Immunity

Going Viral

Going Viral

University of Maine. Biology & Biomedical Sciences – Going Viral. UMaine

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Virus Study at UMaine’s Zebrafish Facility

“You can see the bacteria?”

“Do you want to say the quality value of biofilm formation?”

“Why would isolating them give you a different value?”

“Are there any other explanations?”

“How do you have 1.8 fish?”

Things can get pretty intense in the hallway outside Carol Kim’s microbiology lab in Hitchner Hall at the University of Maine. For the casual observer — especially one without a science background — the barrage of questions is overwhelming, like listening to an auctioneer calling in a foreign language.

But the three undergraduates standing there explaining their senior research projects, pointing to printouts of data and microscopic images of zebrafish taped to the walls, are completely unfazed. They answer Kim’s questions almost as quickly as she asks them. When she challenges them, they challenge back. They are confident. Eloquent. They know their stuff.

Which is exactly the way Kim, a professor of biochemistry, microbiology and molecular biology who directs UMaine’s Graduate School of Biomedical Sciences, likes it.

“I want to set up an environment where students feel comfortable and nurtured,” Kim says. “They have to know I’m going to ask tough questions and they have to be prepared. It’s going to be a lot nicer for me to ask them than to have them present in front of five professors cold. If I can be tough on them and they can answer the questions, they’ll have confidence.”

She wants to maintain rigorous and demanding standards, without the fear factor that can sometimes overwhelm students during their training in the sciences.

Kim is emblematic of a major push on campus to involve undergrads in research, and scenes like the one outside her lab play out across campus every day, especially in the weeks leading up to graduation. At UMaine, hundreds of science, humanities and engineering majors are involved in research, and close collaborations with faculty members are common.

Increasingly, UMaine has become a destination for top students interested in pre-med and biomedical studies, in large part because of the mentoring and rigorous preparation that Kim and her colleagues provide. As a result, many undergraduates are working at a graduate level long before they earn their bachelor’s degree. Like the students in the hall, they know the answers. But more important, they know which questions to ask.

hat curiosity is what drives Kim’s own career, both as an educator and a researcher. And the students who cross paths with her during their time at UMaine have a strong, demanding role model.

Kim has been fascinated by the craftiness of viruses — their resourcefulness and resilience — since grade school. But she’s equally crafty. And her research has moved the entire field of virology forward.

She conducts disease studies with zebrafish, a model organism, to better understand the human innate immune response to infection. She’s the driving force behind UMaine’s Zebrafish Facility, and since she arrived at UMaine in 1998, Kim has received continuous funding for her zebrafish research — more than $4 million in federal grants, primarily from the National Institutes of Health. Among her landmark discoveries is a zebrafish gene that produces interferon, which can inhibit the growth of a virus. She and colleague Rob Wheeler recently received a $60,000 NASA planning grant to study the effects of radiation on innate immune response and the progression of cancer.

Her current research focus provides a better understanding of how bacteria infect and cause inflammation in cystic fibrosis patients. Kim’s studies shed light on the connection between the cystic fibrosis transmembrane conductance regulator, or CFTR, and the innate immune response. That connection may someday be used as the basis for therapeutics that combat bacterial infections in cystic fibrosis.

“I love doing the brainstorming, working with a student to figure out the best question to ask.”
Carol Kim

“Clinical researchers are trying to develop therapeutics for the immediate needs of these patients, and as a result, we’ve seen significant increases in their quality of life. We’re on the other end, with basic research, trying to figure out what’s happening at the molecular and cellular level with the hope of developing those therapies. This project will be ongoing until CF is completely cured, until it’s no longer a problem. It’s going to take a while.”
Another recent collaboration with UMaine physicist Sam Hess and GSBS student Kristin Gabor focuses on immune response to viral infection — not necessarily in CF patients. By using super-resolution microscopy, the researchers are the first ever to view the single-molecule cellular interactions involving antiviral signaling in caveolae, which are flask-like invaginations in the cell membrane. While previous research has shown that viruses exploit caveolae to enter host cells, Kim took it a step further by demonstrating that viruses can evade host cell defenses by disrupting clusters of signaling molecules within the caveolae. Through a combination of fluorescent tagging and super-resolution imaging of viruses and zebrafish cells, Hess’ FPALM (Fluorescence Photoactivation Localization Microscopy) system has allowed Kim and her team to see how individual molecules and clusters move during a viral infection.

