif shared with a family of bunyaviral translation inhibitors, the NSs proteins. We now report the molecular mechanism by which Reaper and NSs proteins inhibit general protein synthesis. Analysis of polysome sucrose gradients shows that Reaper affects the assembly of ribosomal subunits onto the mRNA, suggesting that Reaper inhibits translational initiation. Biochemical purification of the inhibited activity showed that purified initiation factors were insufficient to rescue Reaper translational shut-off. Instead, only purified ribosomes were capable of reversing Reaper's inhibitory activity. Interestingly, both Reaper and NSs bind directly and with high affinity to the 40S ribosomal subunit, specifically to the 40S platform, allosterically affecting its interaction with the 60S subunit and inducing accumulation of 48S complexes (40S bound to mRNA). Toeprinting analysis revealed that the 48S complexes are not stalled at the AUG start codon. Instead, Reaper appears to be inducing 48S complex leaky scanning, with leads to pausing of the 48S at cryptic downstream AUG sites. Therefore, Reaper and NSs might bind to the 40S complex, induce a novel protein expression program through leaky scanning and thereby affect cell fate.
  
  
• Seminar: MACS® Technology: Novel applications for isolation and analysis of cells and biomolecules
  Eleanor C. Brown
  Technical Consultant
  Miltenyi Biotec
  (March 17, 2004)
  Since its introduction in 1989, MACS® Technology is now the gold standard for the separation of cells, in cell research and therapy. Today, MACS® Technology stands for innovative products designed for cell enumeration and analysis, and for the new approaches in isolation and analysis of biomolecules such as DNA, mRNA, and proteins. Miltenyi Biotec's MicroBeads, approximately 50nm in size, are coupled to highly specific monoclonal antibodies, which exhibit significantly higher binding capacity and faster binding kinetics. The MACS® products include a variety of antibody-conjugated MicroBeads targeting many human, mouse, non-human primate cell surface markers, and cytokine secreting antigen-specific T-cells. New antibodies against blood lymphoid and myeloid dendritic cell subsets also allow for novel DC biology research. Join us to find out more about the consistent cell/molecular populations you can isolate obtaining optimal purity and recovery.
  
  
• Seminar: Control without clearance: CD8+ T cell responses in Trypanosoma cruzi infection
  Rick L. Tarleton, Ph.D.
  Distinguished Research Professor
  Center for Tropical & Emerging Global Diseases
  University of Georgia
  (March 8, 2004)
  CD8+ T cells are crucial to the control of T. cruzi infection, presumably as a result of this effector mechanism to detect and facilitate the destruction of parasite-infected host cells. However despite the combined effect of CD8+ T cells and other immune mechanisms, T. cruzi infection appears to be completely cleared only very rarely and results in debilitating disease in a significant number of infected individuals. This seminar will present data on the development, maintenance and target specificity of CD8+ T cells in T. cruzi infection and will address possible reasons why T. cruzi is able to persist despite this and other immune responses.
  
  
• Seminar: The Trichomonas vaginalis Genome Sequence Project at TIGR
  Jane Carlton, Ph.D.
  The Institute for Genomic Research
  (February 12, 2004)
  The genome of the sexually transmitted human pathogen Trichomonas vaginalis is being sequenced at TIGR. T. vaginalis is the most common non-viral sexually transmitted parasite, responsible for approximately 5 million cases of trichomoniasis annually in the United States. In addition to its prevalence, infection with T. vaginalis is emerging as one of the most important cofactors in amplifying HIV transmission, particularly in African-American communities in the US. From the point of view of basic biology, the trichomonads are of particular interest for a variety of reasons not the least of which is their relationship to other luminal parasites such as Entamoeba histolytica and Giardia lamblia and their position at the base of the eukaryotic tree. The genome sequence is currently at 5X coverage, and has already produced a number of surprising results, such as the presence of a large number of identical repeats in the genome, an abundance of transposable elements and a much larger genome size than originally predicted. Given the large evolutionary distance between the human host and this pathogen, the genome sequence of the protist is likely to reveal a number of candidate genes encoding potential chemotherapeutic and vaccine targets specific to the parasite. Details concerning the genome project will be presented, in addition to a description of the studies indicating an increased incidence of HIV transmission in T. vaginalis-infected individuals.
  
  
• Seminar and Workshop: Nikon E800 Fluorescence Microscope: Capabilities and Training
  Danilo Rosado
  Micron Optics
  (October 23-24, 2003)
  
  
• Seminar: Exploring the Proteome: Tools and Technologies
  Christina Pavlick, Ph.D.
  Bio Rad Laboratories
  (June 2003)
  
  
• Seminar/Workshop: Proteomics and Real Time PCR
  Scott Moore
  Bio Rad Laboratories, Life Science Group
  (June 24-25, 2003)
  Proteomics spans the genomic-wide analysis of proteins at the levels of expression, isolation and characterization in order to gain understanding of function within the context the living networks. Some of the compelling reasons for approaching protein analysis at this level include: a) less than 50% correlation between mRNA and protein levels, b) alternative mRNA splicing, c) posttranslational modifications events, and d) broad differences in protein half lives. The complexity of this type of analysis requires multidisciplinary tools and instrumentation, not required in research projects using the classical approach "one protein at a time" analysis.
  
