Histone deacetylases (HDACs) are an ancient class of enzymes that have been conserved throughout evolution and are found in diverse organisms such as animals, plants, fungi, eubacteria and archaebacteria. In C. elegans, twelve HDACs have been identified so far. These HDACs have been grouped into four different classes (Class I, II, III and IV) based on their cofactor requirements and sequence homologies. hda-1 is one of the three Class I HDACs in C. elegans and plays a role in the morphogenesis of several organs including the vulva. This thesis focuses on the role of hda-1 in vulval morphogenesis. The hermaphrodite vulva has twenty-two cells which can be further divided into seven different cell types: VulAs, VulBls, VulB2s, VulCs and VulDs (secondary great granddaughters), YulEs and VulFs (primary great granddaughters). The analysis of expression pattern of hda-1 revealed that hda-1 is expressed in the progeny of both the primary and secondary vulval precursor cells (VPCs). To examine hda-1 mutant phenotype in detail, I examined the expression pattern of five different vulval cell-type specific markers (cdh-3, zmp-1, ceh-2, egl-17 and daf-6) in hda-1 animals. The results revealed that hda-1 is necessary for proper differentiation of multiple vulval cell types. To study the evolutionary conservation of hda-1 function, I examined the role of hda-1 ortholog in C. briggsae. C. briggsae is a close relative of C. elegans and has almost identical vulval morphology. Knocking down Cbr-hda-1 in C. briggsae animals resulted in defective vulval phenotype. Consistent with this result, the expression of two cell- fate specific markers (C. briggsae orthologs of zmp-1 and egl-17) was found to be altered in Cbr-hda-1 RNAi treated animals. Thus, hda-1 function in the vulva appears to be conserved in these two species. To identify the hda-1 targets in vulval morphogenesis in C. elegans, microarray approach was taken. Two genes fos-1 and lin-29 were identified as putative targets and were examined in some detail. Among the targets identified (these still need to be validated), I focused on fos-1 and lin-29 for detailed investigation. The RNAi-mediated knockdown of hda-1 caused alterations in the expression pattern ofthefos-1 transcript,fos-1b. To examine interaction between fos-1 and lin-29, I used double RNAi approach and examinedfos-1 (RNAi), lin- 29 (RNAi), hda-1 (cw2) animals. It was found that fewer animals exhibit defects in vulval morphology in these animals as compared to fos-1 (RNAi), hda-1 (cw2) animals. While this suggests a possible interaction between lin-29 and hda-1 in the vulva, these results need to be validated by doing additional experiments. In summary, the work described in this thesis demonstrates that hda-1 plays an important role in vulval morphogenesis and regulates the expression of several important genes. Also, the function of hda-1 in C. elegans and C. briggsae is evolutionarily conserved. / Thesis / Master of Science (MSc)

Vulval development in C. elegans serves as powerful paradigm to understand the interplay of diverse signal transduction pathways during organogenesis. Previous studies have demostrated that the canonical Wnt signaling pathway plays a pivotal role in the development of the vulva in C. elegans and helps in establishing the 20-10-20 vulval induction pattern of the vulval precursor cells (VPCs). The main focus of my masters research project was to get an understanding of how this vulval induction pattern, established in response to Wnt signaling has evolved in other closely nematode species, particularly C. briggsae. We fmd that the Wnt signaling pathway has evolved to positively as well as negatively regulate the competence of VPCs in C. briggsae. We demonstrate that while mutations inpry-1/Axin in C. elegans result in Multivulva (Muv) phenotype, mutations in the C. briggsae pry-1 gene give rise to a novel MultivulvaVulvaless (Muv-Vul) phenotype. This phenotype is characterized by VPCs anterior to P6.p frequently adopting induced cell fates while those posterior to P6.p frequently adopt a non-induced fate. Furthermore, we also show that the functioning of the Wnt signaling pathway in C. briggsae is dependent upon the activity of key regulators of the Wnt pathway such as the TCFILEF-1 family member pop-1, the f3-catenin bar-] and the hox gene lin-39. Taken together, the fmdings from this study show that while a conserved canonical Wnt pathway confers competence on VPCs in both C. elegans and C. briggsae, the final outcome nonetheless seems to have diversified. / Thesis / Master of Science (MSc)

<p> Caenorhabditis elegans (C. elegans) is a model organism which is known for its transparent body, small body size, high reproductivity and short lifecycle. Several important genes and signal transduction pathways are well conserved in C. elegans. lin//, a LIM homeobox family member, plays a crucial role in the development of the vulva in C. elegans. LIM homeobox genes are a subgroup of Homeobox family that play fundemental role in animal development. In C. elegans lin-If mutant animals fail to form a functional vulva and vulval-uterine connection and consequently exhibit egg-laying defective phenotype. The cell lineage and marker gene expression studies have shown that lin-// is required for the patterning of all primary and secondary lineage vulval cells. lin- II also functions in the nervous system. </p> <p> lin-// expression is mainly observed in the developing vulval cells and in the pi cells which are involved in the formation of vulval-uterine connection. lin-If expression is also seen in VCs and in some of the head and tail neurons. The completed genome sequences of closely related species in Caenorhabditis genus serve as a power tool to do systematic comparative studies. The lin-If regulatory sequences from these species have been compared along with the expression patterns. </p> <p> We looked at the regulation of lin-// in closely related nematode species like C. elegans, C. briggsae, C. remanei and Caenorhabditis n species. </p> <p> Consistent with this. expression of lin-11 is observed in the developing vulval cells. We are interested in understanding evolutionary changes in the regulation and function of lin-II in reproductive system </p> <p> /in-11 is a LIM homeodomain family member which IS involved in several developmental events. lin-11 role is documented in the thermoregulatory circuit specifying AIY interneuron, in chemosensory neurons like A W A and olfactory neurons A WS. During vulval development lin-II expression is dynamically expressed in subset of secondary lineage cells and is broadly expressed in all the cells indicating its role in cell identity and cell fusion of the vulval cells. lin-II is also required for the formation of vulval uterine connection which is the passage to lay eggs in the hermaphrodite. linllloss of function hermaphrodites have change in the axis of the secondary lineage cells during vulval development, uterine Jt cell migration defect, defects in the AIY, A W A and A WS interneurons resulting in egg-laying defect and protruding vulva and neuronal defects and reduced mating efficiency. </p> <p> The expression pattern of lin-If in closely related species is highly similar but not identical. From the sequence comparison of lin-If regulatory sequences a 1 kb conserved block of sequences have been identified which includes the regulatory sequences responsible for the expression of lin-If in vulva and Jt cells. We propose that cisregulatory elements controlling lin-If gene expression are slowly evolving though there is no change in the function which indicates that lin-If plays critical role during the development of the vulva and other tissues. </p> / Thesis / Master of Science (MSc)

Vulval intraepithelial neoplasia (VIN) is a distressing, premalignant condition, frequently associated with type HPV16 infection, with increasing incidence in younger women. Traditional surgical treatment is sub optimal. Several different clinical studies influencing local and/or systemic immunity to HPV have been reported. Imiquimod, an immune response modifier, can stimulate local innate immunity and also drive an adaptive immune response. Therapeutic HPV vaccines are designed to generate cell-mediated immunity against HPV infected cells. The rationale of this study was that local imiquimod treatment, in addition to having a direct effect on VIN could also provide an immunological platform for the therapeutic HPV vaccination to achieve an enhanced and durable response. In this phase II trial, we used a combination of imiquimod and vaccination with TA-CIN (HPV16 E6E7L2 fusion protein). Women with biopsy proven high-grade VIN were recruited. Imiquimod treatment for 8 weeks was followed by three i.m. injections of TA-CIN. The objectives were to measure lesion size, histology, lesion HPV status, symptoms, immune responses before and after treatment as well as safety, toxicity, and tolerability. Lymphoproliferation to HPV antigens was used to analyse immunity to HPV before and after treatment. Local immune factors (CD4, CD8 and T regulatory cells) were assessed by immunofluorescence. 74, 85 and 79% of women had a ≥50% reduction in the size of lesion and 32, 58 and 63% had regression of VIN on histology at weeks 10, 20 and 52 respectively. At baseline, 79% had moderate to severe symptoms compared to 21% at week 52(P=0.01). Of the women who showed histological responses at week 52, 5/10 also cleared their HPV 16 infection. Follow-up for an average of 15 months showed 84% of patients with a ≥50% reduction in lesion size. Treatment was well tolerated. A significant post vaccination increase in proliferation to TA-CIN and its components was associated with histological responders (P=0.008) but not the non-responders (P=0.7). In the group as a whole, significant increases in the number of CD4, CD8 and T regulatory cells (Treg) were evident by week 20 compared to baseline (P=0.03, P=0.01, P= 0.04 respectively); At week 20, the increased CD4 and CD8 density was significantly associated only with the histological responders (P=0.03; P= 0.03) while increased Treg density was associated with only non-responders (P=0.05). Intralesional Treg density was significantly higher in non-responders compared to responders pre and post treatment (P=0.01, 0.05 respectively). This study demonstrated that imiquimod followed by vaccination achieved histological clearance of VIN at 52 weeks in almost 60%. Higher pre-existing and post-treatment levels of Treg cells were associated with a lack of treatment response. Lymphoproliferation of PBMC established that the vaccination was immunogenic and HPV 16 antigen specific. Importantly, these systemic immune responses to HPV 16 antigens were significantly associated with treatment responders.

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