“No one has ever really looked at this,” Kim says. “No one has been able to see it the way we’ve been able to see it.”

To the uninitiated, these may seem like disparate projects, but they all have two things in common: zebrafish and the innate immune response, the body’s first line of defense against infection. Innate immunity deals with how the body reacts immediately after it comes into contact with a pathogen. This happens daily, almost constantly, and it’s why healthy people don’t get sick every time they encounter a new pathogen. It’s why your skin swells when you get a splinter or a paper cut. This is not to be confused with adaptive immunity, which is acquired through vaccination or prior infection. Zebrafish are the ideal model for this research for several reasons, including the fact that they develop rapidly and their embryos are clear, allowing researchers to see the infection as it happens.

Innate immunity is pivotal to understanding how the body defends itself against infection, how viruses and bacteria adapt to the body’s defenses, and how more effective treatments might be developed.

Even one of these accomplishments would be noteworthy. Together, they’re huge. But when asked if there is a single moment that has defined her time at UMaine, she doesn’t miss a beat.

“Every year, when students in our department get into the top graduate schools, the top medical schools, the top dental schools, when they get great jobs it makes me think, ‘Wow, that’s why we’re here.’ UMaine is only the first step, but we hope we had an impact on their lives. We’re very proud of our students”

Working in Kim’s lab has been the highlight of Walter Mowel’s four years at UMaine. Mowel is a Biology major and Pre-Med student from Montpelier, Vt., whose scientific interest lies in infectious diseases. He spent his final semester researching a virus that hasn’t been studied in zebrafish before. And when he stood up for Kim’s hallway inquisition, his enthusiasm for the project outweighed any fear.

“Before this, I spent a lot of time working on an experiment that didn’t work out, but with this project, I was able to get a lot of data that has me really excited,” Mowel says. “It’s so much fun to essentially look at something for the first time. A lot of times, when you set out to conduct a study, you’ll go to a scientific journal or you’ll go online to see what other scientists have done, but in this case, we couldn’t. We’re basically finding something new and that’s been outstanding.”

Aaron Perreault, a Biochemistry major and Honors student from Northfield, N.H., spent his junior and senior years in Kim’s lab studying Pseudomonas aeruginosa, a bacterium that regularly infects cystic fibrosis patients — one of many pathogens that the innate immune response normally quells.

“When children with cystic fibrosis are younger, they get infections with pathogens that make sense, such as Haemophilius influenzae, staph infections, more common lung pathogens. But by the time they’re in their teens, patients all have Pseudomonas infections, which are very rare in healthy individuals,” Kim says. “Why Pseudomonas rather than a more common respiratory pathogen?”

The answer to that question may someday be used to create therapeutics, and Perreault, one of five UMaine students to gain admission to Tufts University School of Medicine as a sophomore through the Tufts Maine Track Early Assurance program since 2009, may someday prescribe those therapeutics.

Though Perreault admits that the biggest lesson he’s learned is that his lab skills aren’t particularly strong, the experience has also taught him the value of analytical thinking and keeping an open mind. His work in Kim’s lab was the basis for his Honors thesis, and in the weeks leading up to his defense, he was no stranger to Kim’s hallway inquisitions.

“It can be intimidating at first, but once you figure out that she’s grilling you to make you a better student, it makes you work harder,” Perreault says.

Kim will do whatever it takes to instill confidence in her students. Sometimes that means an informal inquisition like the one in the hall. Other times, it means meeting on nights or weekends to make sure that her students are prepared to consider every angle when defending their theses or dissertations. And sometimes, it just means handing over the reins.

