Chemical Engineering @ChemengFiles.Com

This work concerns applications of NMR techniques in metabolic engineering studies. As demonstrated here, NMR is a valuable tool in analyzing intracellular metabolic and energetic states. When combined with growth and fermentation studies, it greatly enhances the understanding of cellular responses to particular genetic modifications.

This work concerns applications of NMR techniques in metabolic engineering studies. As demonstrated here, NMR is a valuable tool in analyzing intracellular metabolic and energetic states. When combined with growth and fermentation studies, it greatly enhances the understanding of cellular responses to particular genetic modifications.

Armed with the complete sequence of the human genome and an ever-increasing array of biological techniques, researchers continue to learn more about the genetic basis of diseases. For two decades, scientists and physicians have been developing therapeutic strategies for treating many diseases at the genetic level, creating the field of "gene therapy." For those diseases caused by loss-of-function mutations in a specific gene, delivery of a wild-type copy of that gene to affected cells can reduce or eliminate the disease phenotype. Viruses, having evolved to be extremely effective at delivering nucleic acids (i.e., their own genes for viral production) to cells, have been modified to include therapeutic genes of interest. While such viral gene therapy vectors are the most efficient vectors developed, concerns about their safety and immunogenicity have prompted many to investigate non-viral vector alternatives. Cationic polymers and lipids have emerged as leading non-viral vector materials. Our laboratory has developed a class of cyclodextrin-containing polycations (CDPs) that condense DNA into complexes that can be endocytosed by cells, achieve expression of their genetic payload in those cells, and may be modified to target particular cell types within an animal.

In the past five years, scientists have discovered a new mechanism for the reduction of gene expression in mammalian cells via sequence-specific cleavage of a particular messenger RNA (mRNA); this phenomenon is known as RNA interference (RNAi). Since RNAi is triggered by nucleic acids (small interfering RNA (siRNA) duplexes), I hypothesized that CDPs may be suitable vectors for the delivery of siRNA. In my thesis work, the safety of synthetic siRNA duplexes is examined both in cultured cells and in vivo. Using a number of different siRNA sequences, two different strains of mice, and three different methods of administration, I fail to observe any cytokine (IL-12 or IFN-a) responses, morphological changes, or alterations in complete blood counts (CBCs) or liver enzyme levels.

Armed with the complete sequence of the human genome and an ever-increasing array of biological techniques, researchers continue to learn more about the genetic basis of diseases. For two decades, scientists and physicians have been developing therapeutic strategies for treating many diseases at the genetic level, creating the field of "gene therapy." For those diseases caused by loss-of-function mutations in a specific gene, delivery of a wild-type copy of that gene to affected cells can reduce or eliminate the disease phenotype. Viruses, having evolved to be extremely effective at delivering nucleic acids (i.e., their own genes for viral production) to cells, have been modified to include therapeutic genes of interest. While such viral gene therapy vectors are the most efficient vectors developed, concerns about their safety and immunogenicity have prompted many to investigate non-viral vector alternatives. Cationic polymers and lipids have emerged as leading non-viral vector materials. Our laboratory has developed a class of cyclodextrin-containing polycations (CDPs) that condense DNA into complexes that can be endocytosed by cells, achieve expression of their genetic payload in those cells, and may be modified to target particular cell types within an animal.

In the past five years, scientists have discovered a new mechanism for the reduction of gene expression in mammalian cells via sequence-specific cleavage of a particular messenger RNA (mRNA); this phenomenon is known as RNA interference (RNAi). Since RNAi is triggered by nucleic acids (small interfering RNA (siRNA) duplexes), I hypothesized that CDPs may be suitable vectors for the delivery of siRNA. In my thesis work, the safety of synthetic siRNA duplexes is examined both in cultured cells and in vivo. Using a number of different siRNA sequences, two different strains of mice, and three different methods of administration, I fail to observe any cytokine (IL-12 or IFN-a) responses, morphological changes, or alterations in complete blood counts (CBCs) or liver enzyme levels.

