title: Effects of Drying Conditions on Protein Properties of Blood meal

authors: Damba, Chawangwa

subjects: Effects of Drying Conditions on Protein

abstract: Blood meal is a by-product of the meat industry produced through drying of animal blood. It contains about 85 wt.% proteins. Drying has been used as a method to preserve biomaterials and involves lowering of the water activity of biomaterials. The purpose of this research was to study the drying kinetics of producing blood meal in an oven dryer, to evaluate suitable drying models for describing the drying process and to determine the effects of drying conditions on the physio-chemical properties of blood meal.
Moisture content and drying rates were determined by drying coagulated blood at different temperatures (60 °C, 100 °C and 140 °C) for a constant period of 24 hours. The initial moisture content of coagulated blood was about 60.7 wt% on a wet basis. A drying temperature of 140 °C was found to be the optimal for routine moisture determination for coagulated blood as equilibrium moisture was achieved within 24 hours period. A constant drying rate period was not observed in any of the conditions tested, and the initial increasing rate period as followed by a short transition phase prior to the falling-rate period. Thus, moisture removal from the coagulated blood was governed by a diffusion-controlled process.
The experimental drying data for coagulated blood was used to fit the Lewis, Page, Modified Page, Logarithmic and Henderson and Pabis models and the statistical validity of models tested were determined by non-linear regression analysis. The Page model had the highest R² (0.9999) and lowest χ² (0.0001) and RMSE values. This indicated that Page model adequately described the oven drying behaviour of coagulated blood. 
Blood meal samples were produced by drying coagulated blood at different temperatures (60 °C, 100 °C, 140 °C) to varying moisture contents (5 %, 10 % and 15 %). A drop penetration test using water and/or sodium dodecyl sulphate dissolved in water were used to determine the wettability of the samples produce while thermal analysis techniques such as Thermo-Gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Dynamic Mechanical Analysis (DMA) were used to investigate the thermal properties of bloodmeal. X-ray scattering was used to investigate conformational changes in blood meal proteins during drying. 
Drying conditions had substantial effects on both physicochemical and thermal properties of blood meal. It was established that drying temperature had a more significant effect on the wettability of blood meal than the final moisture content. However, the final moisture content had larger contribution to the thermal stability of blood meal than drying temperature. Blood meal produced at 60 °C to 15% moisture content was the most stable sample while blood meal produced at 140 °C to 10% moisture content was the least stable.
Protein denaturation was observed at 92 °C to 122 °C, depending on moisture content and drying temperature. DMA results revealed that different relaxations occurred when drying coagulated blood. A dry glass transition temperature for samples was observed between 219 °C - 226.8 °C. This suggested that bound water does not act as plasticiser in blood meal. Glass transitions observed in DSC were therefore, considered more accurate and reliable for blood meal samples containing moisture. Drying of coagulated blood was observed to have drastic effect on the structural arrangement of coagulated blood in XRD. Increase in moisture content was observed to have an effect on the β-sheets structure of samples dried at 60 °C and 100 °C. Since bloodmeal produced at 60 °C and 100 °C did not show complete denaturation of proteins, future thermoplastic processing should consider blood meal produced within this temperature range for improved properties.

