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X-linked Kallmann's syndrome, a disease characterised by anosmia (a consequence of hypoplastic/absent olfactory bulbs) and hypogonadotrophic hypogonadism (due to a defect of GnRH neuron migration into the hypothalamus) is caused by mutations in the KAL gene. Anosmin-1, the protein product of KAL, is extracellular and consists of a cysteine rich C-terminal region, followed by four fibronectin type III (Fnlll) domains. It has been observed in vivo as a 100 kDa glycosylated protein and as additional cleaved 55 kDa and 45 kDa fragments in vitro. Its overall function and domain structure is unknown, but current data suggests a role in neuronal and kidney development. In this thesis: 1. A Two Hybrid assay using a foetal brain cDNA library was utilised to study potential protein/protein interactions involving anosmin-1. Although recombinant anosmin-1 was produced, no positive interactors were identified. 2. Computer comparative modelling methodologies were used to study the potential three dimensional conformation of domains of the mature protein and the nature of amino acid substitutions encoded for by three known missense mutations. Two were shown to occur at buried locations and are predicted to affect the domain structure, whilst the third occurs at an exposed position, identifying this region as potentially biologically important. 3. A potential cleavage site (KKKRRK) located within the second Fnlll was studied using a mutant anosmin-1 lacking this site. Cleavage of this protein was studied and compared to that of the wild type. The results exclude this protein motif as the site of this cleavage. 4. A cell adhesion bioassay was developed to compare a wild type and mutated peptide (containing the N267K amino acid substitution) generated from the first Fnlll domain sequence. An adhesive property for both peptides was observed, and significantly, the mutant showed no loss of function when compared with the wild type.

BBSome is a protein complex that facilitates the cargo trafficking within the cilia with the help of other trafficking complexes. Disruption in the integrity of BBSome complex due to mutations in different BBS gene manifest in a wide range of BBS phenotypes which can be overlapping or unique. Bardet Biedl Syndrome (BBS) is a pleiotropic autosomal recessive disorder characterized by obesity, retinopathy, renal malformation, polydactyly, anosmia, and intellectual disability in both humans and mouse models.Bbs7 is part of BBSome complex and is known to directly interact with the chaperonin complex comprised of Bbs6,Bbs10,Bbs12, which is needed for the BBSome assembly. Bbs7 and Bbs10 mouse model on mixed 129/B6 background have been reported to live to adulthood and manifest BBS phenotypes. Here we used the BBS7 and BBS10 null allele on pure B6 background and found out that homozygotes don’t survive past P0 due to olfactory deficits which prevents them to smell their mother and interferes with successful feeding. We also show that there is better survival of Bbs7 and Bbs10 mutants when they are heterozygous for Ttc21b-aln. We also synthesized incipient congenic 129/B6 strain carrying Bbs7 or Bbs10 null allele and noticed more than 50% survival of homozygotes. Overall, we demonstrate that the genetic background of mouse has profound effects on the severity of pathogenicity of a gene and related phenotypes. We conclude that there are modifiers which are either segregated together with Bbs7 and Bbs10 or are lost during segregation which leads to better survival of mutant animals past P7. In future, we will do the linkage analysis to find out these modifiers.

Zinc is both an essential and potentially toxic metal. It is widely believed that oral zinc supplementation can reduce the effects of the common cold; however, there is strong clinical evidence that intranasal (IN) zinc gluconate (ZG) treatment for this purpose causes anosmia, or the loss of the sense of smell, in humans. Using the rat olfactory neuron cell line, Odora, we investigated the mechanism by which zinc is toxic to olfactory neurons. Following treatment of Odora cells with 100 and 200 µM ZG for 0-24 hr, we performed RNAseq and in silico analyses and found up-regulation of pathways associated with zinc metal response, oxidative stress, and ATP production. We also observed that Odora cells could recover from zinc-induced oxidative stress, but ATP depletion persisted with longer exposure to ZG. We also found that ZG exposure caused an increase in NLRP3 and Il-1ß protein levels in a time-dependent manner, suggesting that zinc causes an inflammasome-mediated cell death, pyroptosis, in olfactory neurons. The role of metallothionein (Mt), a metal response protein whose polymorphisms are associated with metal toxicity, particularly in developing nations, was also investigated. In vitro studies using shRNA knockdown of Mt1/2 demonstrated basal elevated level of markers of reactive oxygen species and depressed levels of reduced glutathione; however, the cells were still able to recover from excess zinc and showed a similar LC50 with untransfected Odora cells. In vivo studies utilizing wild-type, MT1/2 knockout mice (KO), and heterozygotes administered ZG by IN instillation showed profound loss of the olfactory mucosa in the nasal cavity. Recovery was monitored, and a lower percentage of the mice were able to smell 28 d after treatment; however, no significant difference was observed in the average thickness of the olfactory epithelium between KO and wild type mice. Both in vitro and in vivo studies showed down-regulation of actinin levels, which may be the trigger for ZG-induced olfactory mucosal damage.