In this thesis, I have developed elastomeric microfabricated cell sorting devices using a micromachining technology, "soft lithography". Inexpensive elastomeric microfabricated devices were designed to replace flow chambers in conventional fluorescence-activatived cell sorters (FACS). Sorting of cells and other particles was accomplished via different means of flow control. My early work of cell sorting on these devices was accomplished using electrokinetic flow. However, in order to alleviate problems associated with electrokinetic flow, the microfabricated cell sorter was integrated with microvalves and micropumps for pneumatic actuation control. The integrated cell sorter has better capabilities of fine-tuning the flow control, manipulating single cells and is less harmful to the cells than electrokinetic flow. Substantial enrichments of beads and cells were accomplished on these devices. Novel sorting algorithms, which can only be implemented in microfabricated devices, were also demonstrated. Compared with conventional FACS, these microfabricated devices allow for more sensitive optical detection for bacterial cells and DNA, innovative sorting schemes and are disposable to eliminate any cross-contamination from previous runs. Ultimately, these elastomeric microfluidic flow cells provide an inexpensive, robust and effective way to perform cell sorting and can be used as stand-alone devices or as a part of an integrated system for diagnostics and/or cytometric measurements.

Presently, the microfabricated cell sorter is enjoying new applications in various fields for high throughput screening, including directed evolution, digital genetic circuits, microbiology and cell biology of gene expression and regulation. In addition, this sorter is not limited only to the detection of optical signals. I have attached the sorter to a high resolution magnetometer, a superconducting quantum interference device (SQUID) microscope, to obtain cytometric data of the magnetic field strengths of magnetotactic bacteria as they flowed through the device.

In this thesis, I have developed elastomeric microfabricated cell sorting devices using a micromachining technology, "soft lithography". Inexpensive elastomeric microfabricated devices were designed to replace flow chambers in conventional fluorescence-activatived cell sorters (FACS). Sorting of cells and other particles was accomplished via different means of flow control. My early work of cell sorting on these devices was accomplished using electrokinetic flow. However, in order to alleviate problems associated with electrokinetic flow, the microfabricated cell sorter was integrated with microvalves and micropumps for pneumatic actuation control. The integrated cell sorter has better capabilities of fine-tuning the flow control, manipulating single cells and is less harmful to the cells than electrokinetic flow. Substantial enrichments of beads and cells were accomplished on these devices. Novel sorting algorithms, which can only be implemented in microfabricated devices, were also demonstrated. Compared with conventional FACS, these microfabricated devices allow for more sensitive optical detection for bacterial cells and DNA, innovative sorting schemes and are disposable to eliminate any cross-contamination from previous runs. Ultimately, these elastomeric microfluidic flow cells provide an inexpensive, robust and effective way to perform cell sorting and can be used as stand-alone devices or as a part of an integrated system for diagnostics and/or cytometric measurements.

Presently, the microfabricated cell sorter is enjoying new applications in various fields for high throughput screening, including directed evolution, digital genetic circuits, microbiology and cell biology of gene expression and regulation. In addition, this sorter is not limited only to the detection of optical signals. I have attached the sorter to a high resolution magnetometer, a superconducting quantum interference device (SQUID) microscope, to obtain cytometric data of the magnetic field strengths of magnetotactic bacteria as they flowed through the device.

Considerable attention has recently been given to the preparation of the ciguatoxins, the major group of toxins implicated with the onset of ciguatera fish poisoning (CFP) – a seafood-borne illness associated with the consumption of reef fish in tropical and subtropical areas. Ciguatoxins are characterized by a very complex polycyclic framework of 13 ether rings ranging from five to nine members. Previous studies directed towards the synthesis of the ciguatoxin skeleton have illustrated the difficulty associated with the construction of medium rings, particularly eight and nine-membered.

We envisaged that an electrophile-induced epoxy-alcohol cascade cyclization, previously developed in our laboratory and utilized in the synthesis of hemibrevetoxin-B, could be used for the construction of medium-ring ethers. As little was known about this cyclization cascade, an investigation has been launched to test the potential of this biomimetic approach in the construction of [6,8]-trans-fused bicyclic ethers and its applicability towards the synthesis of the HIJ rings of the ciguatoxins.

Considerable attention has recently been given to the preparation of the ciguatoxins, the major group of toxins implicated with the onset of ciguatera fish poisoning (CFP) – a seafood-borne illness associated with the consumption of reef fish in tropical and subtropical areas. Ciguatoxins are characterized by a very complex polycyclic framework of 13 ether rings ranging from five to nine members. Previous studies directed towards the synthesis of the ciguatoxin skeleton have illustrated the difficulty associated with the construction of medium rings, particularly eight and nine-membered.

We envisaged that an electrophile-induced epoxy-alcohol cascade cyclization, previously developed in our laboratory and utilized in the synthesis of hemibrevetoxin-B, could be used for the construction of medium-ring ethers. As little was known about this cyclization cascade, an investigation has been launched to test the potential of this biomimetic approach in the construction of [6,8]-trans-fused bicyclic ethers and its applicability towards the synthesis of the HIJ rings of the ciguatoxins.