Former Trainees

Zahra Peg

Project Title

Binding studies of sialic acid containing glycans and sialic acid binding proteins using microarray technology

Project Description

My projects focus on studying interactions between sialic acids containing glycans and their binding proteins by glycan microarray, as well as roles of these interactions in human health and disease processes. Sialic acid-recognizing proteins from novel plants, vertebrates and microbes are obtained from various sources, including collaborations with colleagues from UCSF, University at Buffalo and several other universities in the world. One class of these proteins, the serine-rich repeat (SRR) glycoproteins, for example, are a diverse family of adhesins found in Gram-positive bacteria. My other two major projects are related to two new anti-glycan antibodies found in human. I am investigating their evolution and effect in human health.

Techniques

HPLC, glycan microarray printing and screening, binding assays, ELISA, Tissue culture, flow cytometry, other basic molecular biology techniques.

Headshot of Frederico da Silva

Project Title

Dietary intake of non-human sialic acid Neu5Gc and anti-Neu5Gc antibodies as human unique inflammatory mechanism for the progression of colorectal cancer.

Project Description

Dietary factors have been implicated as important sources of modifiable risk for cancer. Among such factors much evidence links red meat consumption to increased risk of various cancers, especially colon cancer. I am interested in studying the human specific mechanism involving the consumption of a non-human sialic acid Neu5Gc, anti-Neu5Gc antibodies and inflammation in tumorogenesis and progression of colorectal cancer. I am also interested to explore ways in which the elimination of Neu5Gc from human tissues could be exploited as prevention or therapeutic target for cancer.

Techniques

qRT-PCR, Immunofluorescence, Cell proliferation, Western Blot, Flow Cytometry, In vivo studies in mice, Histology of tumor tissues, DMB-HPLC.

Project Title

Microbial Glycan Mimicry and Glycosidases in Inflammatory Responses

Project Description

This project seeks to understand what happens when the glycobiological homeostasis of the mammalian host is perturbed by exogenous factors. A loss-of-function and gain-of-function analyses will be adopted on both sides of the host-pathogen equation. This project pursues a central hypothesis: Group A Streptococcus (GAS) causes serious infections because it has evolved to display its own sugars or modify the sugars of the host in a fashion that mimics or interferes with host glycan-based regulation of innate immunity and inflammation. Specifically, the innate immune and inflammatory functions of phagocytic cells (neutrophils and macrophages) upon challenge with GAS strains that alternatively (a) display a glycan (hyaluronic acid, HA) that mimics common host carboydrates by interacting with the cognate receptor on myeloid cells, CD44 or (b) produces a glycosidase (hyaluronate lyase, HylA) that can target (cleave) the same host carbohydrate, will be investigated. Techniques:Molecular cloning and bacterial genetic manipulation, recombinant protein purification, in vivo mice studies (immunization, infection, blood and organ isolation), ELISA, Western blot, flow cytometry, confocal microscopy, enzyme assays, neutrophil and opsonophagocytosis assays.

Chih-Ming

Project Title

Immunoregulatory Siglecs modulate Inflammasome Activation in Macrophages

Project Description

Pathogenic microorganisms are sensed by the inflammasome, resulting in releasing pro-inflammatory cytokines. Siglecs have been shown to have roles in regulating the immune system. However, their interaction with the inflammasome has not been characterized. My project aims to how siglecs regulate inflammasome activation and how bacterial or exogenous ligands to interact with siglecs to modulate siglecs signaling.

Techniques

Primary cell culture, bacterial infection, western blot, ELISA, PCR, flow cytometry.

Ann Lin Headshot

  • Ann Lin, PhD

Project Title

Identifying the role of human milk oligosaccharides and Tamm-Horsfall glycoprotein during urinary tract infection

Project Description

Urinary tract infection (UTI) is one of the most common and recurrent infection affecting 150 million people worldwide. The first part of my studies involves understanding the role of human milk oligosaccharides (HMO) during UTI. HMOs are comprised of a diverse collection of complex oligosaccharides that are known to protect hosts against microbial infections. The goal is to identify the mechanisms used by HMO to elicit protection in human bladder epithelial cells against Uropathogenic E. coli (UPEC) and Group B Streptococcus infections. The second part of my studies involves characterizing the functions of Tamm-Horsfall glycoprotein (THP), the most abundant protein found in the urine. Lack of THP has been associated with severe bacteriuia, cystitis and pyelonephritis. Using genetic tools in combination with animal models, my goal is to elucidate the importance of THP during UTIs. Techniques:Bacteria infections (adhesion/invasion assays), tissue culture, primary macrophage isolation, in vivo mouse models (ie. oral, subcutaneous injection, blood and organ isolation), IHC/IF microscopy, enzyme assays, real-time qPCR, ELISA, Western blot, immunoprecipitation, protein isolations, molecular cloning, bacterial genetic manipulations, RNA interference, mammalian cell transfections.

