published_or_final_version / Paediatrics / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Traumatic brain injury (TBI) is a devastating health problem based on its high incidence, economic burden, and lack of effective pharmacological treatment. Individuals who suffer an injury often experience lifelong disability. TBI results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components, membrane receptors, and cytosolic proteins, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. A comprehensive analysis using two separate calpastatin transgenic mouse lines was performed to test the hypothesis that calpastatin overexpression will reduce posttraumatic calpain activity affording neuroprotection and behavioral efficacy. Increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a neuron-specific calcium-calmodulin dependent kinase II alpha or a ubiquitous prion protein promoter. Both transgenic lines exhibited enhanced calpastatin expression within the brain, extending into peripheral tissues under the prion protein promoter. hCAST overexpression significantly reduced protease activity confirmed by reductions in acute calpain-mediated substrate proteolysis in the cortex and hippocampus following controlled cortical impact brain injury. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points in either transgenic line. Partial hippocampal neuroprotection observed at a moderate injury severity in neuron-specific calpastatin overexpressing transgenic mice was lost after severe TBI. Greater levels of calpastatin under the prion protein promoter line failed to protect against hippocampal cell loss after severe brain injury. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. However, the reduction in proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury. Augmenting calpastatin levels may be an effective method for calpain inhibition and may have efficacy in reducing behavioral morbidity after TBI and neurodegenerative disorders.

Refuges of non-Bacillus thuringiensis (Bt) cotton are used to delay Bt resistance in several key insect pests. In 2004, I discovered that Bt cotton plants sometimes enter refuges via the seed bag, and hypothesized that this type of gene flow could have important effects on resistance evolution in insect pests. In the research described herein, I investigated the sources of Bt plants in the non-Bt cotton seed supply and assessed the potential implications of this gene flow on pest resistance evolution. I report results from an empirical study of gene flow in 15 non-Bt cotton seed production fields, as well as results from simulation modeling studies of gene flow from one-toxin and two-toxin Bt cotton. The current policy on gene flow from genetically engineered crops in the United States is also reviewed, including the implications of my research findings for policymakers. Key findings of this study included the prominent role of seed-mediated gene flow in the seed-production setting, and the utility of a geographic information system (GIS) ring analysis approach for describing pollen-mediated gene flow in cotton fields. Modeling results indicated that high rates of gene flow of Bt cotton into refuges could have large effects on pest resistance evolution under certain sets of assumptions, particularly in parts of the world where farm-saved seed is planted year after year in cotton fields. It appears that some of these effects could be mitigated by using non-cotton refuges or by using plants that contain linked transgenes that confer multiple toxins. There are no clear regulations in the United States regarding gene flow of Bt cotton into refuge seed or into seed production fields of non-Bt cotton, as Bt cotton has been deregulated following extensive safety testing. Nevertheless, results from this research suggest that limiting gene flow into refuge seed could be important for sustaining the efficacy of Bt cotton against targeted insect pests in regions where refuges are used.

Šio darbo tikslas buvo patikrinti ar įmanoma padidinti mielių Saccharomyces cerevisiae K2 tipo kilerinio preprotoksino geno raiškos galimybes augaluose, naudojant konstitutyvų žiedinio kopūsto mozaikos viruso CaMV 35S promotorių. Šiam tikslui pasiekti buvo atlikta: neonkogeninių agrobakterijų (Agrobacterium tumefaciens) kamieno konjugantų atrinkimas, turinčių augalų transformacijos vektorių pART27-KillK2 su mielių K2 tipo kilerinį preprotoksiną koduojančiu genu; atlikta paprastojo tabako (Nicotiana tabacum L.) transformacija minėtų agrobakterijų pagalba bei selektyvi Nicotiana tabacum L. transgeninio kaliaus atranka. Po sėkmingos Agrobacterium tumefaciens konjugacijos su E.coli kamienu, turinčiu kilerinę plazmidę pART27-KillK2 ir sėkmingos modelinio augalo N.tabacum agrobakterinės lapų eksplantų transformacijos, buvo taikomas PGR metodas bei vykdomas kileriškumo patikrinimas. PGR ir DNR elektroforezės pagalba buvo nustatyta, kad KillK2 genas yra įsiterpęs į augalo genomą ir vyksta silpna šio geno raiška, tačiau žiedinio kopūsto mozaikos viruso CaMV 35S promotoriaus prieš šį geną aptikti nepavyko. Ankstesnių tyrimų metu buvo manoma, kad K2 tipo kilerinio preprotoksino geno informacija transformuotuose augaluose su vektoriumi pGA482-KillK2 yra realizuojama kuomet šio geno transkripcija tiesiogiai reguliuojama mielių alkoholdehidrogenazės ADH1 promotoriumi, tačiau šio darbo metu, atliekant tyrimus, buvo parodyta, kad promotorius ADH1 taip pat nėra prisijungęs prie geno KillK2... [toliau žr. visą tekstą] / The aim of this work was to verify the possibility to increase expression of yeast (Saccharomyces cerevisiae), K2 type killer preprotoxin in plants, using constitutive cauliflower mosaic virus CaMV 35S promoter. In order to achieve it, the following was done: selection of unoncogenic strain of agrobacterium (Agrobacterium tumefaciens) with plant transformation vector pART27-KillK2, containing K2 type killer preprotoxin gene; transformation of plain tobacco (Nicotiana tabacum L.) with mentioned agrobacteria; selection of Nicotana tabacum L. transgenic callus. Following the success of the Agrobacterium tumefaciens conjugation with E.coli strain (containing killer plasmid pART27-KillK2) and successful transformation of model N.tabacum plant with A.tumefaciens, there was used the PCR method to check out the killer effect of it. PCR and DNA electrophoresis showed that KillK2 gene is incorporated into plant genome and there is weak expression of this gene. However there was not found the promoter of couliflower mosaic virus CaMV 35S upstream this gene. In the previous studies, it was considered that the expression of mentioned transformed gene exist only when the transcription of it is directly regulated by alcoholdehidrogenase ADH1 promoter. However after investigation it was shown that promoter ADH1 isn’t connected to beginning of gene KillK2. There is the possibility that both ADH1 and CaMV35S promoters are remote from the beginning of KillK2 gene, or the iniciation is done by... [to full text]

Pax6 encodes a highly conserved transcriptional regulator that is widely expressed during development of the eye, olfactory bulbs and central nervous system. Pax6-/- mice exhibit severe brain defects, lack eyes and nasal structures, and die at birth. Included among the functions of Pax6 are cell adhesion, cell cycle progression, axon guidance and boundary formation. The pallial-subpallial boundary (PSPB) is both a physical and gene expression boundary separating dorsal and ventral telencephalon. Pax6 is required for this boundary to develop. In Pax6-/- embryos, genes which normally have a sharp border of expression at the PSPB become ectopically expressed and the radial glial fasicles that make up the physical component of the boundary fail to form. There is also an increase in the number of interneurons migrating dorsally across the boundary to enter the cortex while corticofugal axons struggle to cross the PSPB and enter the ventral telencephalon. Here a novel tauGFP-Pax6 reporter mouse, DTy54, is described in which cells capable of expressing Pax6 are tauGFP positive. In general the expression pattern of tauGFP corresponds well with the known Pax6 expression pattern in the eye and forebrain and the gradient of cortical Pax6 expression from high rostro-laterally to low caudo-medially is also recapitulated by tauGFP. The cytoskeletal localisation of the tauGFP also labels cellular processes and the axons projecting from Pax6 positive cells such as those forming the optic nerve can be clearly seen. At E10.5 the forebrain expression patterns of tauGFP and Pax6 correspond exactly, but at later stages tauGFP expression can be seen in areas negative for Pax6. This can