O parasita da malária Plasmodium falciparum percebe o ambiente em que se encontra, elaborando respostas celulares adequadas, que envolvem secreção de proteínas, crescimento e diferenciação celular. Fatores relacionados com a geração de segundos-mensageiros e proteínas efetoras da sinalização celular estão descritos na literatura. Porém, a função de receptores responsáveis pela percepção de estímulos extracelulares no parasita é um tema pouco explorado. A identificação in silico de receptores de sete domínios transmembrânicos putativos no genoma de P. falciparum possibilitou a exploração da função dos mesmos. A tese caracteriza funcionalmente dois receptores, PFSR10 e PFSR25. A expressão proteica dos receptores foi demonstrada em fases eritrocíticas de P. falciparum. Os receptores possuem candidatos a parceiros moleculares que executam diversas funções celulares, entre elas invasão do eritrócito, endocitose e exocitose. Os receptores foram transfectados em células de mamíferos e, através de ensaios de dinâmica de cálcio de high-throughput, sugere-se que PFSR10 codifique um receptor que participa na percepção de ATP extracelular e que PFSR25 codifique um sensor de KCl. O trabalho também sugere que KCl modula cálcio citosólico em P. falciparum e que parasitas nocaute para PFSR25 são incapazes de modular cálcio citosólico em resposta a KCl. / The malaria parasite P. falciparum perceives its milieu and elaborates adequate intracellular responses, that involve protein secretion, growth and cell differentiation. Factors related to second messengers generation and effectors of cell signaling are described in the literature. However, the function of receptors responsible for stimulus perception remains elusive. The in silico identification of putative seven transmembrane receptors in the Plasmodium falciparum genome allowed the exploration of their function. In the thesis, two putative receptors were characterized, PFSR10 and PFSR25. The proteic expression of the receptors was demonstrated in erythrocytic stages of P. falciparum. The receptors have putative interaction partners that participate in cellular functions such as invasion, exocytosis and endocytosis.The receptors were transfected in mammalian cells and, through high-throughput calcium dynamics assays, it is suggested that PFSR10 codes for a receptor that participates in extracellular ATP perception and that PFSR25 codes for a KCl sensor. It is also suggested that KCl modulates cytosolic calcium in response to KCl and that knockout parasites for PFSR25 are incapable of modulating cytosolic calcium in response to KCl.

