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Identification of Novel Modulators Towards High Cell
Density and High Producing Chinese Hamster Ovary
Suspension Cell Cultures As Well As Their Application in
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 15

Identification of novel modulators towards high cell density

and high-producing Chinese hamster ovary suspension cell
cultures as well as their application in biopharmaceutical
protein production

B EAT
T HALMANN

λογος
Technischen Fakultät
Biotechnologie an der

Bielefelder Schriften
zur molekularen Biotechnologie
Herausgeber:
AG Zellkulturtechnik (Prof. Dr. Thomas Noll)
AG Zelluläre und molekulare Biotechnologie (Prof. Dr. Kristian Müller)
AG Fermentationstechnik (Prof. Dr. Karl Friehs)
 Identification of novel modulators towards high cell
density and high-producing Chinese hamster ovary
suspension cell cultures as well as their application in
biopharmaceutical protein production

Dissertation

for the completion of the doctoral degree doctor rerum naturalium

at the technical faculty of the Bielefeld University, Germany

Submitted by

Beat Thalmann
(*16.01.1984)
Dipl. Chemist (FH), MSc Biotechnology (FH)
from
Berg am Irchel (ZH), Fischingen (TG) and Pfungen (ZH), Switzerland

2015
Chairperson: Apl. Prof. Dr. techn. Karl Friehs
1st Examiner: Prof. Dr. rer. nat. Thomas Noll
2nd Examiner: Prof. Dr. rer. nat. Kristian Müller
Associate examiner: Dr. rer nat. Heino Büntemeyer

Date of the oral examination: 16.06.2015

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Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind
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c Copyright Logos Verlag Berlin GmbH 2015

Alle Rechte vorbehalten.

ISBN 978-3-8325-4046-3
ISSN 2364-4877

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 Identification of novel modulators towards high cell density and high-producing Chinese hamster ovary
suspension cell cultures as well as their application in biopharmaceutical protein production

The laboratory work was fully established in the period between November 2008 and April
2013 at the Celonic GmbH (Jülich, Germany) according to the current state of the art. All cell
lines that were used are properties of Celonic GmbH. The functionally revealed factors
cgrTtc36 and cgrSnord78 are patented:

Andreas Herrmann, Beat Thalmann, Arndt-René Kelter: Expression System. 05.11.2012;

WO2014/068048 A1
Identification of novel modulators towards high cell density and high-producing Chinese hamster ovary
suspension cell cultures as well as their application in biopharmaceutical protein production
 Identification of novel modulators towards high cell density and high-producing Chinese hamster ovary
suspension cell cultures as well as their application in biopharmaceutical protein production

Dedictated to my daughters Celia Félice and Lucia Florence….

“It’s a magical world … let’s go exploring!”

Calvin and Hobbes, Bill Watterson, 31st December 1995
Identification of novel modulators towards high cell density and high-producing Chinese hamster ovary
suspension cell cultures as well as their application in biopharmaceutical protein production
 Abstract

I Abstract

The present work covers the investigation of growth-promoting, cell density-increasing as well
as productivity-enhancing cellular factors and the application in Chinese hamster ovary
(CHO-K1) production cell lines. The freshly established serum-free progenitor CHO-K1-derived
suspension cell line was chemically mutated achieving a randomly altered cell population,
which enables the selection of cellular clones with superior growth characteristics. Selection
at high osmolality (525 – 600 mOsmol∙kg-1), single cell cloning by limited dilution and clonal
selection under harsh unregulated bioreactor conditions yielding in five high cell
density-growing clones, of which one possessed a 2.5-fold increase in maximal cell density
compared to control. The transcriptome analysis of the superior mutated CHO-S cell clone
with the progenitor clonal control cell line and subsequent verification by quantitative
real-time PCR resulted in six significantly deregulated transcripts. Hereafter, four transcripts
(TPRp, LOC100759461, Gas5 and Ttc36) were validated regarding the influence on growth as
well as production characteristics thereof by transfection of the progenitor and a
hIgG-producing CHO-S cell line. Amongst these, C. griseus Ttc36 showed an immediate effect
in increasing maximal as well as integral viable cell density. Surprisingly, the applied Gas5
variants did not show growth arrest, whilst the isolated over-transcription of intronic
sequence possessing the C/D box snoRNA Snord78 actually exclusively increased the cell
specific productivity and led to a 2.0 to 2.5-fold hIgG titer enhancement. In totally contrast to
Snord78, Ttc36 overexpression did not affect cell specific productivity but the growth rate, cell
density, cell aggregation as well as cell viability in a positive manner. Hence, two cellular
factors with emerging positive impacts on biopharmaceutical recombinant protein production
were revealed and successfully applied in early hIgG production processes. In this thesis,
further insights in results and further discussions on putative mechanisms of these functional
initially discovered cellular factors are provided.

