Northeastern University College of Science

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• In 500 words or less, please tell us about your aspirations as a scientist and how participating in this summer program fits in with your academic and career goals.  Please be sure to include your research interests.

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Research Project Summary: Biofilms are groups of bacterial cells acting as a super-organism encased in a network of self-produced extracellular matrix molecules. Though biofilm assembly is well understood, it is unclear how biofilms disassemble or how this process is regulated. REU students in this project will investigate the transcriptional network in Bacillus subtilis in which SlrR, a transcriptional regulator, is both a positive regulator of genes essential for biofilm assembly and a negative regulator of another set of genes involved in biofilm disassembly. Students will explore similarities between the inactivation of SlrR and that of LexA, the global regulator of the DNA damage response in Escherichia coli. Results will identify the relationship between bacterial DNA damage response and biofilm disassembly, illuminating critical, and potentially targetable, pathways regulating bacterial survival.

Student Involvement in Project: Students will develop hypotheses, and design and execute experiments, to elucidate specific links between biofilm disassembly and the DNA damage response in B. subtilis. They will study the genetics and physiology of biofilm assembly and disassembly.

What Students will Learn: All students will gain facility in hypothesis formation and testing, and in basic molecular biology techniques and bacterial genetic analyses. Further, all students will engage in extensive data analysis, gain experience in presenting results in small and large groups, and learn to coordinate efforts in a complex research team.

Broader impacts of this project: Gaining insights into connections between biofilm disassembly and DNA damage response will provide a global view of the complex networks occurring in bacteria to organize their life strategies. Furthermore, understanding the molecular details of biofilm disassembly can provide insights into novel solutions to prevent bacterial biofilms.

Research Project Summary: Long bone growth is supported by a cartilaginous structure, the growth plate (GP/physis) comprised of a resting zone (RZ), a proliferating zone (PZ) and a hypertrophic zone (HZ). My lab recently discovered a population of long-term stem cells, characterized by expression of FoxA2 transcription factor, in the top compartment of GP RZ. In this project, the student will investigate what signaling pathways control stem cell longevity using transgenic mouse models. We expect to identify genes and signaling pathways that control epiphyseal cartilage growth.

Student Involvement in Project:

Students will develop hypotheses, design, and execute experiments to investigate the role of FoxA2 in the transcriptional control of stem cell long-term self-renewability. Students will delete FoxA2 in stem cells and perform a colony forming unit assay to assess by serial passaging whether stem cells without FoxA2 exhaust faster than stem cells with intact FoxA2 signaling. Finally, students will help design a study to identify FoxA2 specific transcriptional targets.

What Students will Learn: Students will learn the basics of designing, executing, and interpreting experimental results and communicating scientific discoveries to others. They will learn how to work with transgenic reporter mice, how to genotype and select the correct progeny, how to label and isolate stem cells using FACS (florescence-activated cell sorting). They will delete specific genes (FoxA2) in mouse stem cells using CRISPR-Cas9, and they will assess stem cell self-renewability by serial passaging in a colony forming unit assay. Students will learn how to use statistics to determine the statistical significance between samples. All students will learn responsible conduct of research in the process of experimental science.

Broader impacts of this project: Results from these experiments will offer insights in the mechanism of epiphyseal cartilage growth during skeletal development. Our students will gain knowledge and expertise in stem cell biology and skeletal development and growth. They will learn how to work in a team of researchers, how to develop a scientific hypothesis and design an experimental strategy to test this premise. Our goal is to help them become passionate about resear­­­ch, and to enable them to build a scientific story and to present it to their peers.

Research Project Summary: The assembly of the multilayered bacterial envelope, which contributes to drug resistance and resilience against immune attack, is controlled by regulatory networks that respond to and alleviate external stress. The proposed studies will focus on dissecting the regulation networks controlling key elements of envelope biogenesis, including a protective outer membrane, capsule, and cell wall, in the refractory opportunistic pathogen Acinetobacter baumannii.

Student Involvement in Project: Students will develop hypotheses and design experiments to elucidate the signal transduction and regulatory pathways that respond to stress and modify synthesis of the cell envelope in A. baumannii. To test their hypotheses, students will target key proteins in the pathway by directed mutagenesis and molecular cloning and examine their role in modulating envelope synthesis and in mediating the response to stresses including antibiotic exposures.

What Students will Learn: Students will learn how to develop a hypothesis, design experiments that test the hypothesis, garner data through experimentation, analyze data, and interpret results. They will learn techniques in molecular biology including PCR, DNA/RNA extraction, and DNA cloning. They will learn how to manipulate A. baumannii genetically using recombinant DNA technology, CRISPR interference, and bacterial transformation/conjugation. They will learn how to study the bacterial envelope using protein and polysaccharide gel electrophoresis, fluorescence microscopy, and antibiotic resistance tests. Students will also learn how to effectively communicate their scientific findings to experts and non-experts through meetings and presentations. Broader impacts of this project: Students will learn how to use bacterial genetics and molecular biology to answer scientific questions about the biology of antibiotic-resistant bacteria. They will gain an appreciation of the power of bacterial genetics and related techniques and will gain perspectives on strategies to undermine the protective barrier of bacteria and antibiotic resistance, an important societal problem. The students will develop confidence in being able to tackle difficult problems through science and will gain skills in communicating their scientific interests to their peers, ultimately enhancing their ability to enter STEM careers.

