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- Title
- EXPRESSION OF GAL/GALNAC LECTIN OF ENTAMOEBA HISTOLYTICA IN TRANSGENIC CHLOROPLASTS TO DEVELOP A VACCINE FOR AMEBIASIS.
- Creator
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Chebolu, Seethamahalakshmi, Daniell, Henry, University of Central Florida
- Abstract / Description
-
Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the...
Show moreAmebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.
Show less - Date Issued
- 2005
- Identifier
- CFE0000511, ucf:46467
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000511
- Title
- GLOBALLY-ASYNCHRONOUS, LOCALLY-SYNCHRONOUS WRAPPER CONFIGURATIONS FOR POINT-TO-POINT AND MULTI-POINT DATA COMMUNICATION.
- Creator
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Ravi, Akarsh, Yuan, Jiann, University of Central Florida
- Abstract / Description
-
Globally-Asynchronous, Locally-Synchronous (GALS) design techniques employ the finer points of synchronous and asynchronous design methods to eliminate problems arising due to clock distribution, power dissipation, and large area over head. With the recent rise in the demand for System-on-a-Chip (SoC) designs, global clock distribution and power dissipation due to clock distribution are inevitable. In order to reduce/eliminate the effects of the global clock in synchronous designs and large...
Show moreGlobally-Asynchronous, Locally-Synchronous (GALS) design techniques employ the finer points of synchronous and asynchronous design methods to eliminate problems arising due to clock distribution, power dissipation, and large area over head. With the recent rise in the demand for System-on-a-Chip (SoC) designs, global clock distribution and power dissipation due to clock distribution are inevitable. In order to reduce/eliminate the effects of the global clock in synchronous designs and large area overhead in asynchronous designs, an alternative approach would be to utilize GALS design techniques. Not only do GALS designs eliminate the issue of using a global clock, they also have smaller area overhead when compared to purely asynchronous designs. Among the various GALS design approaches proposed till date, this thesis focuses on the working and implementation of Asynchronous Wrapper designs proposed by Muttersbach et al., in [1, 2]. This thesis specifically addresses different approaches to incorporate the wrappers in VLSI circuits, rather than discussing the efficiency and viability of GALS design techniques over purely synchronous or asynchronous approaches. It has been proven by researchers [3] that GALS design approaches bring down power consumption due to the elimination of the global clock by small amounts, but there is also a drop in performance. Since the goal of this thesis is to introduce the reader to GALS design techniques and not prove their efficiency, it is out of the scope of this thesis to validate the results shown in [3]. In our aim to introduce the reader to GALS design techniques, we first provide a comparison of synchronous and asynchronous design approaches, and then discuss the need for GALS design approaches. We will then address issues affecting GALS such as metastability, latency, flow control, and local clock alteration. After familiarizing the reader with the issues affecting GALS, we will then discuss various GALS design techniques proposed till date. We show the use of asynchronous FIFOs and asynchronous wrappers to realize GALS modules. Two wrapper design approaches are discussed: one being the asynchronous wrapper design proposed by Carlsson et al., in [4], and the other being the asynchronous wrapper design proposed in [1, 2]. An in-depth discussion and analysis of the wrapper design approach proposed in [1, 2] is provided based on the state transition graphs (STGs) that characterize the port-controller AFSMs. Various data transfer channel configurations that incorporate the wrapper port-controllers are designed and realized through VHDL codes, with their functioning verified through simulation results. Design examples showing the working of asynchronous wrappers to achieve point-to-point, synchronous-synchronous and synchronous-asynchronous data communication are provided. Finally, a design example to achieve multi-point data communication is realized. This example incorporates a previously proposed idea. We provide a modification to this idea by designing an arbiter that arbitrates between two separate requests coming into a multi-input port. Through the above design examples, the functionality and working of GALS asynchronous wrappers are verified, and recommendations for modifications are made to achieve flexible multi-point data communication.
Show less - Date Issued
- 2004
- Identifier
- CFE0000238, ucf:46245
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000238
