Thesis (M.S.)--Rowan University, 2007. / Typescript. Includes bibliographical references.

Made available in DSpace on 2014-06-12T15:51:12Z (GMT). No. of bitstreams: 2 arquivo4477_1.pdf: 2619648 bytes, checksum: c371ffaaab9afa32bc9f96397ed74514 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / O presente estudo teve por objetivo analisar o impacto do óleo Diesel na microbiota nativa do solo de manguezal de Vila Velha- Itamaracá, analisando a capacidade biodegradadora natural dos microrganismos nativo presente nesta área. Os resultados mostraram que microrganismos solo de manguezal (fungos e bactérias) quando submetidos a impactos da substituição total de sua fonte de carbono sem um prévio contato com o poluente reduziram no número de colônias. Porém quando o solo foi submetido ao impacto com o poluente, durante trinta dias a reação microbiana foi de um aumento populacional, sendo observado a diminuição na diversidade das formas de colônias e surgimento de outras. Nos estudos realizados com a substituição total da fonte de carbono em meio aquoso sem estimulo a aeração confirmou-se através dos ensaios bioquímicos a presença do gênero Pseudomonas e sua participação no processo de biodegradação, através da redução de componentes do óleo Diesel analisado por cromatografia GCMS. Nos ensaios submetidos à agitação obteve-se um aumento populacional superior ao do impacto no solo, apresentando uma diminuição na tensão superficial em 40% no período de vinte dias, neste a redução do poluente em ensaios cromatográficos apresentou-se maior chegando a 99% no composto decano

Accessible point of care diagnosis of Tuberculosis (TB) is an essential development to better manage the global epidemic that infects 10 million people annually. Current diagnostics are centralised, causing patient loss to follow up or delays in treatment. Although serological diagnosis using finger-prick blood has been historically insensitive in the diagnosis of TB, the detection of anti-mycolic acid (MA) antibodies in patient sera has been shown to allow accurate diagnosis in HIV-positive, previously infected and TB exposed patients. MA is a unique lipid antigen of mycobacteria with anti-MA antibodies being formed early upon infection in a T-cell independent pathway. This work aimed to contribute towards the development of a lateral flow immunoassay termed MALIA (Mycolate Antibodies Lateral flow Immuno Assay). This diagnostic has the unique lipid antigen MA as the immobilised capture agent and custom developed monoclonal anti-MA chicken antibodies (gallibodies) as the labelled bio-recognition element. Casein hydrolysate was newly applied as a blocker in enzyme-immuno assay (EIA) to characterise the functionality of the gallibodies in order to circumvent import restrictions on bovine milk products. MA dissolved in hexane and immobilised on nitrocellulose was not detected by the passively conjugated gold labelled gallibody conjugate in the lateral flow test (LFT) format, despite the confirmation with lipid staining and EIA that MA remained immobilised and antigenic on nitrocellulose. Various substrates, blockers and running buffers were explored for the LFT to attempt to detect MA. The method of passive conjugation of the gallibodies to gold nanoparticles was chosen as this is a commonly successful and simple strategy for conjugate preparation in LFTs. Initial characterisation of gold labelled gallibody conjugate suggested that the orientation of the gallibody on the nanoparticle may be favourable for binding by anti-chicken antibody (the control) but not MA. The biological activity (MA binding) of the gold labelled conjugate was probed on alternative methods. The results showed that in EIA, gold labelling caused the loss of MA binding but not anti-chicken immunoglobulin binding. This is possibly due to the extra force in the wash steps caused by the presence of the gold nanoparticle. Interaction of gold labelled gallibody conjugate with antigenic MA nanoparticles in transmission electron microscopy showed loss of biological activity, while dynamic light scattering intensity measurement of the same interaction showed a weak interaction similar to that seen between gold labelled bovine serum albumin conjugate with fatty acid coated nanoparticles. To address the challenges uncovered by this research, gallibodies can be re-engineered to increase functional affinity (by increasing the valency) to be able to compensate for activity losses due to labelling. In addition, labelling of MA, rather than gallibodies may result in a successful, inverted MALIA to meet the ultimate aim to drastically change the face of the TB epidemic at the critical fault line – point of care diagnosis. The promising avenues uncovered by this key explorative research must be pursued to actualise this critically important and non-standard LFT technology. / Dissertation (MSc)--University of Pretoria, 2019. / Biochemistry / MSc / Unrestricted

