Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.

Targeting a small portion of customers for advertising has long been recognized by businesses. In this thesis we proposed a novel approach to promoting products with no prior transaction records. This approach starts with discovering the GP-association rules between customer types and product genres that had occurred frequently in transaction records. Customers are characterized by demographic attributes, some of these attributes have concept hierarchies and products can be generalized through some product taxonomy. Based on GP-association rules set, we developed a comprehensive algorithm to locating a short list of prospective customers for a given promotion product. The new approach was evaluated using the patron¡¦s circulation data from OPAC system of our university library. We measured the accuracy of estimated method and the effectiveness of targeted advertising in different parameters. The result shows that our approach achieved higher accuracy and effectiveness than other methods.

Histone deacetylase (HDAC) inhibition has recently emerged as a novel therapy for cancer treatment. However, currently approved histone deacetylase inhibitors (HDACi) are pan-inhibitors thus inhibiting all 11 zinc dependent HDAC isoforms including those not involved in tumorigenesis. These inhibitors are also associated with various side effects including a potentially fatal cardiotoxicity. To address these issues, isoform selective HDACi were designed and synthesized. The use of 3-hydroxy-pyridin-2-thione (3HPT) as zinc chelation group resulted in small molecules devoid of HDAC1 inhibition but active against HDAC6 and/or 8. Selected 3HPT containing HDACi displayed anticancer activity against various cancer cell lines including DU145, LNCaP and Jurkat. Surprisingly, the lead-compounds were very potent against Jurkat Jγ cells which are resistant to SAHA-induced apoptosis. HDACi were also targeted to cancer cells using folic or pteroic acids as targeting groups. Incorporation of the folic acid into the HDACi pharmacophoric model resulted in inhibitors selective for HDAC6, whereas pteroic-based HDACi inhibited both HDAC1 and 6. Only the pteroic-based inhibitors displayed anticancer activities against folate receptor overexpressing tumors such KB and HeLa. Furthermore, cell-based studies established the inhibition of HDAC1 as the basis for the anticancer activities of the pteroic-based HDACi.

Targeted radionuclide therapy (TRT) holds great promise for the treatment of cancer. In TRT, radioactive nuclides are delivered specifically to tumours by molecules that recognise and bind to structures overexpressed by, or specific to, cancer cells. Human epidermal growth factor receptor like protein 2 (HER-2) is an oncogene product overexpressed in e.g. urological, breast, or ovarian cancers that have been correlated to poor prognosis and resistance to hormonal therapy. There is also evidence that tumour cells retain their HER-2 overexpression in metastases. Trastuzumab and pertuzumab are two humanised monoclonal antibodies targeting different parts of HER-2. This thesis describes the radiolabelling of these antibodies for use in TRT and diagnostics. The thesis also investigates possible methods for modifying uptake and retention of radioactivity delivered with antibodies binding to HER-2. Modification of the cellular retention of 125I by using polyhedral boron anion based linker molecules (DABI and NBI) is investigated, and it is shown that linking 125I to trastuzumab using DABI increases cellular accumulation of radioactivity by 33%. It is also shown that trastuzumab can be efficiently coupled to the positron emitter 76Br by using NBI. Furthermore, it is shown that cellular uptake of 125I can be modified by stimulating EGFR (HER-1) with EGF. When labelled with the alpha emitter 211At, trastuzumab could specifically kill cells in vitro. This cell killing effect could be prevented by saturating the receptors of the target cells with non-radiolabelled trastuzumab. Pertuzumab was radiolabelled with the low energy beta emitter 177Lu without losing affinity or immunocompetence. [177Lu]pertuzumab was specific to HER-2 in vitro and in vivo. This targeting conjugate was shown to increase median time to tumour progression in mice bearing xenografts of the radioresistant SKOV-3 cell line. In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with 177Lu, show promise as TRT agents.

Modern warfare in general, and targeted killing (TK) in particular, challenge conventional legal paradigms. While some contend that targeted killing is a clear violation of law, others argue that it is the law that should adapt to its modern context. In this thesis, I argue in favor of the latter. I will first explain the two dominant paradigms through which one can interpret TK: law enforcement versus armed conflict, going on to argue that an armed conflict paradigm can be legitimately invoked. In sections IV and V, I examine the rights and status of targeted individuals in modern conflict. I will then explore Jeremy Waldron’s objection to TK—that its potential for abuse outweighs its utility. I conclude by arguing that TK, like all warfare, is justified only by the unacceptability of its alternative, and that the justification of all warfare abides under the same pragmatic presumption.

Immuno-nanoparticles of poly(ᴅ,ʟ-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-g-poly(ethylene glycol) (poly(LA-co-TMCC)-g-PEG) have been used to target breast cancer cells through the specific binding of trastuzumab antibodies to over-expressed human epidermal growth factor receptor 2 (HER2). Small interfering RNA (siRNA) and antisense oligonucleotides (AONs) disrupt the synthesis of select proteins. It is hypothesized that oligonucleotides coupled to polymeric immuno-nanoparticles can be used for gene silencing and specifically to target luciferase. The first objective is to demonstrate the capacity to create dual functional micelles with antibodies and oligonucleotides. The second objective is in vitro testing of the nanoparticle for gene silencing activity. Oligonucleotides are conjugated to the nanoparticle by sequential click reactions of Diels Alder chemistry and copper catalyzed azide-alkyne cycloadditions, respectively. A luciferase assay is used to quantify knockdown of luciferase levels in SKOV-3luc cells (HER2+, luc+). When used in conjunction with a targeted drug delivery vehicle, the nanoparticles provide selective interactions with SKOV-3luc cells.

Immuno-nanoparticles of poly(ᴅ,ʟ-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-g-poly(ethylene glycol) (poly(LA-co-TMCC)-g-PEG) have been used to target breast cancer cells through the specific binding of trastuzumab antibodies to over-expressed human epidermal growth factor receptor 2 (HER2). Small interfering RNA (siRNA) and antisense oligonucleotides (AONs) disrupt the synthesis of select proteins. It is hypothesized that oligonucleotides coupled to polymeric immuno-nanoparticles can be used for gene silencing and specifically to target luciferase. The first objective is to demonstrate the capacity to create dual functional micelles with antibodies and oligonucleotides. The second objective is in vitro testing of the nanoparticle for gene silencing activity. Oligonucleotides are conjugated to the nanoparticle by sequential click reactions of Diels Alder chemistry and copper catalyzed azide-alkyne cycloadditions, respectively. A luciferase assay is used to quantify knockdown of luciferase levels in SKOV-3luc cells (HER2+, luc+). When used in conjunction with a targeted drug delivery vehicle, the nanoparticles provide selective interactions with SKOV-3luc cells.

No description available.

No description available.

The oncogenic transcription factor STAT3 is aberrantly activated in over 70% of human tumours, including Multiple myeloma (MM). The present studies use both genetic and chemical tools to validate STAT3 as a therapeutic target, and demonstrate the anti-MM activity of a novel small molecule STAT3 inhibitor, BP-4-018. We show that shRNA-mediated STAT3 knockdown induces apoptosis in human myeloma cell lines (HMCLs). We translate these findings to a therapeutically relevant setting by demonstrating the broad anti-MM activity of BP-4-018 against HCMLs and primary patient samples, and demonstrate that BP-4-018 remains active against HMCLs co-cultured with bone marrow stroma. Inhibiting STAT3 via shRNA knockdown and BP-4-018 suppresses STAT3 transcriptional activity and down-regulates anti-apoptotic and proliferative STAT3 target genes. Finally, we show that BP-4-018 has activity in vivo, both alone and combined with subtherapeutic doses of bortezomib, without significant toxicities. Taken together, these data support the utility of STAT3 inhibitors for MM treatment.