published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The purpose of this study was to determine if resistance exercise altered peripheral BDNF concentration. Eighteen trained male subjects were split into two groups performing varied repetition ranges. DUP-HR and DUP-LR groups trained 3x/week for 8 weeks, and were equated for total volume (repetitions X sets X intensity). Plasma BDNF and interleukin-6 (IL-6) levels were measured prior to and immediately following the first exercise session of weeks 1, 2, 4 and 6. Pre-exercise levels were also assessed prior to the second and third sessions of week 1 and 6. Lastly, resting levels were measured before and after training intervention. No group differences (p>0.05) were detected for either biomarker. An acute BDNF elevation (p=0.018) was detected only in the final week of training. IL-6 elevations were detected at all acute measurements (p<0.01). BDNF and IL-6 percentage change correlated significantly (p<0.05) in week-1. No chronic alterations were observed (p>0.05). / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Ependymin (EPN) is a neurotrophic factor (NTF) that functions in goldfish long-term memory formation and optic nerve elongation (Shashoua, 1976; Shashoua, 1977; Shashoua, 1985). Goldfish EPN, or CMX-8933 (a short goldfish EPN mimetic studied by our lab), surprisingly have several demonstrated effects on mammalian cells, including neuroregenerative effects in a rat stroke model (Shashoua et al, 2003), and the activation of therapeutic superoxide dismutase (SOD) (Parikh, 2003) and transcription factor AP-1 (Adams et al, 2003) in mouse neuroblastoma cells or rat primary cortical neuronal cultures. Among its various functions, AP-1 can function as a master switch in long-term memory consolidation (Sanyal et al, 2002), so it may be a key event in EPN's mechanism of action. AP-1 activation is also a characteristic associated with other full-sized neurotrophic factors, including nerve growth factor and brain-derived nerve growth factor. This thesis was divided into three parts. The purpose of part I was to determine whether our previously observed upregulation of SOD by CMX-8933 is dependent upon (or merely concurrent with) AP-1 activation. Four independent SOD immunoblot experiments demonstrated that pre-treatment of rat primary cortical cultures with trifluoromethyl pyrimidine carboxylate (TFPC), a specific inhibitor of AP-1, significantly (p = 0.0004) decreased cellular levels of SOD by 67% at its IC50 concentration of 1 ìM, and completely inhibited the upregulation at 10 and 100 ìM concentrations. Thus, the CMX-8933-induced upregulation of SOD appears to depend (directly or indirectly) on AP-1 activation. Part II of this thesis included the use of bioinformatics to re-verify exciting recent observations that EPN-like proteins exist in mammals, termed mammalian-ependymin-related proteins or MERPs (Apostolopoulos et al, 2001). If our analyses were convincing, human EPN mimetics would then be designed and tested for AP-1 activation. Computer alignments and hydropathy plots performed with EPN amino acid sequences deduced from gene entries in GenBank verified the existence of mammalian homologs containing highly conserved domains with fish EPN's, suggesting the possibilities of similar protein conformation and function. Two human EPN mimetics were designed, hEPN-1 (8 aa long, corresponding to the same region as CMX-8933) and hEPN-2 (14 aa long, containing CMX-8933 and 6 upstream aa). Several mimetic doses were tested on mouse Neuro-2a cultures for nuclear translocation of c-Jun and c-Fos proteins (comprising the AP-1 particle upregulated by fish CMX-8933). Seven independent c-Jun immunoblot experiments, and five c-Fos experiments, demonstrated a strong (as high as 25-fold) dose-dependent increase in the nuclear titers of the AP-1 proteins. Both peptides had statistically equivalent effects. Thus, human EPN appears to exist, and two mimetics derived from its sequence appear to be biologically active against mouse neuroblastoma cells. Since hEPN-1 and -2 have only a few residues in common with CMX-8933, we hypothesize that the mimetic shape rather than sequence may be important for biological activity. In part III of this thesis, the biological effects of hEPN-1 and hEPN-2 on mouse Neuro-2a cells were studied further using RT-PCR to analyze potential increases in specific mRNAs. mRNAs related to growth, energy production, and protein translation were tested since previous data in our lab (Kaska, 2003) indicated mRNAs for translational elongation factor-2 (EF-2), and ribosomal proteins L19 and S12 were upregulated in rat primary cortical cultures by fish mimetic CMX-8933 (Kaska, 2003). Treatment of Neuro-2a cells with 1.0 ìg/ml hEPN-1 (the highest dose tested for the AP-1 translocation experiments) for 24 hrs appeared to increase (N = 1) mRNAs for ATP Synthase-C, ribosomal protein L19, and translational EF-2, relative to the levels of housekeeper polyubiquitin. Thus hEPN-1 may be involved in processes related to growth. Altogether, the data from this thesis extends our knowledge of fish EPN mimetic CMX-8933 (showing that its induction of SOD requires AP-1), demonstrates that human EPN may exist (bioinformatics), shows that two human EPN mimetics are biologically active (induce AP-1 translocation), and shows that one mimetic hEPN-1 may activate several mRNAs related to growth in mouse Neuro-2a cells.

