Senior Honors Theses 2011-2012

The Division of Biological Sciences Senior Honors Theses Program (BISP 196) is open to undergraduate biology majors who have an overall, and major, GPA of 3.7 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year. The goals of the program are to increase one-one interaction between students and faculty to encourage more biology majors to pursue independent research.

Each student in the program has a faculty mentor who provides guidance to the student during the year. In spring quarter each year, students in the honors thesis program participate in a poster session that showcases their research, and gives them the opportunity to discuss their research with faculty and their fellow students. Below are abstracts of all the outstanding research projects conducted by undergraduates in the program during the 2011-2012 academic year.

Investigating the cytokine activity of the Drosophila Toll Pathway

Allen Ninh, Warren College, Biochemistry and Cell Biology major, Psychology minor, Class of 2012
PI: Steven A. Wasserman Ph.D., Division of Biological Sciences, Section of Cell & Developmental Biology

Comparisons between Drosophila species based on genome-wide transcription profi ling have identifi ed a core set of Toll-induced genes of unknown function that potentially play a role in the Drosophila innate immune response. We are currently developing assays to elucidate the function of these immune effectors and, for those that are likely to act as cytokines, identify their receptors. Ectopic expression of one candidate cytokine is associated with the proliferation of lamellocytes and melanotic tumor formation in larvae and adult flies. Bioinformatics studies suggest that this immune effector may act through a G protein-coupled receptor (GPCR) pathway. In order to study this, I have generated a stable cell line expressing a genetically encoded calcium indicator, GCaMP. Using a synthetic peptide and assaying for calcium infl ux in GCaMP expressing cells, we hope to identify the mechanism by which this immune effector acts. The results of these studies hold promise for providing a better understanding of these novel immune effectors and of their roles in the innate immune response.

Effects of Immunosuppresants Cyclosporin A and FK506 on Central Axon Outgrowth in vitro

Ari D. Kappel, Sixth College, Physiology and Neuroscience major, Cognitive Science minor, Class of 2012
PI: Mark H. Tuszynski, MD, Ph.D., Department of Neurosciences

Spinal cord injury (SCI) and disorders disrupt movement, sensation, and function of the central nervous system leading to lifelong debilitations and often paralysis. Unlike axons in the peripheral nervous system (PNS), injured axons in the adult mammalian central nervous system (CNS) do not regenerate after injury. Novel research in our lab has shown that grafts of embryonic nervous tissue can induce functional recovery of severed axons in the CNS. However, grafts of embryonic tissue require immunosuppression, and any future application of embryonic nervous tissue grafts in a clinical setting will require immunosuppression. In the past, it has been shown that conventional immunosuppressants such as cyclosporin (CsA), and prograf (FK506) inhibit axonal regeneration in the PNS (Tessier-Lavigne et al, 2003). The current research investigates the effects of CsA and FK506 on CNS axons in vitro. Time course information, and concentration dependence data are investigated in an isolated setting in order to elucidate the molecular and cellular effects of common immunosuppressants, CsA and FK506, on CNS axons.

Protective effects of spinach aldolase on muscle adenylate kinase and Phosphofructokinase-1 activities

Brian Jihoon Park, Muir College, Human Biology major, Class of 2012
PI: Dr. Percy Russell, Ph.D., Division of Biological Sciences

From previous studies, a hypothesis was developed that ascorbic acid (AA) inhibits muscle glycolytic enzymes during periods of rest to facilitate the storage of glucose as glycogen. Phosphofructokinase-1 (PFK-1) is considered the enzyme that controls the rate of glycolysis and along with lactate dehydrogenase (LDH) and adenylate kinase (AK), PFK-1 is inhibited by AA. During contraction, AA does not inhibit glycolysis due to the formation of a complex of glycolytic enzymes with contractile muscle proteins that protects them. Muscle aldolase prevents AA inhibitions and its protective properties are considered a microcosm of the complex formed with contractile muscle proteins. It was observed that spinach aldolase appeared to protect muscle PFK-1 activity from AA inhibition. The current study focuses on this ability of a plant enzyme, spinach aldolase, to interact and protect animal enzymes. Specifi cally the studies showed that spinach aldolase protected muscle AK activity loss due to dilution and protected muscle PFK-1 from inhibition by each AA and ascorbyl dipalmitate, an AA derivative shown to be more inhibitory than AA alone.