Project Title

Role of Siglecs in inflammatory responses

Project Description

Sialic acid-recognizing immunoglobulin-like lectins (Siglecs) are cell surface receptors that bind sialic acids on a variety of glycoconjugates. My project focuses on the characterization of two potential “paired receptors”, Siglec-11 and Siglec-16, which possess nearly identical extracellular domains, while their intracellular domains are capable of inducing opposing signals (inhibitory or activating). We aim to define physiological roles of these two receptors in inflammatory states. As a long term goal, we would like to use transgenic mice as models to address the general role of activating and inhibitory Siglecs in modulating the “set-state” of the innate immune response.
Techniques:cloning, cell culture, flow cytometry, ELISA, immunoblot, protein purification, in vivo studies in mice.

  • Nina N. Schommer, PhD

Project Title

Role of Hyaluronidase in host-pathogen recognition events

Project Description

Our project focuses on the role of Hyaluronan in host-pathogen recognition events in the skin. The pathogen Group A Streptococcus (GAS) expresses a HA capsule in a form of molecular mimicry that impairs immune recognition and phagocytic clearance. It is unclear how the interplay of host HA and bacterial HA may impact GAS virulence potential. To understand the respective roles of host and bacterial HA during GAS skin infections, we established a mouse model to conditionally overexpress mammalian hyaluronidase-1.Collaboration with V. Nizet
Techniques:in vivo studies in mice, host-pathogen adherence assays, microscopy, biomolecular techniques.

  • Pranitha Kamat, PhD

Project Title

A mechanistic and pharmacologic evaluation of potential attenuators of chondrogenesis in the context of multiple hereditary exostoses

Project Description

I am working on identifying compounds that can attenuate a disorder called multiple hereditary exostoses (MHE). This disease condition is characterized by cartilage-capped growth plate-like exostoses next to long bones. MHE is an autosomal dominant disorder resulting from heterozygous mutation in Ext1 or Ext2 that encode for copolymearse involved in heparan sulfate biosynthesis. The cells therefore exhibit lack of heparan sulfate on their surface thereby contributing to the exostoses. We hypothesize that restoring the cell surface heparan sulfate could attenuate exostoses. This will be achieved by screening for compounds that can increase heparan sulfate expression on CHO cells. Potential hits will then be tested on isolated human chondrocytes and finally in a mouse model of MHE.
Techniques:Immunostaining, cell culture, confocal microscopy, aortic endothelial cell isolation from pigs.

  • Xander van Wijk, PhD

Project Title

Blood-brain barrier proteoglycans in bacterial meningitis.

Project Description

My project focuses on the role of heparan sulfate proteoglycans (HSPGs) in neonatal meningitis. At least two-thirds of deaths from neonatal meningitis are attributable to Group B Streptococcus (GBS) and Escherichia coli, but several additional bacteria can cause the infection. As many leading bacterial pathogens interact with HSPGs, the aim of my research is to determine whether utilization of HSPGs to cross the blood-brain barrier is a general virulence mechanism in bacterial meningitis. Furthermore, I plan to assess whether a particular HSPG or modification of the heparan sulfate chains is most critical for bacterial adherence, invasion, and translocation.
Techniques:flow cytometry, bacterial adherence/invasion assays, cell culture, isolation of brain microvascular endothelial cells from mice, glycosaminoglycan analysis.

Project Title

Hyaluronan catabolism in innate immune system

Project Description

Studies done in the previous period have shown that fragmentation of hyaluronan (HA) stimulate host defense, repair events and innate immunity. The goal of the proposed research is to investigate the roles of HA by focusing on Hyaluronidase 1 and Hyaluronidase 2, the catabolic enzymes responsible for its breakdown in somatic tissues. Our main hypothesis is that the breakdown of HA by hyaluronidase is essential for recognition of skin injury and trigger danger signals to alarm host damages. Specifically, we plan to use the transgenic mice which we have generated for conditional overexpression of hyaluronidase and mice lacking the enzyme.
Techniques:In vivo studies in mice, flow cytometry, enzyme assays, cell culture, glycosaminoglycan analysis.

Project Title

The role of macrophage heparan sulfate proteoglycans in atherosclerosis and diet-induced obesity.