be seen at E12.5 in the ventral telencephalon and in both the dorsal and ventral telencephalon at E15.5. Pax6 and tauGFP expression colocalise more closely in the diencephalon. In situ hybridization analysis of Pax6 and tauGFP transcripts suggests that many of the discrepancies in expression seen at the protein level are due to a longer protein half-life for tauGFP than for Pax6. The expression of tauGFP allows the PSPB to be accurately dissected. The cells from this region can then be sorted by FACS to isolate cells expressing high levels of tauGFP and enrich for the Pax6 positive population. Microarray analysis of gene expression is this population of cells in Pax6+/+.DTy54+ and Pax6sey/sey. DTy54+ embryos is described here. This analysis identified many genes that show a significant change in expression at the PSPB in the absence of Pax6 expression including Ngn2, Lhx6, Neurod6 and CyclinD1 and 2. The biological processes and molecular functions in which these genes are involved were examined to provide insight into the role of Pax6 in this population of cells. Several processes previously reported to be regulated by Pax6 were identified together with a number of novel processes with which Pax6 has not formerly been associated. Some of these include cell cycle, neurogenesis, transcription and metabolic and signalling pathways. This study has also identified many novel downstream targets of Pax6, such as Sema3G and PlexinA4, which will help to elucidate the genetic basis for the Pax6sey/sey phenotype at the PSPB. The changes in expression levels of Ngn2, Lhx6 and Gsh2, identified by microarray, were validated by in situ hybridization, which showed a good correspondence with the microarray results.

Cell-to-cell communication is one of the fundamental requisites of making multicellular organisms. G protein-coupled receptors (GPCRs) are one of the most abundant receptor-types within vertebrates. They canonically mediate their signal via hetrotrimeric G proteins, and G protein signaling is regulated by regulators of G protein-signaling (RGS). One of these RGS proteins, RGS4, is preferentially expressed in the central nervous system of humans and has been strongly connected to dopaminergic signaling, along with a number of severe neuronal diseases. rgs4 is not well studied in the model organism Danio rerio, the zebrafish, with only two publications. In this project, a newly constructed transgenic line, tg(rgs4:mCherry), with the fluorophore mCherry regulated by the promoter element of rgs4 was characterized in order to investigate fidelity to endogenous rgs4 expression and the utility of the transgenic line. The mCherry expression is apparent by 48 hours post fertilization, and expression is found mainly in neuronal tissue. Cell bodies are visible only in some labeled areas, while other areas show a more diffuse signal indicative of projections. There is only one transgenically labeled area that also unambiguously expresses rgs4; the pronephric tubule. This line is therefore not particularly well suited for rgs4-specifc studies, but this does not discredit the fidelity of the construct. A transgenic line made with a site-directed technique would most likely confer the fidelity of the promoter to the expression of the fluorophore. A way of increasing the labeling resolution includes exchanging the mCherry fluorophore for one with stronger signal and a lower tendency to aggregate, e.g. eGFP. Increasing the resolution of the characterization, e.g. to the level of sub-nuclei or neuronal types, would serve to enhance the utility of the line. As it is, the tg(rgs4:mCherry) zebrafish line has limited uses, and yet it is not without them.

Previous work has shown D1/D2 requisite synergism can still occur in the striatum in the absence of action potentials. Some nonclassical communication such as gap junctions may be allowing the segregated dopamine (DA) receptors to interact to produce stereotyped motor activity. Connexin-32 (Cx32) and connexin-36 (Cx36) were targeted for study due to their abundance in neural tissues and presence in the striatum. Mice lacking either the Cx32 or Cx36 gene and their respective wildtype littermates were compared on a climbing behavior task used to gauge their dopaminergic activity after receiving either saline, D1 agonist, D2 agonist, or both D1 and D2 agonists. The results showed that D1/D2 requisite synergism was still intact in both strains of mice. The Cx32 WT mice displayed significantly greater scores than the KO mice in the D1/D2 treatment. The Cx36 mice did not display a significant genotype difference, but a trend was observed with the KO females having larger scores relative to WT females or to males of either genotype.