Role of Tissue-nonspecific Alkaline Phosphatase and Fetuin in Biomineralization

Bryan Lam, Revelle College, General Biology major, Urban Studies and Planning minor, Class of 2012
PI: Paul A. Price, Ph.D., Division of Biological Sciences

Our goal is to understand the mechanisms by which proteins control the the normal mineralization of bones and teeth and prevent the abnormal mineralization of soft tissues. We studied two proteins which have been implicated in the regulation of biomineralization: tissue-nonspecifi c alkaline phosphatase (TNAP) and fetuin. Fetuin is a 48kDa glycoprotein that is synthesized in the mammalian liver and found in high concentrations in serum and bone and is an important inhibitor of apatite growth in-vitro. TNAP has been identifi ed in previous studies as a component of serum calcifi cation activity and its effect in increasing the rate of mineral formation is observed only exclusively in the presence of fetuin. Apatite crystal formation was investigated using an in-vitro assay that closely resembles the calcium, phosphate and pH of human serum. We have demonstrated that fetuin is a substrate for alkaline phosphatase activity and that de-phosphorylation of fetuin greatly reduces its mineral inhibition activity. The high concentration of alkaline phosphatase and fetuin in bones suggest that bone fetuins may have a more dephosphorylated state. The reduced mineral inhibition activity of fetuin within bone may allow for normal mineralization to occur. Our findings also suggest that the phosphorylation state of serum fetuin may play a role in abnormal soft tissue mineralization such as the formation atherosclerotic plaques.

The role of the homeodomain protein Six6 in pituitary gonadotrope gene expression

Chiara Maruggi, Revelle College, General Biology major, Italian Literature minor, Class of 2012
PI: Pamela Mellon, Ph.D., Department of Reproductive Medicine

The hypothalamus orchestrates the release of FSH and LH from the pituitary through its pulsatile release of GnRH. FSH and LH in turn act on the gonads to stimulate hormonal secretions and gametogenesis. This research aims at better understanding regulation of gonadotrope gene expression through studying the mechanism by which Six6 represses the expression of LHβ, FSHβ, and GnRH receptor (GnRH-R). Six6, a member of the SIX/Sine oculis family of homeobox genes, stimulates progenitor cell development in the growing pituitary, eye, and brain. In mice, absence of Six6 causes a hypo-pituitary phenotype and a striking decrease in fertility. Recent work has shown repression of LHβ, FSHβ, and GnRH-R by Six6 and a relief of repression of the genes when transfecting with Six6 carrying a mutation in the eh1 domain. The region of repression by Six6 is in the 800 to 600 region for GnRH-R, in the -300 to -87 region for LHβ and inside of -95 in FSHβ. Two non-exclusive hypotheses will be explored further: displacement of activators from binding sites and recruitment of TLE co-repressors. The murine TLE (Grg, Groucho-related gene) proteins are co-repressors regulating development. Research has shown that both long-form (Grg4) and short form (Grg5) proteins regulate the GnRH gene by co-localizing with through Oct1 and Msx1 in vivo. Elaborating on these fi ndings, this project also aims at identifying the possible role of TLE/Grg proteins in the repression of LHβ, FSHβ or GnRH-R. Such findings would help increase our knowledge of the mechanisms regulating GnRH release—mechanisms that play a key role in physiology and pathophysiology dealing with development and reproduction.

The Unique Shuttling Pathway of the Peroxisomal PTS2 Receptor, Pex7, in Pichia pastoris

Danielle Hagstrom, Muir College, Molecular Biology major, Class of 2012
PI: Suresh Subramani, Ph.D., Division of Biological Sciences

Peroxisomal matrix protein import relies on two pathways that use one of two peroxisomal targeting signals (PTSs), known as PTS1 and PTS2, present on cargo proteins. The majority of import occurs through the PTS1 pathway, dependent on the receptor Pex5. The PTS2 pathway is dependent on the receptor Pex7 and its co-receptor Pex20. Pex5 and Pex20 bind their respective cargo in the cytosol and shuttle them into the peroxisome matrix to be released. After cargo release, Pex5 and Pex20 are subject to mono-ubiquitination which allows for receptor recycling – a process dependent on the ubiquitin-conjugating enzyme, Pex4, and the AAA ATPases, Pex1 and Pex6. When receptor recycling is blocked, Pex5 and Pex20 are poly-ubiquitinated and degraded by the proteasome, in a process called the RADAR (Receptor Accumulation and Degradation in the Absence of Recycling) pathway. However, we found that the shuttling pathway of Pex7 differs from that of Pex5 and Pex20 as Pex7 is present at low levels in wild-type cells but stabilized in pex mutants affecting normal peroxisomal matrix protein import, suggesting that Pex7 is constitutively degraded during its normal import cycle. Specifi cally, Pex7 is stabilized in the receptor recycling mutants, Δpex4 and Δpex6, due to its inability to enter into peroxisomes, as seen by fractionation and protease protection assays. However, in the mutants affecting peroxisomal membrane proteins containing a RING domain, such as Δpex2, Pex7 can enter peroxisomes but presumably cannot be exported, thus causing accumulation of Pex7. Additionally, the import and export of Pex7 may have a previously unknown dependence on Pex5 and Pex20, which may be mediated by the special cargo, Pex8, which contains both a PTS1 and PTS2. The shuttling pathway of the PTS2 receptor, Pex7, demonstrates a sharp divergence from the previously studied shuttling pathways of Pex5 and Pex20 and could represent a mechanism to regulate Pex7 levels when the PTS2 pathway is not needed.