I
Zusammenfassung

II Zusammenfassung

Die vorliegende Arbeit umfasst die Detektion von wachstumsfördernden, die Zelldichte sowie
Produktivität erhöhenden, zellulären Faktoren und deren Anwendung in CHO-K1 basierenden
Produktionszelllinien (Ovarienepithelzellen von Cricetulus griseus). Eine daraus neu etablierte
Serum-freie Suspensionszelllinie wurde durch chemische Mutagene genotypisch verändert,
um eine zufällig veränderte Zellpopulation mit neuen, verbesserten Eigenschaften zu
generieren. Zur Anreicherung von robusten Subpopulation sowie Einzelzellklonen wurde die
mutierte Zellpopulation bei hoher Osmolalität (525 – 600 mOsmol∙kg-1), durch
Einzelzellklonierung und klonale Selektion mittels repetitiven Langzeitkulturen selektiert.
Daraus resultierten fünf CHO-S Zellklone mit erhöhtem und längerem Wachstum bei hoher
Zelldichte, wobei eine klonale Zelllinie eine bis zu 2.5-fache maximale Zelldichte erreichte.
Durch differentielle Transkriptomanalytik und nachfolgende Verifizierung mittels
quantitativer Echtzeit-PCR von ebendiesem Zellklon sowie einer klonalen Kontrollzelllinie
wurden sechs signifikant deregulierte Transkripte ermittelt. Basierend auf
Transfektionsstudien und Überexpression in der Kontroll- sowie einer hIgG-produzierenden
CHO-S Zelllinie wurden vier Transkripte (TPRp, LOC100759461, Gas5 and Ttc36) validiert.
Unter allen Faktoren erwies die Überexpression von C. griseus Ttc36 sich als überaus
vorteilhaft gleichermaßen in Bezug auf die Erhöhung der maximalen sowie integralen viablen
Zelldichte. Anders als erwartet, verursachte die zelluläre Anhäufung der isolierten Gas5
Varianten keinen Wachstumsstopp, wobei die eigenständige Intron-basierte Transkription der
Gas5-stämmigen C/D box snoRNA Snord78 eine drastische Erhöhung der Zell-spezifischen
Produktivität und des hIgG Titers um das 2.0- bis 2.5-fache zur Folge hatte. Dem gegenüber
führte die Überexpression von Ttc36 nicht zu einer Veränderung der Zell-spezifischen
Produktivität, veränderte hingegen die Wachstumsrate, maximale sowie integrale Zelldichte,
die Zellaggregation und die Viabilität im positiven Sinne. Somit wurden zwei zelluläre Faktoren
mit positiven Eigenschaften für die Durchführung und Wirtschaftlichkeit der Produktion von
biopharmazeutischen, rekombinanten Proteinen gefunden sowie erfolgreich auf ihre
Anwendung in der Produktion von hIgGs getestet. Diese Dissertation gibt Einblicke in die
Auswirkungen auf diverse CHO-S Zelllinien sowie mögliche Mechanismen dieser erstmals
funktionell beschriebenen Faktoren.