Due to the stochasticity, diversity and variability of gene expression, transgenic crop study, is confronted with some uncertainties, such as what kinds of the influence from foreign gene on the transgenic crop, and how to fulfill the monitoring of transgenic crop growth real-/ near real-time efficiently. The influence of foreign gene could be treated as a special source of stress to vegetation. Therefore, it is promising to detect the difference between transgenic and contrast group and so as to monitor the growth of sample to assist to fulfill sample screening work, focusing on the plant biophysical traits or responses to stress by spectral techniques. Hyperspectral remote sensing technique is a kind of practical and field spectroscopy technique, which is simple, rapid, real-/ near real-time, user friendly and cheap. In this study, this technique was employed to indicate the differences between transgenic crop samples and their parents, and to monitor their growth. By the proposed approach, fine spectra of transgenic paddy rice were obtained, and the growth of samples were monitored the by their biophysical traits, finally the screening of cultivars were fulfilled in contrast controlled experiments. The biophysical traits or bio-process were concentrated on rather than on micro-structure or components of proteins. It will be implemented to monitor the growth of the samples real-/ near real-time, helping researchers know their samples clearly and screen samples efficiently. / In order to develop and validate this approach, 6 experiments in different fields were conducted, including three kinds of genomes and their transgenic samples. They were classified as the experiment-repeat experiments and the gene-repeat experiments. Moreover, a three-month experiment was also conducted for evaluating the capability of the approach to monitor the sample growth under the condition of an artificial stress (herbicide stress). Morphologic and parameterized features of foliar spectra of samples were applied to indicate the growth of the samples. / In the future, more factors should be considered. They are mainly: much more effective communication with biological researchers should be conducted; more research methods should be introduced, the study scope should be extended to the whole bands (350-2500nm) and more foliar chemicals should be involved as indicators of the growth status of the samples, etc. ii / The results proved this approach proposed was not a substitute to the popular methods for gene detection and crop assessment, but an important, helpful and efficient complement to make the crop breeding study under control and efficient as much as possible. By the approach, the researcher could know their samples clearly and real-/near real- time. / Li, Ru. / Advisers: Jinsong Chen; Hui Lin. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Henry, Kam Yin Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 148-160). / ZEBRAFISH (BRACHYDANIO RERIO) / ACKNOWLEDGEMENTS / LIST OF CONTENTS / ABSTRACT / ABBREVIATION / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Transgenic fish --- p.1 / Chapter 1.2 --- Zebrafish --- p.4 / Chapter 1.3 --- The grass carp GH gene and protein / Chapter 1.3.1 --- The genomic sequence --- p.5 / Chapter 1.3.2 --- The cDNA sequence --- p.7 / Chapter 1.3.3 --- The grass