The mitochondrion is the powerhouse of the cell. Therefore, it is critical to the homeostasis of the cell that populations of mitochondria that are damaged or in excess are degraded. The process of targeted elimination of damaged or excess mitochondria by autophagy is called mitophagy. In this report, analysis of the mitophagy regulators PINK1/PARKIN and BNIP3L and their roles are assessed in the lens. PARKIN, an E3 ubiquitin ligase, has been shown to play a role in directing damaged mitochondria for degradation. While BNIP3L, an outer mitochondrial membrane protein, increases in expression in response to excess mitochondria and organelle degradation during cellular differentiation. We have shown that PARKIN is both induced and translocates from the cytoplasm to the mitochondria in human epithelial lens cells upon oxidative stress exposure. In addition, our findings also show that overexpression of BNIP3L causes premature clearance of mitochondria and other organelles, while loss of BNIP3L results in lack of clearance. Prior to this work, PARKIN mediated mitophagy had not been shown to act as a protective cellular response to oxidative stress in the lens. This project also resulted in the novel finding that BNIP3L-mediated mitophagy mechanisms are required for targeted organelle degradation in the lens. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica / d by in vivo micro dialysis. Based on these observations, we conclude that calpain cleavage of fGAD65 occurs under pathological conditions. / by Chandana Buddhala. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Male C7BL/6J mice were implanted with bilateral dorsal CA1 guide cannulae. After confirming that intrahippocampal microinfusion of muscimol impaired hippocampal function, demonstrated by impaired performance in the Morris water maze, the influence of intrahippocampal muscimol was tested in the Novel Object Recognition paradigm. During a test session 24 h after the last habituation/sample session, mice were presented with one familiar object and one novel object. Successful retention of object memory was inferred if mice spent more time exploring the novel object than the familiar object. Results demonstrate that muscimol infused into dorsal CA1 region prior to the test session eliminates novel object preference, indicating that the hippocampus is necessary for the retrieval of this non-spatial memory - a topic that has garnered much debate. Understanding the similarities between rodent and human hippocampal function could enable future animal studies to effectively answer questions about diseases and disorders affecting human learning and memory. / by Lisa Rios. / Thesis (M.A.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Huntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal huntingtin (Htt). However, it diffuses in the cytosol of cells expressing mhtt. Palmitoylation of GAD65 is required for GAD65 trafficking, and I demonstrated the palmitoylation of GAD65 is reduced in the HD model. Overexpression of huntingtin-interacting protein 14 (HIP14), the enzyme that palmitoylates GAD65, rescues GAD65 palmitoylation and vesicle-associated trafficking. This data suggests that impairment of GAD65 palmitoylation by mhtt may alter its localization and lead to altered inhibitory neurotransmission in HD. / by Daniel Rush. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Ischemic stroke has a multiplicity of pathophysiological mechanisms. Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exerts a diverse range of neuroprotection against ischemic stroke. Several lines of evidence demonstrated the contribution of endoplasmic reticulum (ER) in apoptotic cell death involving ischemia. Cell culture of undifferentiated PC12 cells were subjected to 10mM glutamate and selected doses of G-CSF (25ng/ml, 50ng/ml, 100ng/ml and 250ng/ml) for 24 hours. Cell viability, expression of the G-CSF receptor and expression level of CHOP were assessed in vitro. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). Rats were subcutaneously injected with G-CSF (n= 15; 50ug/kg body weight) 24 hours post-MCAO for 4 days. Vehicle treated rats were administered 5% dextrose for 1 day (n=4) or 4 days (n=16). Sham-operated rats (n=9) were not subjected to MCAO. Neurological deficit and infarct volume were measured while expression levels of pAKT, Bcl2, Bax, Bak, cleaved caspase-3, GRP78, ATF4, ATF6, p-p38MAPK, pJNK, CHOP and HSP27 were analyzed by western blotting. In vitro G-CSF receptor was expressed on undifferentiated PC12 cell, and an optimal dose of 50 ng/ml G-CSF significantly protected these cells against glutamate-induced cytotoxicity (P < 0.05). G-CSF significantly down-regulated (P < 0.01) the ER stressinduced pro-apoptotic marker CHOP in vitro. In vivo, G-CSF reduced infarct volume to 50% while significantly improved neurological deficit compared to vehicle rats. G-CSF significantly (P < 0.05) up-regulated pro-survival proteins pAKT and Bcl2 while downregulating pro-apoptotic proteins Bax, Bak and cleaved caspase 3 in the ischemic brain. It also significantly (P < 0.05) downregulated the ER intraluminal stress sensor GRP78, proteins of ER stress induced intracellular pathway; ATF4, ATF6, p-p38MAPK, pJNK and the ER stress induced apoptotic marker CHOP, which suggests that ER stress is being ameliorated by G-CSF treatment. G-CSF also reduced the level of HSP27, providing additional evidence of cellular stress reduction. G-CSF treatment increased cell survival by attenuating both general pro-apoptotic proteins and specific effector proteins in the ER stress induced apoptotic pathways. Our data has provided new insight into the anti-apoptotic mechanism of G-CSF, especially as it relates to ER stress induced apoptosis in ischemia. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25. / by Daniel C. Chauss. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

This study evaluated the stability of oral bacteria in healthy subjects and documented community shifts in smokers and oral/periodontal disease by employing PCR-RFLP, DGGE and sequence analysis of the 16S rDNA gene from metagenomes and plate-wash (cultured) bacteria of oral wash from 15 participants,. A stable core of bacterial DNA fingerprint was detected within and between subjects and did not change over time when analyzed in smokers and healthy non-smokers. Signature bands in smokers, non-smokers and periodontal disease subjects were evident suggesting the presence of potential indicators of health and poor oral health. Taxon diversity was higher in smokers including members of the genera Rothia, Synechococcus, Neisseria, Thiomargarita and Pyrobaculum but highest in periodontal disease. The two techniques successfully aligned the subjects within appropriate categories (based on their oral microbial genetic patterns)confirming their diagnostic suitability. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

The ubiquitin ligase Highwire is responsible for cell-autonomously promoting synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects in synaptic function and extra branching at the axon terminal, corresponding to transient branching that occur in the course of giant synapse formation during metamorphosis. The MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner, suggesting different molecular mechanisms downstream of the MAP kinase pathway govern function and pruning. A novel role for Highwire is revealed, regulating the giant fiber axon’s ability to respond to external cues regulated by Fos. When expression of the transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection