published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

For the majority of social species, group composition is dynamic, and individuals are interconnected in a heterogeneous social network. In this study I investigate the mechanisms underpinning social structure in the endangered southern resident killer whale (Orcinus Orca) population using a long term dataset, and explore the consequences of these. My results demonstrate that resource availability may be an important determinant of social network structure. A significant relationship between the connectivity of the social network and salmon abundance occurred, with a more interconnected network in years of high salmon abundance. As networks are non-random, highly connected individuals may play a key role in population processes such as information and disease transmission. While associations occurred both within and between matrilines, females had a significantly higher number of associates than males, as did older individuals of both sexes. Older males played a more important role in interconnecting the network. The attributes of group leadership were then investigated in matrilines and in individuals. Leadership was not a factor of size or mean age of matriline. However, there was a significant relationship between leadership score and the matriline sex ratio. Individually, females had higher leadership scores than males, and there was a positive correlation between leadership score and age in both sexes. I suggest that the oldest females have the highest 4 leadership scores due to increased ecological knowledge that comes with a prolonged lifespan. Using multi-generational records for two populations of killer whales, I show that both reproductive and post-reproductive mothers increase the survival of offspring, particularly in older male offspring. This is consistent with theoretical predictions, and may explain why female killer-whales have evolved the longest post-reproductive lifespan of all non-human animals. Given the role that individuals of high network centrality can play in population processes, understanding the driving forces behind social network structure is vital when designing effective conservation and management plans.

A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy FACULTY OF HEALTH SCIENCES UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG 2014 / Breast tumour progression involves complex interactions between malignant cells and the tumour microenvironment. It is increasingly apparent that immunity is a critical determinant for tumour progression. T regulatory (TREG) lymphocytes, which dominate tumour infiltrating lymphocyte populations, are implicated in facilitating tumour immunoediting processes and suppressing Natural Killer (NK) cell anti-tumour function. To investigate such cellular interaction, experimentation traditionally involves using reductionist 2-dimensional culture systems that do not recapitulate the spatial dimensions of the in vivo microenvironment. Three-dimensional (3D) culture systems, conversely, recreate these dimensions, allowing tumour cells to assume a phenotype more representative of the tumour microenvironment. Given that immunity is a critical factor in determining tumour progression, a novel 3D culture system was established to investigate the interactions between TREG lymphocytes, NK cells and hormone-dependent (MCF-7) or hormone-independent (MDA-MB-231) breast cancer cells. Lymphocyte subpopulations were magnetically isolated, with the efficacy of the sorting procedure verified using flow cytometry. To generate 3D cultures, cell populations were resuspended in growth factor-reduced Matrigel and cultured for 72 hours. This culture system proved effective for RNA extraction for downstream applications; for immunolocalisation of selected tumour biomarkers (ER-α, TGF-β, MUC-1 and EGFR) for qualitative analysis; and for acquisition of cytokine data (IL-1β, IL-2, IL-6, TNF-α, IFN-, CCL2, CCL4 and CXCL8) for quantitative multivariate statistical analysis. Immune mediation was shown to induce the disruption of cell-cell associations, altering the expression of biomarkers and secreted cytokine profiles. Collectively, these results reflect tumour cell subversion of NK cell and/or TREG lymphocyte function to promote tumour progression by generating an inflammatory microenvironment. While hormone-dependent and hormone-independent breast cancer cells differed in their specific response to immune mediation, the mechanisms by which they elicited responses resulted in similar outcomes – that of enhanced evasive and invasive capacity. It is necessary to further elucidate the relationship between the investigated cytokines, biomarkers and immune cells, to understand their interactions and potentially provide more information for therapeutic intervention, given that these factors may contribute to tumours not responding favourably to combined modalities of therapy.

Type 1 innate lymphocytes comprise two developmentally divergent lineages, type 1 helper innate lymphoid cells (hILC1s) and conventional NK (cNK) cells. All type 1 innate lymphocytes (ILCs) express the transcription factor T-bet, but cNK cells additionally express Eomesodermin (Eomes). We show that deletion of Eomes alleles at the onset of type 1 ILC maturation using NKp46-Cre imposes a substantial block in cNK cell development. Formation of the entire lymphoid and non-lymphoid type 1 ILC compartment appears to require the semi-redundant action of both T-bet and Eomes. To determine if Eomes is sufficient to redirect hILC1 development to a cNK cell fate, we generated transgenic mice that express Eomes when and where T-bet is expressed using Tbx21 locus control to drive expression of Eomes codons. Ectopic Eomes expression induces cNK cell-like properties across the lymphoid and non-lymphoid type 1 ILC compartments. To investigate if T-bet is sufficient to direct type 1 ILC development into the hILC1 lineage, we also generated transgenic mice in which Tbx21 locus control drives expression of T-bet codons. Enforced T-bet expression, however, does not appear sufficient to induce hILC1-like attributes among type 1 ILCs. Subsequent to their divergent lineage specification, hILC1s and cNK cells possess substantial developmental plasticity elicited by the absence or presence of Eomes.

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression.

Although Icelandic and Norwegian killer whales are thought to have been in contact prior to the collapse of the herring stock in the 1960s, the Northeast Atlantic killer whales currently seem to show high site fidelity. So far, photoidentification data have suggested movement of a few individuals between East Iceland and North Scotland, and two calls have been shown to be shared by the Icelandic and Norwegian populations. Based on previous and newly analysed call samples, the aim of this study was to describe the geographic variation in the vocal repertoire of the Northeast Atlantic killer whales. Recordings have been conducted off Southwest Iceland in the summers 2004, 2008 and 2009 using sound recording tags attached using suction cups (Dtags), a 4-element vertical hydrophone array and a 2-element towed hydrophone array. From the 57 hours of recording analysed, 1742 calls were classified. In total, 56 distinct call categories composed of 35 call types and 31 subtypes were identified. This discrete call repertoire contained less biphonic calls but more calls composed of buzzes and/or clicks than the Norwegian repertoire. The reasons for these differences remain unknown. One Icelandic call subtype was defined as a compound call, a type of call that is common in the Norwegian population. The comparison of the different vocal repertoires of Northeast Atlantic showed four good or likely call matches in herring-eating killer whales (one between Southwest Iceland and Shetland, one between East Iceland and Norway, and two between Shetland and Norway). No matches were found between Southwest Iceland and East Iceland. I suggest that the four shared calls are most likely to have come from a common ancestral pod and have been transmitted through vocal learning. Over time, geographic isolation of the groups induced by changes in the migratory patterns of the herring might have been accompanied by divergence in their call repertoires.

A study was conducted using data compiled from two sources to test the hypothesis that killer whales display seasonal variability in their occurrence in the Galapagos Marine Reserve (GMR), Ecuador. Three questions arise from this hypothesis: 1) do killer whale sightings display temporal variability; 2) are sightings spatially associated with resources; and 3) if sightings are spatially associated with resources, does the spatial association change temporally? I combined and evaluated two sets of GMR killer whale sighting data (n=154) spanning a twenty-year time frame collected via opportunistic sightings by an observer network and shipboard line-transect surveys. I tested for a (a) correlation between the total annual sightings and bi-annual seasonality (upwelling versus non-upwelling); (b) correlation between the total annual sightings and the Multivariate El Nino Southern Oscillation Index (MEI); (c) correlation between sightings, the MEI, and seasonality; (d) spatial association between sightings and resources; and (e) spatial change in sightings with seasonality. Sightings were roughly equally distributed between non-upwelling (56%) and upwelling seasons (July-December). No direct correlation was found between sightings and the MEI. Sightings occurred more often than expected by chance during the peak upwelling months of August-November when the MEI was within one standard deviation of the average (binomial z=2.91, p<0.05). Sightings were spatially associated with areas of high chlorophyll a values (binomial z=4.46, p&lt;0.05), pinniped rookeries (binomial z=6.03, p&lt;0.05), and areas with high combined resource value (binomial z=5.36, p&lt;0.05). The spatial distribution of sightings did not shift with seasonality, with the exception that sightings occurred less often than expected in areas of low combined resource value during the upwelling period (binomial z=-3.17, p&lt;0.05). Though variability in observer effort should be considered when evaluating these data, these results do not suggest a strong pattern of seasonal occupancy or that killer whales are responsive to El Nino Southern Oscillation events. Further research is needed to determine if killer whales in the GMR comprise a single resident population, multiple resident and transient populations, or if killer whales observed in the GMR are part of a population inhabiting the eastern tropical Pacific region, which visit the area at various times.