carp GH protein --- p.7 / Chapter 1.4 --- Functional aspects of promoter regions / Chapter 1.4.1 --- PEPCK --- p.9 / Chapter 1.4.2 --- RSV-LTR --- p.10 / Chapter 1.4.3 --- hMT-IIA --- p.10 / Chapter 1.4.4 --- MMTV-LTR --- p.11 / Chapter 1.5 --- Eukaryotic gene expression in cultured cells / Chapter 1.5.1 --- COS-7 and HepG2 cells --- p.11 / Chapter 1.5.2 --- Transfection system --- p.12 / Chapter 1.5.3 --- Fate of DNA after transfection --- p.13 / Chapter 1.6 --- Electroporation and microinjection as tools for gene transfer / Chapter 1.6.1 --- Electroporation: Theory and operation --- p.13 / Chapter 1.6.2 --- Microinjection: Design of microinjector --- p.16 / Chapter 1.6.3 --- Fate of DNA after gene transfer in embryos / Transient expression --- p.16 / Stable transformation --- p.17 / Inheredity of transgene --- p.17 / Chapter 1.7 --- The aims of the present study --- p.18 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Electrophoresis of DNA / Chapter 2.1.1.1 --- Agarose gel electrophoresis --- p.19 / Chapter 2.1.1.2 --- PAGE --- p.20 / Chapter 2.1.2 --- Purification of DNA --- p.21 / Chapter 2.1.3 --- Recovery of DNA fragments / Chapter 2.1.3.1 --- Electroelution --- p.22 / Chapter 2.1.3.2 --- Geneclean kit --- p.23 / Chapter 2.1.4 --- Standard recombinant DNA techniques / Chapter 2.1.4.1 --- Dephosphorylation --- p.24 / Chapter 2.1.4.2 --- Kinasing --- p.24 / Chapter 2.1.4.3 --- Ligation --- p.24 / Chapter 2.1.4.4 --- Filling in reaction --- p.25 / Chapter 2.1.4.5 --- Transformation --- p.25 / Chapter 2.1.5 --- Minipreparation of plasmids --- p.26 / Chapter 2.1.6 --- Large preparation of plasmids / Chapter 2.1.6.1 --- Qiagene kit --- p.27 / Chapter 2.1.6.2 --- CsCl density gradient centrifugation --- p.27 / Chapter 2.1.7 --- DNA sequencing --- p.29 / Chapter 2.1.8 --- "Extraction of DNA from embryos, fry and fish" / Method 1 --- p.32 / Method 2 --- p.32 / Chapter 2.1.9 --- Probe labelling / Chapter 2.1.9.1 --- End-labelling --- p.33 / Chapter 2.1.9.2 --- Random priming --- p.33 / Chapter 2.1.10 --- CAT assay --- p.33 / Chapter 2.1.11 --- Polymerase chain reaction(PCR) --- p.35 / Chapter 2.1.12 --- Radioimmunassay(RIA) of FGH --- p.36 / Chapter 2.1.13 --- Dot blotting --- p.38 / Chapter 2.1.14 --- Southern blotting --- p.39 / Chapter 2.2 --- "Linkers, primers and probes" / Chapter 2.2.1 --- Primers --- p.41 / Chapter 2.2.2 --- Linkers --- p.45 / Chapter 2.2.3 --- Probes --- p.47 / Chapter 2.3 --- Construction of chimeric growth hormone genes / Chapter 2.3.1 --- Sources of plasmids --- p.50 / Chapter 2.3.2 --- General principles --- p.50 / Chapter 2.3.3 --- PEPCKgcGHcDNA --- p.51 / Chapter 2.3.4 --- RSVgcGHcDNA --- p.54 / Chapter 2.3.5 --- hMTgcGHcDNAcDNA --- p.56 / Chapter 2.3.6 --- MMTVgcGHcDNA --- p.58 / Chapter 2.3.7 --- "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.60 / Chapter 2.4 --- Expression of chimeric genes in cultured cells / Chapter 2.4.1 --- Culturing of COS-7 and HepG2 cells --- p.66 / Chapter 2.4.2 --- Expression of chimeric genes in COS-7 and HepG2 cells --- p.67 / Chapter 2.5 --- Zebrafish / Chapter 2.5.1 --- "Culturing, Spawning and hatching" --- p.67 / Chapter 2.6 --- Electroporation and microinjection for gene transfer / Chapter 2.6.1 --- Electroporation / Chapter 2.6.1.1 --- Tuning up electroporation --- p.69 / Chapter 2.6.1.2 --- Evidence of gene