When Steve Altman was at UMaine — he earned a bachelor’s in microbiology in 2002 and a master’s in molecular biology in 2003 — he worked on basic immunology in zebrafish. Kim gave him a lot of wiggle room with his experiments, but she also challenged him to try things that might be outside his comfort level, and that continues to influence the way he does science.

“Some of my friends worked in labs where the principal investigator told them what to do,” recalls Altman, who now conducts Alzheimer’s research for Amgen in Cambridge, Mass. “With Carol, it was a little bit more open-ended. She gave me guidance, but she also allowed me to make decisions on my own. That stuck with me.”

Inspiring the next generation of doctors and researchers is what gets Kim out of bed in the morning. She wants them to get jazzed about how crafty bacteria and viruses are. She wants them to experience the thrill of seeing something for the first time. And the best way to do that is through research.

“I love doing the brainstorming, working with a student to figure out the best question to ask,” Kim says. “Seeing them come in to check on their fish, to see what their results are, that’s exciting to me.”

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Battle Lines

Battle Lines

Staples, B. (2013) Battle Lines. UMaine Today

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A UMaine biomedical lab looks for answers in the transformation of a peaceful yeast to fatal fungus

“We’re using zebrafish to ask really specific questions that cannot be
answered another way,” Wheeler says. “These questions have been inaccessible
for a long time. We hope to be able to better utilize existing therapies and be
able to develop better therapies.”

LIFE-AND-DEATH battles rage in Robert Wheeler’s lab at the University of Maine. The combatants — zebrafish and Candida albicans— fight to the bitter end in glass-bottom microplates. Similar perilous battles are being fought inside humans. The C. albicans fungus is a leading cause of hospital-acquired infection that annually kills several thousand patients nationwide. During the staged scuffles in Wheeler’s lab in Hitchner Hall, anesthetized zebrafish are injected with Candida and placed in a gelatinous material called agarose.
A laser microscope captures and magnifies the struggles inside the zebrafish blood vessels in real time in high-definition color detail. The microplate clashes provide the assistant professor of microbiology with the ability to view how immune cells fight the microbe, identify genes involved in virulence, test new drugs and learn how gene perturbations affect hostpathogen interaction.

In March 2012, Wheeler received a three-year, more than $421,600 grant from the National Institutes of Health to ask and answer these questions in the project: “Genetics & Visualization of Innate Host Response to Candida albicans Infection In Vivo.” The goal is that the resulting answers will save human lives.

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How Pathogens Become Killers

Wheeler Awarded $500,000 to Study How Common Pathogens Become Killers

University of Maine. (2014) Wheeler Awarded $500,000 to Study How Common Pathogens Become Killers. UMaine News.

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How does a normally peaceful agent break through a previously
impenetrable barrier and become a potential killer?

Robert Wheeler has just received a five-year, $500,000 fellowship from the Burroughs Wellcome
Fund (BWF) to figure that out.

The University of Maine Assistant Professor of Microbiology will study how and why Candida albicans — the most common human fungal pathogen — transforms from an innocuous yeast in the digestive tract of a person with a healthy immune system to a potentially fatal fungus in vital organs of a person whose immune system has been compromised.

“This award marks a new high point in my research career,” says Wheeler, one of 12 scientists nationwide to receive the 2014 Investigators in the Pathogenesis of Infectious Disease Award. After internal competitions at colleges and universities, each institution may nominate two investigators; this year, 144 scientists were put forward.

T“This provides substantial funding that we can use to pursue high-risk projects with the potential to change our perspective on how dangerous infections begin.”

The goal, he says, is to improve diagnosis and therapy of fungal infection due to better understanding of the interactions between host and pathogen cells.

Wheeler’s lab will explore the host-fungal dialogue at mucosal surfaces where C. albicans — the leading cause of hospital-acquired infection that annually kills several thousand patients in the U.S. — is normally kept in check. “We expect that this will allow us to understand how the healthy immune system normally inhibits infection and how C. albicans invades past the epithelial wall,” he wrote in his application.