II
 Contents

III Contents

I Abstract ............................................................................................................. I

II Zusammenfassung ............................................................................................ II

III Contents .......................................................................................................... III

1 Aim of the work ................................................................................................ 1

2 Introduction ...................................................................................................... 2

2.1 Short overview of Chinese hamster ovary cell lines ............................................... 2

2.2 Unspecific mutations: A short survey of their use in cell line generation ............. 3

2.3 Selection methods for cell line generation of distinct phenotypes ........................ 4

2.4 Identification of unknown regulatory cellular factors ............................................ 5

2.4.1 Proteomics............................................................................................................... 6

2.4.2 Transcriptomics ....................................................................................................... 7

2.5 Pathway engineering towards high cell density cultures........................................ 8

2.5.1 Cell cycle engineering .............................................................................................. 8

2.5.2 Modulation of cell death factors ............................................................................. 8

2.5.3 Relevance of heat shock proteins, molecular chaperons and detoxifying enzymes

……. ........................................................................................................................ 10

2.5.4 Hypothermic factors .............................................................................................. 11

2.5.5 Metabolic engineering towards high cell densities .............................................. 12

2.5.6 Increasing the maximal cell density by pathway engineering – A summary ........ 13

2.6 Towards high cell density growing CHO cell lines: A theoretical approach .......... 14

3 Material and methods .................................................................................... 17

3.1 Cultivation ............................................................................................................. 17

3.2 Sampling ................................................................................................................ 19

III
Contents

3.2.1 Cell culture supernatant recovery for analytical purpose .................................... 19

3.2.2 Cell pellet recovery for RNA and genomic DNA isolation ..................................... 19

3.3 Manual cell counting ............................................................................................. 21

3.4 Cryopreservation and thawing .............................................................................. 21

3.5 Next generation sequencing ................................................................................. 22

3.6 Quantitative real-time PCR analysis ...................................................................... 22

3.6.1 Total RNA isolation ................................................................................................ 22

3.6.2 cDNA generation ................................................................................................... 22

3.6.3 Quantitative real-time PCR analysis ...................................................................... 23

3.7 Preparative Methods............................................................................................. 26

3.7.1 Genomic DNA isolation ......................................................................................... 26

3.7.2 Preparative PCR ..................................................................................................... 27

3.7.3 Custom gene synthesis .......................................................................................... 29

3.7.4 Restriction digest ................................................................................................... 29

3.7.5 DNA dephosphorylation ........................................................................................ 30

3.7.6 DNA phosphorylation ............................................................................................ 30

3.7.7 Isolation and Purification of DNA Fragments ........................................................ 30

3.7.8 Ligation .................................................................................................................. 30

3.7.9 Transformation of competent E.coli DH5α cells ................................................... 30

3.7.10 E.coli cultivation .................................................................................................... 31

3.7.11 Plasmid DNA preparation ...................................................................................... 32

3.7.12 Linearisation of vectors and sterile DNA precipitation ......................................... 32

3.8 Vectors and cell line generation ............................................................................ 33

3.8.1 Overview on basal vectors and their derivatives .................................................. 33

IV
 Contents

3.8.2 Nucleofection of CHO-S cell lines .......................................................................... 35

3.8.3 Selection of positively transfected cells and further cultivation .......................... 35

3.8.4 Limited dilution and clonal cell line generation .................................................... 36

3.9 Analytics ................................................................................................................ 37

3.9.1 Fixation of cells and DAPI staining ........................................................................ 37

3.9.2 Agarose Gel Electrophoresis ................................................................................. 37

3.9.3 Plasmid DNA Sequencing ...................................................................................... 39

3.9.4 Determination of glucose concentration .............................................................. 39

3.9.5 hIgG ELISA .............................................................................................................. 40

3.9.6 SDS-PAGE ............................................................................................................... 42

3.10 Data compilation and computation ...................................................................... 43

3.11 Material ................................................................................................................. 44

3.12 Terms and acronyms ............................................................................................. 46

3.13 Cell line terminology.............................................................................................. 46