text: Effects of Drying Conditions on Protein Properties of Blood meal Blood meal is a by-product of the meat industry produced through drying of animal blood. It contains about 85 wt.% proteins. Drying has been used as a method to preserve biomaterials and involves lowering of the water activity of biomaterials. The purpose of this research was to study the drying kinetics of producing blood meal in an oven dryer, to evaluate suitable drying models for describing the drying process and to determine the effects of drying conditions on the physio-chemical properties of blood meal.
Moisture content and drying rates were determined by drying coagulated blood at different temperatures (60 °C, 100 °C and 140 °C) for a constant period of 24 hours. The initial moisture content of coagulated blood was about 60.7 wt% on a wet basis. A drying temperature of 140 °C was found to be the optimal for routine moisture determination for coagulated blood as equilibrium moisture was achieved within 24 hours period. A constant drying rate period was not observed in any of the conditions tested, and the initial increasing rate period as followed by a short transition phase prior to the falling-rate period. Thus, moisture removal from the coagulated blood was governed by a diffusion-controlled process.
The experimental drying data for coagulated blood was used to fit the Lewis, Page, Modified Page, Logarithmic and Henderson and Pabis models and the statistical validity of models tested were determined by non-linear regression analysis. The Page model had the highest R² (0.9999) and lowest χ² (0.0001) and RMSE values. This indicated that Page model adequately described the oven drying behaviour of coagulated blood. 
Blood meal samples were produced by drying coagulated blood at different temperatures (60 °C, 100 °C, 140 °C) to varying moisture contents (5 %, 10 % and 15 %). A drop penetration test using water and/or sodium dodecyl sulphate dissolved in water were used to determine the wettability of the samples produce while thermal analysis techniques such as Thermo-Gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Dynamic Mechanical Analysis (DMA) were used to investigate the thermal properties of bloodmeal. X-ray scattering was used to investigate conformational changes in blood meal proteins during drying. 
Drying conditions had substantial effects on both physicochemical and thermal properties of blood meal. It was established that drying temperature had a more significant effect on the wettability of blood meal than the final moisture content. However, the final moisture content had larger contribution to the thermal stability of blood meal than drying temperature. Blood meal produced at 60 °C to 15% moisture content was the most stable sample while blood meal produced at 140 °C to 10% moisture content was the least stable.
Protein denaturation was observed at 92 °C to 122 °C, depending on moisture content and drying temperature. DMA results revealed that different relaxations occurred when drying coagulated blood. A dry glass transition temperature for samples was observed between 219 °C - 226.8 °C. This suggested that bound water does not act as plasticiser in blood meal. Glass transitions observed in DSC were therefore, considered more accurate and reliable for blood meal samples containing moisture. Drying of coagulated blood was observed to have drastic effect on the structural arrangement of coagulated blood in XRD. Increase in moisture content was observed to have an effect on the β-sheets structure of samples dried at 60 °C and 100 °C. Since bloodmeal produced at 60 °C and 100 °C did not show complete denaturation of proteins, future thermoplastic processing should consider blood meal produced within this temperature range for improved properties.

year: 2017

title: The structural and mechanistic characterisation of Thermophilic RNA Ligases

authors: Kamal, Aliyah

subjects: RNA Ligase

abstract: MicroRNAs (miRNAs) are short regulatory RNAs that play critical roles in post-transcriptional gene regulation. Dysregulation of miRNA expression can be implicated in numerous diseases, including cancer, making miRNAs valuable diagnostic and prognostic biomarkers. Next-generation sequencing (NGS) enables quantitative profiling of miRNA populations, but a key limitation lies in the adapter ligation step required for library preparation. Current commercial RNA ligases, typically active between 14-37 ℃, display strong biases based on RNA sequence and secondary structure resulting in a skewed representation of miRNA populations. At low temperatures, miRNA secondary structures can obscure 5’-phosphate, and 3’-hydroxyl ends, preventing efficient ligation. Thermophilic RNA ligases with high thermostability and distinct substrate preferences may therefore reduce ligation bias and improve NGS accuracy. The archaeal Paleococcus pacificus RNA ligase (PpaRnl) represents a valuable model for exploring the structural and mechanistic features of thermophilic RNA ligases. 
This study aimed to further investigate the substrate preference of a PpaRnl mutant; K238G, a point mutation of lysine at position 238, a key residue in the coordination of the AMP and in turn the binding of the Mg2+ ions in PpaRnl. Alongside the structural and mechanistic characterisation of several PpaRnl mutants to explore the role of a newly identified Mg2+ binding site (Mg2+ B) close to the active site. Key residues for coordinating the Mg2+B ion were mutated in the PpaRnl enzyme (D94A, D248A, E155A, and the double mutant D94A-D248A) as characterisation of these mutants would allow for a deeper understanding of this mechanism. 
Mutants were expressed in Escherichia coli and purified using a combination of immobilised metal affinity chromatography (IMAC) and gel-filtration chromatography. Expression and purification outcomes varied significantly between constructs, while some mutants were successfully obtained in sufficient yields, others demonstrated poor or inconsistent expression, often not even seeing improvement after optimisation of growth conditions and large-scale culture volumes. In particular, the E155A mutant displayed extremely slow growth, while the D94A-D248A double mutant produced negligible results across expression trials. Further characterisation of the mutants, K238G, D94A, and D248A, had shown that these enzymes retain robust activity between 60-65 ℃, with measurable activity extending up to 80 ℃.  Distinct sequence and cofactor preferences were observed. When combined with adapters, these mutants efficiently produced RNA ligation products with minimal side reactions and, in several cases, displayed improved activity compared to the wild-type enzyme. 
The structures of PpaRnl D94A and D248A were solved via X-ray crystallography and although there was similarity in the structures compared to that of the wild-type enzyme and K238G, it was found that a number of the residues coordinating the ligands present within the active-site had changed their orientation. This resulted in the Mg2+B ion not being present in D94A, and no AMP cofactor, Mg2+A or Mg2+B present within the D248A mutant.
Overall, this work highlighted the variability in expression and crystallisation outcomes among the second Mg2+ binding site mutants of PpaRnl with notable differences observed in expression, activity, and structure, highlighting that the Mg2+B ion and its coordination have an impact of the efficiency of the enzyme. This unique mechanism is still not fully understood but its importance and impact has been highlighted within this research, reiterating the importance of construct specific optimisation in structural studies and the potential utility of engineered thermophilic RNA ligases, as tools to reduce ligation bias in NGS based miRNA sequencing. Allowing for the possible discovery of biomarkers within viral diseases, and cancer.