What happens at the earliest stages of active infection is one of the biggest mysteries about opportunistic pathogens, he says. And solving that mystery is imperative as infections complicate treatment of diseases, including leukemia, that require suppressing the immune system.

Wheeler’s lab will use zebrafish models of candidiasis at multiple levels — holistic, cellular and molecular genetic — to investigate the interaction between fungal cells and host cells during the earliest stages of infection. The integrated approach will utilize a new set of tools to address questions that have previously been inaccessible, he says.

His lab already has conducted pioneering studies with transparent zebrafish, which model infections caused by bacterial and fungal pathogens of humans. The resulting findings, he says, “opened the door to a deeper understanding of host and pathogen activity at the beginning stage of infection.”

Wheeler credits the previous scientific breakthroughs, and the work on the grant, to the talented, highly motivated and hard-working students and post-doctoral fellows in the laboratory. “The award is based on the pioneering work that they have done to change our perspective on fungal infection over the last five years,” he says.

With this fellowship, Wheeler says his lab will seek to exploit “that opening to discover the mechanistic underpinnings of the dialog between C. albicans and innate immunity at the epithelial barrier.”

On a personal level, Wheeler says he’s humbled to join the creative group of scientists that have previously held or currently hold BWF grants. “It pushes me to further excel and tackle the most important problems in infectious disease,” he says.

Wheeler’s peers lauded both his prior research and his potential.

Aaron Mitchell, professor in the Department of Biological Sciences at Carnegie Mellon University, says Wheeler has “been an insightful innovator for his entire scientific career.”

This award, Mitchell says, will allow Wheeler to build upon his initial findings “to look at the way that the host manipulates the pathogen, and how the pathogen manipulates the host. The remarkable zebrafish toolbox will allow Rob to look for key features of host defense that we can strengthen to thwart the pathogen before it gets a foothold.”

Joseph Heitman, chair of the Department of Molecular Genetics and Microbiology at Duke University Medical Center, says Wheeler’s research on how “Candida albicans … shields its immunogenic cell surface from immune surveillance in a variety of ways, which can in part be circumvented by drugs that unveil immunogenic signals” has blazed trails.

Heitman says the award will allow Wheeler, a “highly creative and innovative” investigator, to continue to be a leader in the field.

Gerald Fink, the Herman and Margaret Sokol Professor at the Whitehead Institute/Massachusetts Institute of Technology, says the award “recognizes [Wheeler’s] preeminence as a leader in the battle to combat Candida, a feared human fungal pathogen … for which we have no satisfactory protection.”

Fink anticipates Wheeler’s research will “provide critical insights into our natural immunity from Candida infections, which is the first step towards developing antifungal agents.”

And Deborah Hogan, associate professor in the Department of Microbiology and Immunology in the Geisel School of Medicine at Dartmouth College, says, “Ultimately, this work is likely to provide important insight into better ways to prevent and fight these often dangerous infections” in babies, in people undergoing chemotherapy and in those with suppressed immune systems.

The first installment of the award will be sent to UMaine on July 15, according to BWF, an independent private foundation based in North Carolina that supports research to advance biomedical sciences.

Victoria McGovern, senior program officer at BWF, says Wheeler’s selection was “based on the scientific excellence and innovation” of his proposal, as well as the strength of the scholarship at UMaine and Wheeler’s accomplishments as a researcher.

Wheeler says he’s pleased the award showcases UMaine and the laboratory to the national research community and he’s excited for opportunities to be in “contact with a number of the best and brightest infectious disease investigators in the U.S., through yearly meetings and a number of networking opportunities at national conferences.”

 

“The University of Maine is very proud of Dr. Wheeler’s achievement,” says Carol Kim, UMaine vice president for research.

“The BWF is a very prestigious award and identifies Rob as a leader in his field.”

 

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