4 Results ............................................................................................................ 47

4.1 Generation of a clonal CHO cell line with capability growing at HCD ................... 47

4.1.1 Adaption of CHO-K1 cells to CM1035 ................................................................... 47

4.1.2 Chemical mutagenesis and mutated cell regeneration ........................................ 48

4.1.3 Selection of CHO-S-E400 towards growth at high osmolality and glutamine

absence …… ........................................................................................................... 52

4.1.4 Single cell cloning of CHO-S-E400HyOsm .................................................................. 54

4.1.5 Selection of CHO-S-E400HyOsm clones .................................................................... 55

4.1.6 Single cell cloning of 25-CHO-S.............................................................................. 57

4.1.7 Transfection and productivity of CHO-S-E400/1.104 and 25-CHO-S/2.5C8 ......... 58

V
Contents

4.1.8 Accumulating cell culture CHO-S-E400/1.104 and 25-CHO-S/2.5C8 .................... 60

4.1.9 Optimised fedbatch cultures and sampling for factor identification.................... 61

4.2 Factor identification and verification .................................................................... 63

4.2.1 Transcriptomics by HiSeq 2000 ............................................................................. 63

4.2.2 Factor verification using quantitative RT-PCR ....................................................... 64

4.3 Factor validation in non-producing and producer cell lines ................................. 69

4.3.1 Validation of cgrTPRp in non-producing 25-CHO-S/2.5C8 cells ............................ 70

4.3.2 Validation of LOC100759461/TPR repeat-containing protein C10orf93-like ....... 71

4.3.2.1 Structural and homological relationship of C10orf93-like species ......................... 71

4.3.2.2 Validation of cgrC10orf93-like in 25-CHO-S/2.5C8 and K20-3 cell line .................. 72

4.3.3 Validation of cgrGas5 variants and cgrSnord78 .................................................... 75

4.3.3.1 Identification, isolation and characterisation of cgrGas5 variants ......................... 75

4.3.3.2 Validation of cgrGas5 variants in 25-CHO-S/2.5C8 ................................................... 79

4.3.3.3 Isolation and validation of cgrSnord78 in 25-CHO-S/2.5C8 as well as K20-3 cell line

………. ...................................................................................................................... 82

4.3.3.4 Single cell cloning of K20-3/cgrSnord78 ..................................................................... 86

4.3.3.5 Application of K20-3/cgrSnord78 variants in a production fedbatch process ...... 86

4.3.4 Validation of cgrTtc36 ........................................................................................... 92

4.3.4.1 Amplification of cgrTtc36 .............................................................................................. 92

4.3.4.2 Validation by overexpression in non-producing cell line 25-CHO-S/2.5C8 ............ 93

4.3.4.3 Further effect validation by overexpression in hIgG-producing cell line K20-3 .... 94

4.3.4.4 Clonal variations in K20-3/cgrTtc36 ............................................................................ 98

4.3.4.5 Application of K20-3/cgrTtc36 variants in a production fedbatch process ......... 104

4.3.4.6 An excursus: The co-expression of cgrTtc36 with cgrSnord78 and cgrTPRp ....... 107

VI
 Contents

5 Discussion ......................................................................................................110

5.1 Increase of genomic variation by mutagenesis and the selection process ........ 110

5.1.1 Mutagenesis and recovery of the 25-CHO-S cell line .......................................... 110

5.1.2 Selection towards growth at high cell densities ................................................. 111

5.2 Transcriptomic analysis and data verification..................................................... 113

5.3 The tetratricopeptide repeat-containing proteins .............................................. 117

5.3.1 The effect upon the overexpression of cgrTPRp ................................................. 117

5.3.2 The putative function of cgrTtc36 ....................................................................... 118

5.3.2.1 Prediction of secondary as well as tertiary structure, modifications and cellular

localisation of cgrTtc36 ............................................................................................... 120

5.3.2.2 The interaction of cgrTtc36 with Hsp70 and the impact thereof .......................... 129

5.3.2.3 The mechanism behind cgrKmtTtc36 fusion protein formation and action........ 132

5.3.2.4 Putative mode of inhibition of cgrTtc36-mediated effects .................................... 140

5.3.3 The putative localisation as well as function of cgrC10orf93-like/cgrTtc40-like

…………... ............................................................................................................... 146

5.3.4 The inhibition of cell aggregation in cgrTtc36 and cgrC10orf93-like/cgrTtc40-like

overexpressing cell lines...................................................................................... 150

5.4 Arguments on cgrGas5 and its intronic sequence cgrSnord78 ........................... 159

5.4.1 Insights into the function and structure of cgrGas5 ........................................... 159

5.4.2 Possible function and targets of cgrSnord78 ...................................................... 163

5.5 Optimisation and industrial relevance of cgrSnord78 and cgrTtc36 .................. 172

6 Conclusion and perspectives ..........................................................................176

7 References .....................................................................................................178

8 Abbreviations ................................................................................................207

VII
Contents

VIII
Other documents randomly have
different content
 Communication - Instructor Guide
Second 2023 - Laboratory

Prepared by: Professor Johnson

Date: August 12, 2025

Chapter 1: Study tips and learning strategies

Learning Objective 1: Research findings and conclusions
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Key terms and definitions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Current trends and future directions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 3: Diagram/Chart/Graph]
Learning Objective 4: Best practices and recommendations
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 5: Interdisciplinary approaches
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 5: Diagram/Chart/Graph]
Definition: Practical applications and examples
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Comparative analysis and synthesis
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 7: Interdisciplinary approaches
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 8: Diagram/Chart/Graph]
Example 8: Key terms and definitions
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 9: Diagram/Chart/Graph]
Key Concept: Practical applications and examples
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 10: Diagram/Chart/Graph]
Part 2: Learning outcomes and objectives
Definition: Historical development and evolution
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Experimental procedures and results
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 12: Best practices and recommendations
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Literature review and discussion
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 14: Ethical considerations and implications
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Study tips and learning strategies
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 17: Comparative analysis and synthesis
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 18: Case studies and real-world applications
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 19: Diagram/Chart/Graph]
Key Concept: Key terms and definitions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Methodology 3: Best practices and recommendations
Key Concept: Assessment criteria and rubrics
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 21: Diagram/Chart/Graph]
Definition: Best practices and recommendations
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Theoretical framework and methodology
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Literature review and discussion
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Note: Comparative analysis and synthesis
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Key terms and definitions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Ethical considerations and implications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Comparative analysis and synthesis
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Ethical considerations and implications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Assessment criteria and rubrics
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 30: Diagram/Chart/Graph]
Review 4: Current trends and future directions
Example 30: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Research findings and conclusions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Assessment criteria and rubrics
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Note: Problem-solving strategies and techniques
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 34: Diagram/Chart/Graph]
Key Concept: Study tips and learning strategies
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Study tips and learning strategies
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 36: Diagram/Chart/Graph]
Key Concept: Case studies and real-world applications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 37: Interdisciplinary approaches
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Learning outcomes and objectives
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Current trends and future directions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Conclusion 5: Experimental procedures and results
Important: Study tips and learning strategies
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Critical analysis and evaluation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 42: Diagram/Chart/Graph]
Key Concept: Statistical analysis and interpretation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Important: Key terms and definitions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 44: Theoretical framework and methodology
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 45: Diagram/Chart/Graph]
Example 45: Best practices and recommendations
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Note: Case studies and real-world applications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 47: Critical analysis and evaluation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Interdisciplinary approaches
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 49: Diagram/Chart/Graph]
Practice Problem 49: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Exercise 6: Case studies and real-world applications
Important: Fundamental concepts and principles
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 51: Research findings and conclusions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Key terms and definitions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 53: Diagram/Chart/Graph]
Remember: Practical applications and examples
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 54: Diagram/Chart/Graph]
Key Concept: Experimental procedures and results
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
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