Melanoma is one of the most aggressive and inherently resistant cancers and the most dangerous skin cancer. While it accounts for fewer than 5% of skin cancer cases, 80% of skin cancer related deaths are attributed to melanoma. While resection remains the gold standard for melanoma treatment, surgery is only effective in providing local control of the disease if the cancer is detected in the early stages. Once melanoma enters the later stages, and particularly in the metastatic phase, recurrence is probable, and no adequate treatment exists. Previous work in this group has shown that photodynamic therapy (PDT) presents an opportunity to induce cell death in melanoma cells through the production of ROS and singlet oxygen at doses high enough to overwhelm the resistance mechanisms of the cancer. In this study, we investigated the use of recombinant SNAP-tag-based antibody fusion proteins as a means of delivering phototoxic molecules directly to cancer cells expressing the CSPG4 and PD-L1 cell surface receptors. SNAP-tag is an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase. It reacts autocatalytically and in a strictly 1:1 coupling chemistry with substrates that have been modified with benzylguanine (BG). Through genetic fusion of this self-labelling protein with a tumour targeting antibody, we developed a recombinant immunoconjugate able to carry BG-modified photosensitizers to selectively target and eliminate malignant melanoma cells. Conjugation of the SNAP-tag fusion protein with the fluorescent dye Alex Fluor 488 showed that anti-CSPG4-SNAP binds specifically to melanoma cells expressing the CSPG4 surface antigen. Binding was tested across a range of cell lines presenting melanoma in its radial and vertical growth phases, in the metastatic growth phase, in its chemoresistant form, and in both its pigmented and unpigmented forms. This binding data thus confirms CSPG4 as a suitable targeted for this treatment strategy. Conjugation of the fusion protein with the BGmodified photosensitizer IRDye 700DX (IR700) has produced no phototoxicity as of yet. In light of the convincing binding analysis, it is concluded that inefficient solubilization of the lyophilized product resulted in inadequate conjugation of BG-IR700 with SNAP-tag. Nonetheless, steps have been planned to resolve the problem in future ongoing work on this project, and we remain confident in the applicability of this technology. The results for the PD-L1 fusion protein were inconclusive. In summary, SNAP-tag technology offers a simple and efficient method for immunofluorescent detection of cancerous cells. These fusions proteins are versatile as they 1) can contain any antibody targeting a tumour-associated or tumour-specific antigen of choice and 2) can be endowed with a wide variety of substrates, as long as the latter contains the BG moiety.

The native polyamines (putrescine, spermidine, and spermine) are low molecular weight amines that exist as polycations at physiological pH. These polycations interact with DNA, RNA, and influence many cellular processes. Intracellular polyamine levels are maintained via a balance of polyamine biosynthesis, catabolism, and transport. Ornithine, an amino acid obtained from L-arginine, is decarboxylated by ornithine decarboxylase (ODC) to form putrescine. The biosynthesis of the higher polyamines, spermidine and spermine, requires the addition of an amino-propyl group donated by decarboxylated S-adenosylmethionine (dc-SAM), which itself is derived from the amino acid L-methionine (Met). Tumor cells are heavily reliant on methionine because it is required for the translation of proteins, methylation of biomolecules and anabolic processes like polyamine biosynthesis. Pancreatic ductal adenocarcinoma cells (PDAC) are heavily reliant upon polyamines, and one way to affect the growth of PDAC cells is to reduce their intracellular methionine pools. LAT1 is the principal transporter of methionine into human cells and this thesis describes the discovery of a new methionine efflux agonist, which uses LAT1 to export methionine into the extracellular space. As expected, this agonist reduced both intracellular methionine and polyamines levels and inhibited cell growth of L3.6pl human pancreatic cancer cells. The agonist contains two chiral centers. Utilizing organic synthesis, the four possible stereoisomers (RR, SS, SR, and RS) were synthesized and. evaluated for their ability to inhibit L3.6pl cell growth, deplete intracellular methionine levels, and reduce intracellular polyamine pools. A success here may lead to new drugs which target pancreatic cancers via methionine starvation.

Transplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate regeneration following myocardial infarction (MI), however, the role of exosomes derived from ES cells (ES-Exo) in activating resident cardiac stem cells (CSCs) to undergo cardiac differentiation is not established. In the present study, Stem cell antigen 1 positive (Sca-1+ve) CSCs were isolated, incubated with exosomes, and evaluated for differentiation into the major heart cell types in vitro. Observations of in vitro cardiac differentiation were further established in an in vivo model of MI. Ligation of the coronary artery, or a sham surgery was performed in C57BL/6 mice 8-12 weeks of age. Mice were split among four study groups: sham, MI, MI + H9c2-Exo (a cell line control), & MI + ES-Exo. ES-Exo were transplanted via intramyocardial (IM) injection immediately following coronary artery ligation. At day 14 (D14), chocardiography was used to evaluate cardiac function. Differentiation into the major heart cells was determined by sarcomeric α-actin (cardiomyocytes) and smooth muscle α-actin (vascular smooth muscle cells) immunostaining. Hematoxylin and Eosin and Masson’s Trichrome staining assessed cardiomyocyte hypertrophy and fibrosis, respectively. Immunostaining for major heart cellular markers revealed significant activation of resident Sca-1+ve CSCs to undergo cardiac differentiation after ES-Exo treatment. Cardiomyocyte hypertrophy and myocardial fibrosis were significantly increased following coronary artery ligation. Results from histological staining revealed significantly decreased levels of hypertrophy and fibrosis in hearts transplanted with ESExo following coronary ligation. In summary, our findings advocate ES-Exo as a viable treatment option to repopulate the myocardium with viable heart cells, attenuate cardiac remodeling, and rescue cardiac function.

Translational frameshifting is a process that leads to the production of multiple different proteins from a single mRNA. Programmed translational frameshifting is typically utilized by retroviruses to increase genome coding capacity and to control the ratio between proteins necessary for different infection stages. In E. coli, translational frameshifting regulates the production of several essential proteins, including the replication/clamp loader subunits tau (τ) and gamma(γ), which are products of the gene dnaX. In E. coli, τ is an essential auxiliary factor of DNA polymerase III holoenzyme. τ can dimerize Pol III and interact with the DnaB helicase, which allows synthesis of leading and lagging DNA strands. The role of γ is unknown since it does not share similar properties with τ apart for clamp loading; but, γ is implicated in DNA repair and likely has key role in polymerase switching. In this study, the effects of ribosomal availability in the cell on the dnaX frameshift frequencies (regulation of τ/γ) were investigated. It was found that lower ribosomal availability in the cell resulted in lower frameshift frequencies on reporters containing the dnaX frameshift motif. Also, E. coli strains with tagged genomic dnaX and disrupted ompT were generated. Although, it is generally accepted that τ/γ are produced at equimolar concentrations, it was discovered that endogenous γ was produced at substantially higher concentrations than τ when cells were grown in defined media (EZ MOPS). This finding shows that that the ratio of τ/γ can be regulated. These findings suggest there are yet uncharacterized roles of γ, such as polymerase switching, that may be the reason for its production in many bacteria.

Ribosomes translate messenger RNA (mRNA) three nucleotides at a time until translation is terminated at a stop codon. However, during all translation, frameshifting can occur, leading to the formation of proteins with amino acid sequences that differ from the in- frame product. Spontaneous frameshifting can be harmful to an organism. For instance, antibiotics such as streptomycin inhibit bacterial growth by increasing misreading and frameshifting. However, programmed translational frameshifting (which can induce high levels of frameshifting) can be used in some instances to control the ratio of specific proteins (as seen with the dnaX gene) or to increase the density of genomic information. This study explored the effects of endogenous small molecules on the IS3-frameshift-motif that is found in the transposase genes of many mobile elements. Using a cell-free protein synthesis system and a luminescent frameshift reporter, it was discovered that the addition of a small molecule extract derived from E. coli significantly decreased frameshifting, suggesting that it contains molecules that can alter translational fidelity. These experiments also revealed that the addition of the translation inhibitor chloramphenicol to translation assays at sub-inhibitory concentrations, reduced frameshift efficiency. During the studies, the role of the stability of luminescent protein reporters on the reported frameshifting levels was also explored, which is omitted in many studies. Finally, a method was developed that allows for the isolation of molecules that weakly associate with ribosomes, which opens the door for more detailed investigations of chemicals that alter translational fidelity. In conclusion, these studies provide new insight on the potential modulation of translational frameshifting by endogenous small molecules, and they set the stage to reveal the important players in this important biochemical process.

