This dissertation describes a study utilizing bioinformatics to analyze homologues of a molecular chaperone, glucose-regulated protein 78 (grp 78), also known as BiP. The selected homologous proteins originate from organisms of infinitely diverse genera. Comparisons of protein sequence yielded the first clues of a common ancestry among these proteins. Furthermore, protein molecular weights, isoelectric points, N-terminal amino acids and half-lives of a known homolog and a non-homologous protein were examined. Additionally, electroporation, a state-of-the-art plasmid insertion technique, was explored using Chlamydomonas reinhardtii, a green alga, as the recipient of a parent plasmid, pSP124S. Distinctive hypertonic solutions and three separate field strengths were used in the plasmolysis of the cell wall of C. reinhardtii and subsequent electroporation, respectively. The number of transformants was tallied to evaluate which electroporation condition would yield the most transformed colonies. We had two discrete hypotheses: 1) that a structurally and functionally similar protein to glucose-regulated protein 78 exists across a wide spectrum of organisms and 2) that Chlamydomonas reinhardtii could be successfully transformed with pSP124S under certain electroporation conditions. The bioinformatics investigation revealed that analogous proteins to Human GRP 78 existed in Mus musculus (mouse), Rattus norvegicus (rat), Gallus domesticus (chicken), Gallus domesticus (chicken), Mesocricetus auratus (golden hamster), Bos taurus (cow), Xenopus laevis (frog), and Spinacia oleracea (spinach). Moreover, these homologous proteins more likely have a common evolutionary origin.

The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), a non-protein amino acid, first attracted attention in correlation to reports of high incidence of the unusual neurological disease amyotrophic lateral sclerosis/Parkinsonism-dementia (ALS/PDC) among the people of Guam in the South Pacific Ocean. Experimental studies have revealed that BMAA causes neuronal cell death. The neurotoxin is suggested to act via excitotoxicity through interaction with glutamatergic receptors. More importantly, BMAA is suggested to misincorporate in the synthesis of proteins, and contribute to protein misfolding and/or deleterious aggregation, which are hallmarks of several neurodegenerative disorders. A selective uptake of BMAA in the rat neonatal hippocampus can interfere with brain development, causing learning and memory impairments in adult rats. The aim of the present study was to investigate the effects of BMAA in human neuroblastoma SH-SY5Y cells. These cells were exposed to BMAA (10 μM, 50 μM, 100 μM or 500 μM) for 72 hours, and the expression of five selected proteins, including heat shock protein-27 (HSP-27), lysosomal associated membrane protein-1 (LAMP-1), CCAAT-enhancer-binding protein homologous protein (CHOP), Golgi associated plant pathogenesis related protein-2 (GLIPR-2), and glucose regulated protein-78 (GRP-78). They were carried out with immunohistochemistry (IHC). Results revealed an increased expression of all selected proteins, which indicates an uptake and shows the effects of BMAA in the cell cultures. Taken together, BMAA caused cellular stress, including endoplasmic reticulum (ER) stress that is correlated with HSP-27, LAMP-1, CHOP, GLIPR-2, and GRP-78. Further studies are needed in order to support the results. The experiments require being repeated using the same biomarkers as well as a combination of them with other biomarkers to elucidate the effects of BMAA.