Research Project Summary: The DNA damage response (DDR) in A. baumannii depends on the expression of the recA gene encoding the cells’ main recombinase. In bacteria RecA is a multifunctional protein with roles in recombination and in induction of the DDR. Formation of single stranded DNA (ssDNA) is the hallmark of DNA damage due to disturbances in DNA replication. The ssDNA is bound by RecA forming the RecA nucleoprotein filament or RecA* that promotes the cleavage of a global DDR repressor, which in turn regulates the expression of the recA gene. Remarkably, in A. baumannii this elegant circuitry is only partially functional. The recA gene is regulated by an unknown transcription factor in response to DNA damage. Moreover, the levels of cellular RecA vary in response to DNA damage. There are cells with low and cells with high RecA. Our research has discovered a 5’ untranslated region (UTR) in the recA message that is responsible for the 2-cell phenotypes. Mutations in the recA 5’UTR that destabilize a potential structure result in induction of gene expression but only a low RecA cell type. This and other findings have led us to believe that the structure of the 5’ UTR is stabilized by an unknown regulator. We seek both recA regulators. Student Involvement in the project: students will develop hypothesis, design, and execute experiments to identify in vivo the recA regulators. Students will use as a host A. baylyi, a close relative of A. baumannii that does not form two-cell types because its recA gene lacks the 5’UTR. Students will use an A. baumannii UTR-recA fluorescent reporter and introduce it by transformation in A. baylyi and observe whether there is one or 2-cel types. The results of this experiment will illuminate the next steps. The student will help design the following experiments for which we already have several molecular tools.

What students will learn: They will learn the basics of designing, executing, and interpreting experimental results and communicating scientific discoveries to others. They will also learn about the bases of gene regulation and signaling cascades in a unique bacterial DDR. Students will learn how to use statistics to determine the statistical significance between samples. All students will learn about responsible conduct of research in the process of experimental science. Broader

Impacts of this project: results from these experiments will allow us to gain insights into the recA regulators, which will in turn provide a glimpse about how cells form phenotypically different types of cells with the same genetic information. The students will gain valuable knowledge about biological resilience in response to injuries in the DNA and the approaches used by different bacteria to do so. They will also learn about the process of science starting from observation to hypothesis and experimentation to science communication. Through the process students will learn that resilience is key to the scientific endeavor.  

Research Project Summary: Contractile non-muscle and smooth muscle cells perform many important biological functions, especially in tubular structures found in the gut, lungs, reproductive and cardiovascular systems. Remarkably, contractability is constant, a good example of biological resilience. However, how cells regulate and coordinate their contractility in vivo is not well understood. We use a combination of techniques to explore the function of the gene regulatory networks required for contractility of cells in the reproductive system of the nematode C. elegans and use in vivo imaging to analyze calcium transients. Students will design projects to identify regulators of calcium signaling in the C. elegans reproductive system.

Student Involvement in Project: The student will select candidate genes to explore for function in the C. elegans spermatheca. They will develop hypotheses, design, and execute experiments to elucidate the role of these genes in regulating calcium signaling and contractility in the nematode. Using video imaging of nematodes expressing the calcium sensor, students will quantitatively analyze the role of their selected genes in this process.

What Students will Learn: All students will learn responsible conduct of research, the process of science, beginning with hypothesis development and testing. Students will gain proficiency in C. elegans genetics, biochemistry, and quantitative imaging.

Broader impacts of this project: Results of the experiments will help us understand contractile tubes, which is relevant to understanding the pathogenesis of hypertension and asthma in which the tubes have lost their resilience. 

Research Project Summary: The axolotl salamander has extraordinary regenerative abilities. Upon amputation of a limb, the axolotl can regenerate its entire limb to generate an exact replica of the missing structure. Although this process of biological resilience has been studied for over two hundred years, the cellular and molecular basis for limb regeneration is still incomplete. Recent advances in genome sequencing, tissue staining, and imaging has facilitated the discovery of which cells contribute to the regenerating limb and the genes involved in the process. Student involvement in the project: In this project, students will build upon previous screening experiments in our laboratory that has identified genes that are likely involved in the regeneration process. The students will perform tissue staining and imaging of regenerating limb tissue that was previously collected by the laboratory.

What Students will Learn: A recent method to stain mRNAs in tissues called hybridization chain reaction has become a powerful approach for imaging RNAs in fixed tissues. Our laboratory has adopted this method for the axolotl salamander to perform multiplexed imaging of mRNAs in salamander tissue sections and whole mount tissues. The students will use protocols developed in the laboratory to image mRNAs in tissue sections and whole mount tissues that are expressed in the regenerating limb. These experiments will provide key information on the cell types that express candidate genes, providing insight into their possible function.

Broader impacts of this project: The students will gain experience in modern staining, imaging, and image analysis techniques. The students will perform confocal imaging of tissue sections and lightsheet imaging of whole mount tissues. They will then perform image analysis include segmentation of the cells in the images using machine learning and quantitative measurements of gene expression in 2D and 3D tissues. This modern approach to a classic question in developmental biology will provide valuable experience in experimental design, execution of wet bench experiments, and modern computational analytical tools.