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections worldwide and is also responsible for trachoma, the leading cause of preventable blindness. Despite being susceptible to most antibiotics, Chlamydia trachomatis is an enormously successful pathogen capable of causing serious health issues. Chlamydia trachomatis harbors a type III secretion system which delivers chlamydial proteins (a.k.a. effectors) into the mammalian host cell during the initial stages of infection and throughout the subsequent intracellular developmental cycle. One of the early secreted effectors is called translocated early phosphoprotein (TepP). In order to gain a better understanding of TepP, we employed Drosophila melanogaster as a model organism to identify novel gene functions. Transgenic fruit flies engineered to express TepP in designated tissues were used to identify macroscopic and/or microscopic phenotypic changes to Drosophila development. The fruit fly model allowed us to explore potential roles for TepP in disease progression. Using this in vivo model, a border cell migration defect was observed in the Drosophila egg chambers in response to the Chlamydia trachomatis effector protein TepP. Through knockdown and overexpression studies as well as qPCR we identified a likely interaction in the PVF1/PVR pathway between TepP and Crk to cause this migration defect. Development of this in vivo model provides a platform for rapid and efficient investigation of bacterial effector proteins of unknown function and interrogation of the mechanisms by which these proteins interact with host pathways to contribute to disease. By developing a better understanding of how chlamydial effectors contribute to disease we are able to identify novel targets to explore for prevention of C. trachomatis infections.

In recent years, there is a growing interest in developing metal based antimicrobial nanomaterials suitable for agricultural and biomedical applications. For centuries, Copper (Cu) biocide has been used for protecting a wide variety of crops from devastating bacterial and fungal diseases. However, prolonged and aggressive use of Cu led to the development of resistance and accumulation in soil. The latter has been linked to aquatic toxicity due to soil Cu leaching. Furthermore, copper build up in soil causes phytotoxicity and reduces uptake of micronutrient Zn through the root system. In biomedical field, Cu has been historically used as an antimicrobial agent in wound dressing. In healthcare facilities, Cu based touch surfaces are shown to significantly reduce antimicrobial infection rates. Emerging biomedical applications include wound healing, sensing and even potential use of nano-Cu as cancer therapeutic. However, Cu cytotoxicity is a serious concern. There is a strong need for developing advanced Cu based composite material that will retain antimicrobial properties as reduced Cu load until a suitable alternative becomes available. In this thesis, the objective is to develop a Cu coated Iron Oxide nanocomposite. The idea is to distribute Cu over the high surface area of biocompatible Iron Oxide nanoparticle (IONP) to improve Cu bioavailability. In the design of Cu-IONP nanocomposite, we have introduced N-Acetyl Cysteine (NAC, an antioxidant biomolecule) that anchors IONP to Cu. The composite was synthesized using a co-precipitation technique. Characterization of the Cu/NAC-IONP nanocomposite was done using Atomic Absorption Spectroscopy (AAS), Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron microscopy and X-Ray Photoelectron Spectroscopy. Characterization results support the formation of composite and NAC as a bidentate ligand conjugating Cu to IONP. Antimicrobial activity of Cu/NAC-IONP nanocomposite was studied using ASTM published protocol. The Cu/NAC-IONP nanocomposite shows higher Colony Forming Unit Percent reduction when compared with NAC-Iron Oxide and no treatment. This suggests that antimicrobial activity of Cu is retained in the nanocomposite. Additionally, AAS study revealed an interesting property of the nanocomposite that the Cu release is strongly dependent on incubation temperature. Cu ion release is increased with the increase in incubation temperature. This new finding may lead to design of a delivery system where Cu release can be controlled by tuning temperature.