Ependymin (EPN) is a goldfish brain neurotrophic factor (NTF) previously shown to function in a variety of cellular events related to long-term memory formation and neuronal regeneration. Because of these functions, EPN and other NTFs have potential applications for treating neuro-degenerative conditions, including stroke. In previous experiments, our lab in collaboration with Victor Shashoua of Ceremedix Inc (Boston, MA), designed short synthetic peptide CMX-8933 (a proteolytic cleavage product of EPN) and CMX-9236 (an EPN-Calmodulin combination peptide) that mimic the action of full-length EPN. In a rat stroke model, administration of these peptides i.v. significantly lowered brain ischemic volume (Shashoua et al., 2003). Because oxidative stress is one of the primary mediators of cell damage following a stroke, we hypothesized that NTFs, and in particular our therapeutic peptides, may act in part by reducing neuronal oxidative stress. Thus, the purpose of this thesis was to determine whether CMX-8933 and CMX-9236 increase the cellular titers of anti-oxidative enzymes. A hybridization array was used as a“hypothesis generator" to obtain candidates for further analysis. This approach applied to rat primary brain cortical cells treated with CMX-8933 identified superoxide dismutase (SOD) as strongly upregulated. SOD immunoblots on whole cell lysates, and RT-PCR on total cellular RNA, were used to confirm this observation. In time-course and dose-response experiments, treatment of rat primary cortical cultures with either peptide showed an optimal 8.5 fold (N = 5, p < 0.001) increase in SOD protein, while administration of CMX-8933 to murine neuroblastoma cells caused a 6.5 fold (N = 3, p = 0.001) increase in SOD mRNA levels. Previous work in other laboratories indicated that systemic (i.v.) administration of full-length NTFs allows only an inefficient delivery across the blood brain barrier (BBB). We hypothesized that our short synthetic peptides may cross the BBB more efficiently. Immunoblot analysis of brains and hearts excised from mice treated i.v. with various doses of CMX-8933 confirmed the elevated SOD titers (10 fold in brain, and 8 fold in heart, at a 6 mg/kg dose for 5 hr; N = 5, p < 0.001). Furthermore, we hypothesized that conjugation of CMX-8933 to BBB carrier DHA, a natural neuronal membrane fatty acid shown previously to enhance the delivery of dopamine to the brain (Shashoua and Hesse, 1996), might further enhance the NTF therapy. Delivery of DHA-8933 increased SOD expression by 3 fold (N = 4, p < 0.001) relative to non-conjugated CMX-8933. Recently, the use of special incubators that allow the culture of cells under low oxygen conditions (anoxia) has been used as an in vitro model for stroke. When we tested our peptides in this new in vitro model, surprisingly SOD was upregulated 3 fold (N = 3, p = 0.003) in rat primary cortical cells cultured for 24 hr under oxygen deprivation, compared to normoxic conditions. This implies that these rat cultures may have an endogenous cellular system for responding to oxygen stress, a finding worthy of further investigation. Treatment of anoxic cells with CMX-8933 increased SOD levels another 2.8 fold (N = 3, p < 0.001) compared to the levels for anoxia alone (for a total of 8.5 fold relative to normoxic cells). Altogether, the data from this thesis illustrate that small NTF EPN peptide mimetics increase the cellular titers of the mRNA and protein for the anti-oxidative enzyme SOD, which may be an important step in their known therapeutic benefits.

Abstract not available

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 100-132).

Thesis (M.Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 100-132). Also available in print.

published_or_final_version / Anatomy / Master / Master of Philosophy