text: The structural and mechanistic characterisation of Thermophilic RNA Ligases MicroRNAs (miRNAs) are short regulatory RNAs that play critical roles in post-transcriptional gene regulation. Dysregulation of miRNA expression can be implicated in numerous diseases, including cancer, making miRNAs valuable diagnostic and prognostic biomarkers. Next-generation sequencing (NGS) enables quantitative profiling of miRNA populations, but a key limitation lies in the adapter ligation step required for library preparation. Current commercial RNA ligases, typically active between 14-37 ℃, display strong biases based on RNA sequence and secondary structure resulting in a skewed representation of miRNA populations. At low temperatures, miRNA secondary structures can obscure 5’-phosphate, and 3’-hydroxyl ends, preventing efficient ligation. Thermophilic RNA ligases with high thermostability and distinct substrate preferences may therefore reduce ligation bias and improve NGS accuracy. The archaeal Paleococcus pacificus RNA ligase (PpaRnl) represents a valuable model for exploring the structural and mechanistic features of thermophilic RNA ligases. 
This study aimed to further investigate the substrate preference of a PpaRnl mutant; K238G, a point mutation of lysine at position 238, a key residue in the coordination of the AMP and in turn the binding of the Mg2+ ions in PpaRnl. Alongside the structural and mechanistic characterisation of several PpaRnl mutants to explore the role of a newly identified Mg2+ binding site (Mg2+ B) close to the active site. Key residues for coordinating the Mg2+B ion were mutated in the PpaRnl enzyme (D94A, D248A, E155A, and the double mutant D94A-D248A) as characterisation of these mutants would allow for a deeper understanding of this mechanism. 
Mutants were expressed in Escherichia coli and purified using a combination of immobilised metal affinity chromatography (IMAC) and gel-filtration chromatography. Expression and purification outcomes varied significantly between constructs, while some mutants were successfully obtained in sufficient yields, others demonstrated poor or inconsistent expression, often not even seeing improvement after optimisation of growth conditions and large-scale culture volumes. In particular, the E155A mutant displayed extremely slow growth, while the D94A-D248A double mutant produced negligible results across expression trials. Further characterisation of the mutants, K238G, D94A, and D248A, had shown that these enzymes retain robust activity between 60-65 ℃, with measurable activity extending up to 80 ℃.  Distinct sequence and cofactor preferences were observed. When combined with adapters, these mutants efficiently produced RNA ligation products with minimal side reactions and, in several cases, displayed improved activity compared to the wild-type enzyme. 
The structures of PpaRnl D94A and D248A were solved via X-ray crystallography and although there was similarity in the structures compared to that of the wild-type enzyme and K238G, it was found that a number of the residues coordinating the ligands present within the active-site had changed their orientation. This resulted in the Mg2+B ion not being present in D94A, and no AMP cofactor, Mg2+A or Mg2+B present within the D248A mutant.
Overall, this work highlighted the variability in expression and crystallisation outcomes among the second Mg2+ binding site mutants of PpaRnl with notable differences observed in expression, activity, and structure, highlighting that the Mg2+B ion and its coordination have an impact of the efficiency of the enzyme. This unique mechanism is still not fully understood but its importance and impact has been highlighted within this research, reiterating the importance of construct specific optimisation in structural studies and the potential utility of engineered thermophilic RNA ligases, as tools to reduce ligation bias in NGS based miRNA sequencing. Allowing for the possible discovery of biomarkers within viral diseases, and cancer.

year: 2025-09-14

title: Māori whānau experiences of a neonatal intensive care unit: Waikato hospital

authors: Thompson, Keri

subjects: Maori whanau narratives

abstract: This thesis topic emerged from my personal experience of having a premature baby in the Neonatal Intensive Care Unit (NICU) at Waikato hospital.  My whānau and I felt like aliens; culturally inept, and totally alone during our time in the NICU.  It was not until I spoke to other Māori mothers that I realised our stories were similar.   