transfer by electroporation / Chapter 2.6.1.2.1 --- CAT assay --- p.71 / Chapter 2.6.1.2.2 --- Dot blot --- p.71 / Chapter 2.6.1.2.3 --- PCR and Southern blotting of PCR products --- p.72 / Chapter 2.6.1.2.4 --- Southern blotting of fish total DNA --- p.73 / Chapter 2.6.2 --- Microinjection / Chapter 2.6.2.1 --- Handling of microinjection --- p.74 / Chapter 2.6.2.2 --- Evidence of gene transfer by microinjection / Chapter 2.6.2.2.1 --- CAT assay --- p.75 / Chapter 2.6.2.2.2 --- PCR and Southern blotting of PCR products --- p.75 / Chapter 2.7 --- Phenotypic alteration of fish generated from electroporated eggs / Chapter 2.7.1 --- Electroporation and handling of fish generated from electroporation --- p.75 / Chapter 2.7.2 --- Measurement of phenotypic change in fish generated from electroporation --- p.77 / Chapter 2.8 --- Detection of transgene and expression of exogenous DNA / Chapter 2.8.1 --- Transgene detection --- p.78 / Chapter 2.8.2 --- Expression of exogenous DNA --- p.79 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Construction of Chimeric growth hormone genes / Chapter 3.1.1 --- Confirmation of integrity of chimeric genes / PEPCKgcGHcDNA --- p.80 / RSVgcGHcDNA --- p.81 / hMTgcGHcDNA --- p.81 / MMTVgcGHcDNA --- p.81 / "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.82 / Chapter 3.1.2 --- Yield of chimeric genes from CsCl density gradient centrifugation --- p.82 / Chapter 3.2 --- Chimeric gene expression in COS-7 and HepG2 cells / Chapter 3.2.1 --- Expression of chimeric genes in COS-7 cells --- p.89 / Chapter 3.2.2 --- Expression of chimeric genes in HepG2 cells --- p.93 / Chapter 3.3 --- Transfer of chimeric genes into embryos / Chapter 3.3.1 --- Electroporation / Chapter 3.3.1.1 --- Monitoring of electroporation --- p.94 / Chapter 3.3.1.2 --- Evidence for gene transfer / Chapter 3.3.1.2.1 --- CAT assay --- p.98 / Chapter 3.3.1.2.2 --- Dot blotting --- p.98 / Chapter 3.3.1.2.3 --- PCR and Southern blotting of PCR product --- p.101 / Chapter 3.3.1.2.4 --- Southern blotting of DNA from fish generated from electroporation --- p.106 / Chapter 3.3.2 --- Microinjection / Chapter 3.3.2.1 --- CAT assay --- p.109 / Chapter 3.3.2.2 --- PCR --- p.109 / Chapter 3.4 --- Phenotypic alterations of fish / The first experiment --- p.112 / The second experiment --- p.113 / The third experiment --- p.113 / The fourth experiment --- p.122 / Chapter 3.5 --- Detection of transgene and expression of exogenous DNA / Chapter 3.5.1 --- Transgene --- p.128 / Chapter 3.5.2 --- Possible expression of exogenous DNA --- p.129 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Chimeric growth hormone genes --- p.132 / Chapter 4.2 --- Expression of chimeric growth hormone genes in COS-7 and HepG2 cells --- p.134 / Chapter 4.3 --- Transfer of exogenous DNA into embyros --- p.136 / Chapter 4.4 --- Phenotypic alteration of fish developed from electroporated eggs --- p.139 / Chapter 4.5 --- The possible integration and expression of exogenous DNA --- p.143 / Chapter 4.6 --- Conclusions --- p.145 / Chapter 4.7 --- Suggestions for further studies --- p.146 / REFERENCES --- p.148 / Chapter APPENDIX I --- Restriction maps / PEPCKgcGH / PEPCKgcGHcDNA / RSVgcGH / RSVgcGHcDNA / hMTgcGH / hMTgcGHcDNA / MMTVgcGHcDNA / pBH1.2 / pMSG-CAT / pUC19 / hMT-IIA / PBC12BI / RSVCAT / pUC101 / pSEl/S2 / PUCSE2/S1 / pUCS2