Project Description

Cardiovascular diseases (CVD) are the leading cause of death in Western societies. CVD-related deaths are primarily caused by complications of atherosclerosis, a disease initiated via focal infiltration and retention of lipoproteins in the subendothelial matrix of arteries due to a combination of aggregation and interaction with proteoglycans produced by the arteries. Little is know about the in vivo contribution of macrophage-derived and -associated proteoglycans during atherosclerosis development and progression. We want to determine the in vivo impact of reduced sulfation of macrophage proteoglycans on atherosclerosis using conditional mouse models lacking macrophage-specific Ndst1 functionality on an LDLR-deficient background. Atherosclerotic lesion quantity and quality will be analyzed in conjunction with macrophage lesion infiltration and lesion apoptosis and efferocytosis. Secondly, we want to evaluate the importance of proteoglycans for macrophage foam cell conversion, another key event in atherogenesis. Using macrophage cell cultures from wild-type or mutant mice we want to determine the importance of biosynthetic genes involved and the array of proteoglycans expressed before and after conversion. Finally, possible difference in macrophage behavior will be evaluated in the context of diet-induced obesity.
Techniques:Primary macrophage and stromal vascular fraction isolation, histology, atherosclerosis en face preparations, adipogenesis assays, blood lipid analysis, lipoprotein isolation, reverse cholesterol transport assays, binding and uptake studies, mRNA profiling, cell culture, glycosaminoglycan analysis, in vivo studies in mice.

Picture of Dr. Lingquan Deng for UCSD Programs of Excellence in Glycosciences

Project Title

Glycan microarray studies of sialic acid binding proteins and cloning of anti-Neu5Gc antibodies from humans

Project Description

One of my projects focuses on studying interactions between sialic acids and their binding proteins by glycan microarrays, as well as roles of these interactions in human health and disease processes. Sialic acid-recognizing proteins from novel plants, vertebrates, and microbes are obtained from various sources, including collaborations with colleagues from UCSF, Uni at Buffalo, Harvard and within UCSD. One class of these proteins, the serine-rich repeat (SRR) glycoproteins, for example, are a diverse family of adhesins found in Gram-positive bacteria. In collaboration with our colleagues from UCSF, we are now seeking to better define the molecular basis for interactions between the adhesins and their sialic acid-containing receptors, and assess effects of these interactions on virulence of infective endocarditis in an animal model. The other major project I am working on deals with cloning of monoclonal anti-Neu5Gc antibodies from humans and studying their biological significance.
Techniques:Organic synthesis, NMR, HPLC, surface conjugation chemistry, glycan microarray printing and screening, binding assays, flow cytometry, other basic molecular biology techniques.

 

  • Karl Markus Roupé PhD

Project Title

Molecular mimicry of human hyaluronan by Group a Streptococcus and its immuno-modulatory effects through host receptors

Project Description

As a Wenner-Gren post doc fellow I am currently studying the interactions between the capsule of group a Streptococci (GAS) and epithelial and inflammatory cells. GAS is able to cover itself with a capsule consisting of hyaluronan identical to human hyaluronan apart from the length of the polysaccharide chains. It is known that by doing so GAS improves the chances of avoiding immune recognition. However less is known about the actual effects of capsule-binding to the various receptors for hyaluronan present on epithelial and professional phagocytic cells. High molecular weight hyaluronan is known to be able to convey anti-inflammatory signals through some of these receptors. I am therefore investigating whether the molecular mimicry of human hyaluronan by group a streptococcus, apart from being an efficient cloaking device for GAS, could be a way for GAS to actively manipulate our immune defenses.
Techniques:Microbiology, Phagocytosis assays, Flow cytometry, Antimicrobial peptide assays, Neutrophil extracellular trap assays, Murine wound and infection models, Microarray analysis, and Labeling of hyaluronan.

  • Masaya Yamaguchi DDS, PhD

Project Title

Molecular analysis of Streptococcus pyognes hyaluronidase

Project Description

My project focuses on hyaluronidase of Streptococcus pyogenes (group A streptococci; GAS). GAS is a Gram-positive pathogen and causes various diseases, such as pharyngitis, rheumatic fever and toxic shock like syndrome. GAS has hyaluronan capsule which is known as one of the major virulence factors. On the other hand, GAS has hyaluronidase (HylA). Most GAS has inactive HylA. However, some GAS has active HylA which can degrade its own hyaluronan capsule. In addition, HylA contributes to GAS survival in human blood. Now we are examining how HylA contributes to GAS survival in blood and whether HylA works as a GAS virulence factor in vivo.
Techniques:Genetic engineering of bacteria, Bacterial adhesion and invasion assay, Bactericidal assay, Cell culture, Human neutrophil isolation, NETs formation assay, BMDM isolation, in vivo studies in mice.

  • Yuka Yamaguchi DDS, PhD

Project Title

Molecular analysis of Streptococcus pyognes hyaluronidase

Project Description

Yuka helps Masaya’s project as a volunteer scholar. She assisted constructing recombinant proteins and Streptococcus pyogenes mutant strains using several techniques, such as bacterial genomic DNA isolation, colony direct PCR, and immobilized metal ion affinity chromatography. Purified recombinant active HylA of S. pyogenes showed high hyaluronidase activity and hylA mutant strain significantly reduced its own the activity. In addition, she performed a part of mice i.p. or skin infection assay using S. pyogenes and blood collection from those infected mice.
Techniques:Genetic engineering of bacteria, ELISA, Bactericidal assay, Cell culture, Human neutrophil isolation, NETs formation assay, in vivo studies in mice.