I sought to understand how other Māori whānau members made sense of their own experiences, how they coped/adjusted to the experience of having a premature baby in an NICU and at home, and the sources of support they utilised. This study did not seek to delineate parents' experiences but attempted to allow whānau members to share their stories and explore their experiences of having a premature or special-care baby in an NICU.

The event of childbirth and the placing of a natural event into a pathological illness model through necessity has repercussions for the ways in which women experience and make sense of the event.  Knowing this, six whānau stories were collected comprising of four individual interviews with the mothers and two whānau hui (focus groups).  My use of a narrative methodology and kaupapa Māori approach enabled all the participants the opportunity to share their stories on how they make sense of their own experiences without the frame of pathological illness. 

The findings present each whānau story autonomously, to highlight the key events that   occur once the birth of a premature/special-care infant is admitted into an NICU. The participants' narratives followed their transitions through an NICU and return home. The narrative analysis and discussion confirmed that this research had been successful in creating a space for the mothers and their whānau members to share their stories.  The discussion combined their narratives to reveal that the effective coping strategies did not remove the sources of stress but rather lessened the impact of the stresses.  In situations where sources of stress were unable to be removed or reduced, participants perceptions of ineffective coping translated into narratives of negative perceptions of the NICU, and birthing experience. 

The importance of positive relationships within an NICU, and in particular, the role of the nurse as a key facilitator, highlighted that efficacious communication resulted in positive experiences for Māori whānau.  Whānau support was identified as one of the most important coping strategies for mothers in the NICU.  Those participants who were encouraged to maintain connections with whānau members reported more positive experiences overall.

Three levels of implications were identified in this research. Firstly, that the participants'narratives provided a range of coping strategies for future Māori parents and their whānau who enter an NICU.  Secondly, there are implications for NICU policy, and best practice particularly when supporting Māori in an NICU environment.  Lastly, the narratives provided meaningful insights that contribute to gaps in local, national, and international literature.  Overall, I realised that just like me, these mothers, and other whānau members, constructed narratives that are meaningful accounts to be shared with others.

text: Māori whānau experiences of a neonatal intensive care unit: Waikato hospital This thesis topic emerged from my personal experience of having a premature baby in the Neonatal Intensive Care Unit (NICU) at Waikato hospital.  My whānau and I felt like aliens; culturally inept, and totally alone during our time in the NICU.  It was not until I spoke to other Māori mothers that I realised our stories were similar.   

I sought to understand how other Māori whānau members made sense of their own experiences, how they coped/adjusted to the experience of having a premature baby in an NICU and at home, and the sources of support they utilised. This study did not seek to delineate parents' experiences but attempted to allow whānau members to share their stories and explore their experiences of having a premature or special-care baby in an NICU.

The event of childbirth and the placing of a natural event into a pathological illness model through necessity has repercussions for the ways in which women experience and make sense of the event.  Knowing this, six whānau stories were collected comprising of four individual interviews with the mothers and two whānau hui (focus groups).  My use of a narrative methodology and kaupapa Māori approach enabled all the participants the opportunity to share their stories on how they make sense of their own experiences without the frame of pathological illness. 

The findings present each whānau story autonomously, to highlight the key events that   occur once the birth of a premature/special-care infant is admitted into an NICU. The participants' narratives followed their transitions through an NICU and return home. The narrative analysis and discussion confirmed that this research had been successful in creating a space for the mothers and their whānau members to share their stories.  The discussion combined their narratives to reveal that the effective coping strategies did not remove the sources of stress but rather lessened the impact of the stresses.  In situations where sources of stress were unable to be removed or reduced, participants perceptions of ineffective coping translated into narratives of negative perceptions of the NICU, and birthing experience. 

The importance of positive relationships within an NICU, and in particular, the role of the nurse as a key facilitator, highlighted that efficacious communication resulted in positive experiences for Māori whānau.  Whānau support was identified as one of the most important coping strategies for mothers in the NICU.  Those participants who were encouraged to maintain connections with whānau members reported more positive experiences overall.

Three levels of implications were identified in this research. Firstly, that the participants'narratives provided a range of coping strategies for future Māori parents and their whānau who enter an NICU.  Secondly, there are implications for NICU policy, and best practice particularly when supporting Māori in an NICU environment.  Lastly, the narratives provided meaningful insights that contribute to gaps in local, national, and international literature.  Overall, I realised that just like me, these mothers, and other whānau members, constructed narratives that are meaningful accounts to be shared with others.

year: 2009