Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Alzheimer's disease (AD) is a large and growing problem and while we today lack a full understanding of this disease, we know that the protein tau and the amyloid fibrils it forms play a central role in its development. We also know that these fibrils can have different morphologies in different diseases and that fibrils produced in vitro not necessarily adopt any of the morphologies found in patients. This means there is a need for more pathologically relevant fibrils in vitro to be able to understand this disease better. One approach to satisfy this need is to use fibrils found in patients as seeds and thus transfer their morphology to recombinantly purified protein. To facilitate this process this study has attempted to develop a way to differentiate between different fibril morphologies using a FRET based system. This involves fluorescent fusion proteins (tau-EXFPs) and fluorescent amyloid probes as well as seeding experiments with pseudo wild type tau (PWT) and tau with the P301L mutation. Greater differences in terms of fibrillation rates and ThT fluorescence between PWT and P301L was shown than previously reported between WT and P301L. They were also shown to differ in fibril morphology in TEM. The ThT fluorescence intensity was to a certain degree transferable from PWT to P301L by seeding. Furthermore, this study confirms that the tau-EXFP fusion protein can be incorporated into amyloid fibrils and strongly suggests that a FRET effect between EXFP and BTD14 (as well as X34 and ThT) can be achieved. It also demonstrates differences in FRET efficiency between PWT and P301L fibrils using FLIM. These results indicate that a FRET based approach could be a useful method to discern different fibril morphologies from each other, but further measurements and optimization are needed before this method could be reliably applied. The fusion proteins could also be used to investigate tau spreading in vivo, e.g. in D. melanogaster. To find suitable FRET partners to the fusion proteins, a ligand screen was conducted. This could be used as an alternative to the FRET method. With the right selection of fluorescent amyloid probes, a unique fingerprint for each fibril morphology could maybe be generated and fulfill the same intended function as the FRET method.

How are neuromodulatory networks organised to adapt sensory discrimination for different contexts? I hypothesised that neurons within a sensory circuit express different neuromodulatory receptors for differential modulation. Here I aimed to use the simple and genetically amenable Drosophila larval Mushroom Body (MB) calyx, a higher order processing area involved in learned odour discrimination, as a model to map octopamine (OA) neuromodulatory circuitry. I first identified olfactory projection neurons (PNs), a GABAergic feedback neuron and cholinergic extrinsic neurons as putative postsynaptic partners to OA neurons in the MB calyx using GFP reconstitution across synaptic partners. Next, I used novel EGFP-tagged OA receptors generated from recombination-mediated cassette exchange with MiMIC insertions in receptor genes to visualise endogenous expression patterns of OA receptors. Most notably, this is the first report of α2-adrenergic-like OA receptor localisation in any insect. For the first time, I showed that the α1-adrenergic-like OAMB localised to PN presynaptic terminals in the calyx; while Octβ1R localised diffusely in the calyx, resembling the innervation pattern of MB neuron dendrites. I detected EGFP-tagged Octα2R and Octβ2R in some PN cell bodies but not in neuron terminals - suggesting that Octα2R and Octβ2R may be expressed in some PNs, provided the misfolded fusion proteins are retained in the cell bodies of the neurons they are normally expressed in. Furthermore, I found that Octα2R and GABAAR fusion proteins localised to OA cell bodies but not to neuronal terminals, suggesting that OA neurons are subjected to inhibition, again given that these are not artefacts of the fusion proteins. To obtain tools to study OA modulation in the larval calyx, I then confirmed the expression patterns of driver lines that more specifically labelled calyx-innervating OA and extrinsic neurons, and tested the efficacy of three OAMB receptor knockdown lines. This initial attempt of mapping OA receptors, while subjected to further verification and development, is consistent with my hypothesis that a single neuromodulatory source can regulate multiple neuronal types in the same circuit through the distribution of different types of neuromodulatory receptors. This provides a new perspective in how the anatomical organisation of neuromodulation within a sensory network may translate to flexible outputs.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

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