  
• Seminar: Genome Sequencing of Human Malaria parasite Plasmodium falciparum
  Malcom Gardner, Ph.D.
  The Institute of Genomic Research (TIGR)
  (April 2003)
  
  
• Seminar: Broadened Immune Responses in Humans Immunized Sequentially with Malaria DNA and Recombinant Protein
  Ruobing Wang, Ph.D.
  The Institute of Genomic Research
  (April 2003)
  
  
• Seminar: Chimerivax TM-WN: Safety and Efficacy of a Live-Attenuated Yellow Fever/West Nile Virus Chimeric Vaccine
  Juan Arroyo, Ph.D.
  Acambis, Inc., Boston, MA
  (March 2003)
  The 17D vaccine strain of yellow fever (YF) virus was used to produce a chimeric live virus containing the capsid and non structural genes of YF and the envelope genes (prME) of West Nile virus. We used the same approach to produce YF/Japanese encephalitis (JE) and dengue vaccines, both in clinical trials, with phase III studies for ChimeriVax -JE initiating later this year. The yellow fever/West Nile chimera containing wild type (wt) NY99 strain prM and E proteins did not cause encephalitis in young mice after peripheral inoculation in contrast to the parent WN NY99 strain; it was also less neurovirulent than commercial YF 17D vaccine (or YF-VAX®). The amino acid sequence comparison of the YF/JE and YF/WN envelope (E) proteins defined sites (designated F, V, and R) for further attenuation of the YF/WN virus. In a variety of animal models, including mice, hamsters, horses, and monkeys, the vaccine proved to be safe and immunogenic and/or protective. Point mutations in the E protein effectively reduced the neurovirolence of the YF/WNwt chimera. Mice given a single subcutaneous inoculation of graded doses of YF/WNwt or the triple mutant YF/WNFVR developed high levels of neutralizing antibodies and were protected against a 100 LD50 challenge with virulent WN NY99. Rhesus monkeys vaccinated with a single dose of vaccine constructs containing single F, double VR, or triple FVR mutation developed viremias similar to YF-VAX® in duration but significantly lower in magnitude. The lower viscerotropism (or viremia) supports the safety profile of the vaccine constructs. Post vaccination antibody titers were measured in these rhesus monkeys by PRNT50 and titers average above 40 at day 15 and 380 at day 30. Challenge by IC inoculation with WN NY99 showed 100% efficacy with no clinical symptoms or detectable viremia. In separate safety studies, the neurovirolence profile in monkeys did not exceed that of YF-VAX®. During vector transmission studies, Culex and Aedes mosquitoes showed no replication of YF/WN virus after oral exposure. ChimeriVax TM-WN is a promising single dose, safe and immunogenic live attenuated vaccine against West Nile. A vaccine lot was manufactured for Phase I/II clinical trials initiating in May 2003 to asses safety and dose range responses.
  
  
• Seminar: The F1 Filamentous Bacteriophage: It's Structure, Lifecycle and Uses as a Biological Tool
  Rafael Tosado Acevedo, Ph.D.
  Duke University Medical Center, Department of Biochemistry
  (November 2002)
  The f1 bacteriophage is a member of the F1 class of filamentous bacteriophages that infect E. coli strains containing the F conjugative plasmid. The bacteriophage contains a circular single-stranded intermediate. The sinlge-stranded DNA is encapsulated by a cylindrical capsid composed of phage-encoded proteins that form a phage particle of approximately 6 nm in diameter and 900 nm in length. The f1 genome encodes 11 genes products. Three of these products are required for DNA replication, five comprise the bacteriophage capsid, and three are involved in encapsulating the newly-synthesized DNA. Assembly is a membrane-associated event in which the capsid proteins are assembled around the DNA as it is extruded through the membrane. The assembly process does not kill a bacterial host and results in titers of up to 1012 phage particles per ml. The structure together with its life cycle has allowed the f1 bacteriophage to be used in molecular biology and protein chemistry research. The ease of isolating the single-stranded genome and the double replicative intermediate has allowed the formation of substrates to study DNA repair and replication. The lack of constraint on the size of the single-stranded DNA that is encapsulated has made it a valuable cloning vehicle. Its structure and mode of assembly has been exploited to display foreign peptides and proteins on its surface. The structure and life cycle of the bacteriophage will be discussed in relation to its use as a biological tool.
  
  
• Seminar: A Chimeric Dengue/West Nile Vaccine
  Robert J. Putnak, Ph.D.
  Walter Reed Army Institute of Research
  (October 2002)
  Brief overview of the dengue virus research ongoing in the Department of Virus Diseases, WRAIR, to include vaccine development efforts and the effort to re-establish a human challenge model for dengue. The remainder of the presentation will concentrate on a collaborative research effort with the U.S. National Institute of Health, National Institute of Allergy and Infectious Diseases, to develop a chimeric West Nile vaccine using live -attenuated chimeric West Nile/Dengue -4 virus .
  
  
• Seminar and Workshop: Real Time PCR: iCycler iQ Detection System
  Frank Bizouarn, Field Application Specialist
  Bio Rad Laboratories
  (September 2002)
  
  
• Seminar: Experience the Proteome: A Practical Application
  Christina Pavlick, Ph.D.
  Proteomics Field Application Specialist, Bio Rad Laboratories
  (September 2002)
  
  
• Seminar: Alternative Splicing of Messenger RNA's, a Potent Engine of Viral and Cellular Proteome Diversity
  Mariano García Blanco, M.D., PhD.
  Duke University Medical Center, Department of Molecular Genetics and Microbiology
  (August 27-28, 2002)
  
  
• Seminar: Regulation of CD8+ T cell Responses: Role of CD4+T cells
  Fidel Zavala, M.D.
  New York University, School of Medicine
  (June 3, 2002)
  CD8+ T cells play a major role in immunity against intracellular pathogens such as virus, bacteria, and parasites. These cells recognize microbial antigens in the surface if infected cells and quickly develop effector mechanism to eliminate the infection. This resulting CD8+ T cell response is the result of a process which is strongly regulated by antigen presentation limiting the number of T cells that become activated while other mechanisms -mediated by CD4+ T cells- provide signals that promote the proliferation of activated CD8+ T cells and/or prevent their death.