From Undergrad to Ph. D., My Lessons

Although the road to becoming a scientist is often riddled with many hurdles, the rewards of self-discovery and potential to revolutionize the world, are priceless. It is said that a PhD is pursued by those who are driven to do something original and those who are have the passion to discover the unknown; but also those that have dedication, persistence and resilience. My journey began at the intersection of College Ave and SW 30th St back in 2004. As with many other Biology majors, I was unsure where my career path should lead to, but I knew it had to center around science. The turning point came in 2005, when I was challenged by an outstanding professor and mentor in Dr. Emily Schmitt to present a poster at the NSU Undergraduate Research Symposium. It served to awaken my appetite and passion for acquiring knowledge. Today I am a post-doctoral fellow in the Dermatology department of University of Miami Miller School of Medicine trying to understand the molecular mechanisms underlying wound healing and I am also an adjunct professor in the Halmos College of Natural Sciences. But on my road to acquiring a PhD, I’ve learned that a PhD is one of the toughest things to accomplish in the world (in educational terms); it is intellectually challenging, emotionally taxing and physically tiring. So then why do it? During my presentation, I will share some highlights of my profession as well as give you some answers to this question

Effect of EGF-Receptor Tyrosine Kinase Inhibitor on Rab5 Function During Endocytosis

Tyrosine autophosphorylation within the cytoplasmic tail of EGF-receptor is a key event, which in turn recruits several factors including Shc, Grb2 and Rin1 that are essential activities for receptor-mediated endocytosis and signaling. In this study, we demonstrated that treatment with AG1478, an EGF-receptor kinase inhibitor, blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of EGF. We also found that EGF-receptor catalytically inactive mutant failed to activate Rab5 upon EGF stimulation. Additionally, endosomal co-localization of Rab5 and EGF-receptor was inhibited by AG1478. Interestingly, AG1478 inhibitor did not block the formation of enlarged Rab5-positive endosomes in cells expressing Rab5 GTP hydrolysis defective mutant (Rab5:Q79L). AG1478 inhibitor also blocked the in vitroendosome fusion in a concentration-dependent manner, and more importantly, Rab5:Q79L mutant rescued it. Furthermore, addition of Rin1, a Rab5 guanine nucleotide exchange factor, partially restored endosome fusion in the presence of AG1478 inhibitor. Consistent with these observations, we also observed that Rin1 was unable to localize to membranes upon EGF-stimulation in the presence of AG1478 inhibitor. These results constitute first evidence that the enzymatic activity of a tyrosine kinase receptor is required endosome fusion via the activation of Rab5

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions

Selective and specific activation of Rab5 during endocytosis of receptor tyrosine kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis

The role of cyclin-dependent kinase 5 (Cdk5) in Neurodegeneration

The purpose of this literature research project is to investigate the role of Cyclin- dependent kinase 5 (Cdk5) in neurodegenerative diseases by summarizing its function in brain development, learning and memory. Cdk5 is a proline directed protein kinase that phosphorylates serine and threonine residues. Additionally the role of Cdk5 in the regulation of NMDA receptor, N-cadherin-mediated adhesion as well as the drugs that inhibit their normal functions will be examined. Recent studies regarding neurodegenerative pathology of diseases such as Alzheimer’s, Parkinson’s and Amyotrophic lateral sclerosis reveled a crucial role of this kinase in central nervous system development, particularly in the normal positioning of neurons, axonal guidance, synaptic plasticity, dopamine signaling, cell adhesion and vesicle trafficking as well as neurodegenerative and neurofibrillary tangle diseases. Cdk5 is regulated by the binding of either p35 or p39, which serve as activators of Cdk5 which proteolytically cleave the two regulatory subunits into p25 and p29 respectively. Accumulation of the p25 subunit has been specifically identified in the neurons of Alzheimer’s as well as other neurodegenerative diseases where the accumulation of p25 serves as a competitive activator of Cdk5 altering the normal substrate specificity of Cdk5 which then causes a hyperphosphorylation of tau protein thus reducing its ability to associate with microtubules inducing cytoskeletal disruption, morphological degeneration and apoptosis indicating that the aberrant activation of tau is neurotoxic

The Effects of Trolox (Vitamin E) on Gene Expression During Oxidative Stress in Saccharomyces Cerevisiae

Cellular oxidative stress contributes to the production of reactive oxygen species (ROS) during metabolic processes, which may cause many illnesses including cancers. Hydrogen peroxide (H2O2) forms radicals that may react with various cellular components such as DNA, proteins, and lipids. This research focuses on the use of Trolox (vitamin E) as an antioxidant in the model organism Saccharomyces cerevisiae(yeast). The abilities of Trolox to decrease cellular damage caused during oxidative stress is investigated through two known oxidative stress genes and four genes of unknown function (suspected to have a role in controlling oxidative stress). Additionally, separate cultures will be exposed to two different concentrations of H2O2 (3% and 30%) to determine its effects on the six genes. RNA is extracted from the Trolox and H2O2treated yeast cultures. Quality and quantity of the RNA is measured using UV- spectrophotometry at the absorbance wavelengths of A260nm and A280nm as well as the A260/A280 ratio. Gel electrophoresis is used to analyze the quality of the RNA extracted by visualizing the large and small ribosomal subunit bands. A reverse transcription reaction is performed to convert mRNA to cDNA, and then Polymerase Chain Reaction (PCR) is performed to test for the presence and relative quantity of targeted genes. The size and relative brightness of the resulting bands are checked against the molecular size standard to determine the potential effects of Trolox and H2O2 treatment on selected gene expression